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Analysis of protein-protein interaction networks is becoming important for inferring the function of uncharacterized proteins. A recent study using this approach has identified new proteins and interactions that might be involved in the pathogenesis of the neurodegenerative disorder Huntington's disease, including a GTPase-activating protein that co-localizes with protein aggregates in Huntington's disease patients.

The impact of the serotonin transporter (5-HTT) gene-linked polymorphic region (5-HTTLPR) on anxiety-related behavior and related cerebral activation has facilitated the understanding of neurobiological mechanisms of anxiety. However, the influence of the 5-HTTLPR genotype on hippocampal neuronal development and neurochemistry, which is relevant to anxiety behavior, has not been investigated. In 38 healthy subjects, absolute concentrations of N-acetylaspartate (NAA) were measured as a main surrogate parameter for hippocampal neurochemistry on a 3-T scanner. A significantly lower hippocampal NAA concentration in s allele carriers was observed as compared to l/l genotype. Other metabolites (choline, creatine + phosphocreatine, glutamate) were unaffected by genotype. The hippocampal NAA concentration was negatively correlated with trait anxiety scores (STAI). Metabolites measured in the anterior cingulate cortex (reference region) were not associated with genotype. The results are in accordance with the recently reported relationship between hippocampal neuronal development and anxiety behavior in adult animals and show an association between human limbic neurochemistry and genetically driven serotonergic neurotransmission relevant to anxiety.

Netrin-1 receptors UNC5H (UNC5H1-4) were originally proposed to mediate the chemorepulsive activity of netrin-1 during axonal guidance processes. However, UNC5H receptors were more recently described as dependence receptors and, as such, able to trigger apoptosis in the absence of netrin-1. They were also proposed as putative tumor suppressors. Here, we show that UNC5H2 physically interacts with the serine/threonine kinase death-associated protein kinase (DAP-kinase) both in cell culture and in embryonic mouse brains. This interaction occurs in part through the respective death domains of UNC5H2 and DAP-kinase. Moreover, part of UNC5H2 proapoptotic activity occurs through this interaction because UNC5H2-induced cell death is partly impaired in the presence of dominant-negative mutants of DAP-kinase or in DAP-kinase mutant murine embryonic fibroblast cells. In the absence of netrin-1, UNC5H2 reduces DAP-kinase autophosphorylation on Ser308 and increases the catalytic activity of the kinase while netrin-1 blocks UNC5H2-dependent DAP-kinase activation. Thus, the pair netrin-1/UNC5H2 may regulate cell fate by controlling the proapoptotic kinase activity of DAP-kinase.

Two novel fluorescent cyanine-AMP conjugates, F550/570 and F650/670, have been synthesized to serve as transcription initiators under the T7 phi2.5 promoter. Efficient fluorophore labeling of 5' RNA is achieved in a single transcription step by including F550/570 and F650/670 in the transcription solution. The current work makes fluorescently labeled RNA readily available for broad applications in biochemistry, molecular biology, structural biology and biomedicine. In particular, site-specifically fluorophore-labeled large RNAs prepared by the current method may be used to investigate RNA structure, folding and mechanism by various fluorescence techniques. In addition, F550/570 and F650/670 may replace [gamma-32P]ATP to prepare 5' labeled RNA for RNA structural and functional investigation, thereby eliminating the need for the unstable and radio-hazardous [gamma-32P]ATP.

<h4>Background</h4>There is growing evidence that mildly increased amounts of iron in the liver can increase hepatic injury, particularly if combined with other hepatotoxic factors, such as alcohol use, use of porphyrogenic drugs, or chronic viral hepatitis. In the present study, the association of hepatitis C virus (HCV) infection with serum measurements of iron status was assessed in the US population.<h4>Methods</h4>We analyzed data from a total of 14,462 participants in the Third National Health and Nutrition Examination Survey. We excluded subjects who were aged <12 years, subjects for whom measurements of serum levels of iron or ferritin or the results of liver function tests were missing, and subjects who had a serum transferrin saturation of > or =50% (to help exclude subjects with hemochromatosis).<h4>Results</h4>Mean serum levels of ferritin and iron (+/- standard error) were significantly higher among subjects with HCV infection (100+/-3 ng/mL and 229+/-17 microg/dL, respectively) than among subjects without liver disease (83+/-0.3 ng/mL and 101+/-2.1 microg/dL, respectively) (P<.0001). Serum levels of ferritin were directly and significantly correlated with serum levels of alanine aminotransferase, aspartate aminotransferase, and gamma-glutamyl transpeptidase (r=0.25, r=0.24, and r=0.28, respectively; P<.0001), whereas platelet counts were inversely correlated with serum levels of ferritin (r=-0.12; P<.0001).<h4>Conclusion</h4>HCV infection is significantly associated with higher serum levels of ferritin and iron in the US population.

BACKGROUND: DNA self-assembly methods have played a major role in enabling methods for acquiring genetic information without having to resort to sequencing, a relatively slow and costly procedure. However, even self-assembly processes tend to be very slow when they rely upon diffusion on a large scale. Miniaturisation and integration therefore hold the promise of greatly increasing this speed of operation. RESULTS: We have developed a rapid method for implementing the self-assembly of DNA within a microfluidic system by electrically extracting the DNA from an environment containing an uncharged denaturant. By controlling the parameters of the electrophoretic extraction and subsequent analysis of the DNA we are able to control when the hybridisation occurs as well as the degree of hybridisation. By avoiding off-chip processing or long thermal treatments we are able to perform this hybridisation rapidly and can perform hybridisation, sizing, heteroduplex analysis and single-stranded conformation analysis within a matter of minutes. The rapidity of this analysis allows the sampling of transient effects that may improve the sensitivity of mutation detection. CONCLUSIONS: We believe that this method will aid the integration of self-assembly methods upon microfluidic chips. The speed of this analysis also appears to provide information upon the dynamics of the self-assembly process.

Proliferation of mammalian cardiomyocytes ceases around birth when a transition from hyperplastic to hypertrophic myocardial growth occurs. Previous studies demonstrated that directed expression of the transcription factor E2F1 induces S-phase entry in cardiomyocytes along with stimulation of programmed cell death. Here, we show that directed expression of E2F2 and E2F4 by adenovirus mediated gene transfer in neonatal cardiomyocytes induced S-phase entry but did not result in an onset of apoptosis whereas directed expression of E2F1 and E2F3 strongly evoked programmed cell death concomitant with cell cycle progression. Although both E2F2 and E2F4 induced S-phase entry only directed expression of E2F2 resulted in mitotic cell division of cardiomyocytes. Expression of E2F5 or a control LacZ-Adenovirus had no effects on cell cycle progression. Quantitative real time PCR revealed that E2F1, E2F2, E2F3, and E2F4 alleviate G0 arrest by induction of cyclinA and E cyclins. Furthermore, directed expression of E2F1, E2F3, and E2F5 led to a transcriptional activation of several proapoptotic genes, which were mitigated by E2F2 and E2F4. Our finding that expression of E2F2 induces cell division of cardiomyocytes along with a suppression of proapoptotic genes might open a new access to improve the regenerative capacity of cardiomyocytes.

We explore the hypothesis that pathology of Huntington's disease involves multiple cellular mechanisms whose contributions to disease are incrementally additive or synergistic. We provide evidence that the photoreceptor neuron degeneration seen in flies expressing mutant human huntingtin correlates with widespread degenerative events in the Drosophila CNS. We use a Drosophila Huntington's disease model to establish dose regimens and protocols to assess the effectiveness of drug combinations used at low threshold concentrations. These proof of principle studies identify at least two potential combinatorial treatment options and illustrate a rapid and cost-effective paradigm for testing and optimizing combinatorial drug therapies while reducing side effects for patients with neurodegenerative disease. The potential for using prescreening in Drosophila to inform combinatorial therapies that are most likely to be effective for testing in mammals is discussed.

Linkage between developmental dyslexia (DD) and chromosome 6p has been replicated in a number of independent samples. Recent attempts to identify the gene responsible for the linkage have produced inconsistent evidence for association of DD with a number of genes in a 575-kb region of chromosome 6p22.2, including VMP, DCDC2, KIAA0319, TTRAP, and THEM2. We aimed to identify the specific gene or genes involved by performing a systematic, high-density (approximately 2-3-kb intervals) linkage disequilibrium screen of these genes in an independent sample, incorporating family-based and case-control designs in which dyslexia was defined as an extreme representation of reading disability. Using DNA pooling, we first observed evidence for association with 17 single-nucleotide polymorphisms (SNPs), 13 of which were located in the KIAA0319 gene (P<.01-.003). After redundant SNPs were excluded, 10 SNPs were individually genotyped in 223 subjects with DD and 273 controls. Those SNPs that were significant at P</=.05 were next genotyped in a semi-independent sample of 143 trios of probands with DD and their parents, to control for possible population stratification. Six SNPs showed significant evidence of association in both samples (P</=.04-.002), including a SNP (rs4504469) in exon 4 of the KIAA0319 gene that changes an amino acid (P=.002; odds ratio 1.5). Logistic regression analysis showed that two SNPs (rs4504469 and rs6935076) in the KIAA0319 gene best explained DD status. The haplotype composed of these two markers was significantly associated with DD (global P=.00001 in the case-control sample; P=.02 in trios). This finding was largely driven by underrepresentation of the most common haplotype in cases (P=.00003 in the case-control sample; P=.006 in trios; 1-degree-of-freedom tests). Our data strongly implicate KIAA0319 as a susceptibility gene for dyslexia. The gene product is expressed in brain, but its specific function is currently unknown.

Evidence is accumulating that hepcidin, a liver regulatory peptide, could be the common pathogenetic denominator of all forms of iron overload syndromes including HFE-related hemochromatosis, the most prevalent genetic disorder characterized by inappropriate iron absorption. To understand the mechanisms whereby hepcidin controls iron homeostasis in vivo, we have analyzed the level of iron-related proteins by Western blot and immunohistochemistry in hepcidin-deficient mice, a mouse model of severe hemochromatosis. These mice showed important increased levels of duodenal cytochrome b (Dcytb), divalent metal transporter 1 (DMT1), and ferroportin compared with control mice. Interestingly, the level of ferroportin was coordinately up-regulated in the duodenum, the spleen, and the liver (predominantly in the Kupffer cells). Finally, we also evidenced a decrease of ceruloplasmin in the liver of hepcidin-deficient mice. We hypothesized that the deregulation of these proteins might be central in the pathogenesis of iron overload, providing key therapeutic targets for iron disorders.

There are increasing evidences regarding the association between iron overload and extra-hepatic malignancies. We studied the prevalence of 12 hereditary hemochromatosis (HH) gene mutations (C282Y, V53M, V59M, H63D, H63H, S56C, Q127H, E168Q, E168X, W169X and Q283P in the HFE gene and Y250X in the TFR2 gene) and its correlation with the iron status in 82 adult patients with acute leukemia (AL); 48 patients (58.5%) were affected by acute myeloid leukemia (AML) and 34 patients (41.5%) by acute lymphoblastic leukemia (ALL); 27 patients (32.9%) had at least one HH gene mutation (6 heterozygous for C282Y, 6 homozygous for H63D, 13 heterozygous for H63D and 2 heterozygous for S56C). Mean serum ferritin levels at diagnosis were increased (822.5+/-811.4 microg/L). However, there was no difference between patients positive or negative for the HH gene mutations. Similarly, we did not observe any statistically significant difference as far as iron status between AML and ALL patients. Our study does not support the evidence of an association between hemochromatosis gene mutations and iron overload in AL patients.

Guanine nucleotide-exchange proteins activate ADP-ribosylation factors by accelerating the replacement of bound GDP with GTP. Mammalian brefeldin A-inhibited guanine nucleotide-exchange proteins, BIG1 and BIG2, are important activators of ADP-ribosylation factors for vesicular trafficking. To identify proteins that interact with BIG2, we used cDNA constructs encoding BIG2 sequences in a yeast two-hybrid screen of a human heart library. Clone p2-5-3, encoding a form of human exocyst protein Exo70, interacted with BIG2 amino acids 1-643 and 1-832, but not 644-832, which was confirmed by coimmunoprecipitation of in vitro-translated BIG2 N-terminal segments and 2-5-3. By immunofluorescence microscopy, endogenous BIG2 and Exo70 in HepG2 cells were visualized at Golgi membranes and apparently at the microtubule-organizing center (MTOC). Both were identified in purified centrosomes. Immunoreactive Exo70 and BIG2 partially or completely overlapped with gamma-tubulin at the MTOC in cells inspected by confocal microscopy. In cells incubated with brefeldin A, most of the BIG2, Exo70, and trans-Golgi protein p230 were widely dispersed from their perinuclear concentrations, but small amounts always remained, apparently at the MTOC. After disruption of microtubules with nocodazole, BIG2 and Exo70 were widely distributed in cells and remained only partially colocalized with p230, BIG2 more so than Exo70. We conclude that in HepG2 cells BIG2 and Exo70 interact in trans-Golgi network and centrosomes, as well as in exocyst structures or complexes that move along microtubules to the plasma membrane, consistent with a functional association in both early and late stages of vesicular trafficking.

We have combined genome-wide transcription factor binding and expression profiling to assemble a regulatory network controlling the myogenic differentiation program in mammalian cells. We identified a cadre of overlapping and distinct targets of the key myogenic regulatory factors (MRFs)--MyoD and myogenin--and Myocyte Enhancer Factor 2 (MEF2). We discovered that MRFs and MEF2 regulate a remarkably extensive array of transcription factor genes that propagate and amplify the signals initiated by MRFs. We found that MRFs play an unexpectedly wide-ranging role in directing the assembly and usage of the neuromuscular junction. Interestingly, these factors also prepare myoblasts to respond to diverse types of stress. Computational analyses identified novel combinations of factors that, depending on the differentiation state, might collaborate with MRFs. Our studies suggest unanticipated biological insights into muscle development and highlight new directions for further studies of genes involved in muscle repair and responses to stress and damage.

The oncogenic transcription factor c-Jun plays an important role in cell proliferation, transformation and differentiation. All identified c-Jun-interacting proteins are localized to the nucleus or cytoplasm and function in their intact forms. Here we show that the pleckstrin homology domain-containing protein CKIP-1 (casein kinase 2-interacting protein-1) functions as a plasma membrane-bound AP-1 regulator. During apoptosis, CKIP-1 is cleaved by caspase-3 and translocated to the cytoplasm and then to the nucleus. C-terminal fragments of cleaved CKIP-1 strongly repress AP-1 activity. Importantly, CKIP-1 overexpression promotes apoptosis by forming a positive feedback loop between CKIP-1 and caspase-3. RNA interference of CKIP-1 or overexpression of c-Jun attenuates the sensitivity to apoptosis, indicating a novel role of CKIP-1 in apoptosis. CKIP-1 is the first case of a c-Jun-interacting protein that regulates AP-1 activity via caspase-3-dependent cleavage and translocation.

<h4>Background</h4>Multiple Endocrine Neoplasia type 1 (MEN1, OMIM 131100) is an autosomal dominant disorder characterized by endocrine tumors of the parathyroids, pancreatic islets and pituitary. The disease is caused by the functional loss of the tumor suppressor protein menin, coded by the MEN1 gene. The protein sequence has no significant homology to known consensus motifs. In vitro studies have shown menin binding to JunD, Pem, Smad3, NF-kappaB, nm23H1, and RPA2 proteins. However, none of these binding studies have led to a convincing theory of how loss-of-menin leads to neoplasia.<h4>Results</h4>Global gene expression studies on eight neuroendocrine tumors from MEN1 patients and 4 normal islet controls was performed utilizing Affymetrix U95Av2 chips. Overall hierarchical clustering placed all tumors in one group separate from the group of normal islets. Within the group of tumors, those of the same type were mostly clustered together. The clustering analysis also revealed 19 apoptosis-related genes that were under-expressed in the group of tumors. There were 193 genes that were increased/decreased by at least 2-fold in the tumors relative to the normal islets and that had a t-test significance value of p < or = 0.005. Forty-five of these genes were increased and 148 were decreased in the tumors relative to the controls. One hundred and four of the genes could be classified as being involved in cell growth, cell death, or signal transduction. The results from 11 genes were selected for validation by quantitative RT-PCR. The average correlation coefficient was 0.655 (range 0.235-0.964).<h4>Conclusion</h4>This is the first analysis of global gene expression in MEN1-associated neuroendocrine tumors. Many genes were identified which were differentially expressed in neuroendocrine tumors arising in patients with the MEN1 syndrome, as compared with normal human islet cells. The expression of a group of apoptosis-related genes was significantly suppressed, suggesting that these genes may play crucial roles in tumorigenesis in this syndrome. We identified a number of genes which are attractive candidates for further investigation into the mechanisms by which menin loss causes tumors in pancreatic islets. Of particular interest are: FGF9 which may stimulate the growth of prostate cancer, brain cancer and endometrium; and IER3 (IEX-1), PHLDA2 (TSS3), IAPP (amylin), and SST, all of which may play roles in apoptosis.

Type IV hemochromatosis is associated with dominant mutations in the SLC40A1 gene encoding ferroportin (FPN). Known as the "ferroportin disease," this condition is typically characterized by high serum ferritin, reduced transferrin saturation, and macrophage iron loading. Previously FPN expression in vitro has been shown to cause iron deficiency in human cell lines and mediate iron export from Xenopus oocytes. We confirm these findings by showing that expression of human FPN in a human cell line results in an iron deficiency because of a 3-fold increased export of iron. We show that FPN mutations A77D, V162delta, and G490D that are associated with a typical pattern of disease in vivo cause a loss of iron export function in vitro but do not physically or functionally impede wild-type FPN. These mutants may, therefore, lead to disease by haploinsufficiency. By contrast the variants Y64N, N144D, N144H, Q248H, and C326Y, which can be associated with greater transferrin saturation and more prominent iron deposition in liver parenchyma in vivo, retained iron export function in vitro. Because FPN is a target for negative feedback in iron homeostasis, we postulate that the latter group of mutants may resist inhibition, resulting in a permanently "turned on" iron exporter.

Huntington's disease (HD) is caused by polyglutamine expansion (exp) in huntingtin. Here, we used a yeast artificial chromosome (YAC) transgenic mouse model of HD to investigate the connection between disturbed calcium (Ca2+) signaling and apoptosis of HD medium spiny neurons (MSN). Repetitive application of glutamate elevates cytosolic Ca2+ levels in MSN from the YAC128 mouse but not in MSN from the wild-type or control YAC18 mouse. Application of glutamate results in apoptosis of YAC128 MSN but not wild-type or YAC18 MSN. Analysis of glutamate-induced apoptosis of the YAC128 MSN revealed that (i) actions of glutamate are mediated by mGluR1/5 and NR2B glutamate receptors; (ii) membrane-permeable inositol 1,4,5-trisphosphate receptor blockers 2-APB and Enoxaparin (Lovenox) are neuroprotective; (iii) apoptosis involves the intrinsic pathway mediated by release of mitochondrial cytochrome c and activation of caspases 9 and 3; (iv) apoptosis requires mitochondrial Ca2+ overload and can be prevented by the mitochondrial Ca2+ uniporter blocker Ruthenium 360; and (v) apoptosis involves opening of mitochondrial permeability transition pore (MPTP) and can be prevented by MPTP blockers such as bongkrekic acid, Nortriptyline, Desipramine, Trifluoperazine, and Maprotiline. These findings describe a pathway directly linking disturbed Ca2+ signaling and degeneration of MSN in the caudate nucleus in HD. These findings also suggest that Ca2+ and MPTP blockers may have a therapeutic potential for treatment of HD.

Huntington's disease (HD) arises from an expanded polyglutamine (polyQ) in the N-terminus of the huntingtin (htt) protein. Neuronal degeneration and inclusions containing N-terminal fragments of mutant htt are present in the cortex and striatum of HD brain. Recently, a model of polyQ aggregate structure has been proposed on the basis of studies with synthetic polyQ peptides and includes an alternating beta-strand/beta-turn structure with seven glutamine residues per beta-strand. We tested this model in the context of the htt exon-1 N-terminal fragment in both mammalian cell culture and cultured primary cortical neurons. We found our data support this model in the htt protein and provide a better understanding of the structural basis of polyQ aggregation in toxicity in HD.

Huntington's disease is a neurodegenerative disease resulting from a CAG (glutamine) trinucleotide expansion in exon 1 of the Huntingtin (Htt) gene. The role of the striatum-enriched A2A adenosine receptor (A2A-R) in Huntington's disease has attracted much attention lately. In the present study, we found that expression of mutant Htt with expanded poly(Q) significantly reduced the transcript levels of the endogenous A2A-R in PC12 cells and primary striatal neurons. Cotransfection of various promoter constructs of the A2A-R gene and an expression construct of poly(Q)-expanded Htt revealed that the Htt mutant suppressed the core promoter activity of the A2A-R gene. Stimulation of the A2A-R using CGS21680, forskolin, and a constitutively active cAMP-response element-binding protein (CREB) mutant elevated the reduced promoter activity of the A2A-R gene by mutant Htt. Moreover, the effect of CGS was blocked by an A2A-R-selective antagonist (CSC), two inhibitors of protein kinase A, and two dominant negative mutants of (CREB). The protein kinase A/CREB pathway therefore is involved in regulating A2A-R promoter activity. Consistently, an atypical CRE site (TCCAGG) is located in the core promoter region of the A2A-R gene. Electrophoretic gel mobility shift assay and mutational inactivation further demonstrated the functional binding of CREB to the core promoter region and showed that expression of poly(Q)-expanded Htt abolished the binding of CREB to this site. Stimulation of the A2A-R restored the reduced CREB binding caused by the mutant and concurrently reduced mutant Htt aggregation. Collectively, the poly(Q)-expanded mutant Htt suppressed expression of the A2A-R by inhibiting its core promoter at least partially by preventing CREB binding.

PRIP-1 was isolated as a novel inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] binding protein with a domain organization similar to phospholipase C-delta1 (PLC-delta1) but lacking the enzymatic activity. Further studies revealed that the pleckstrin homology (PH) domain of PRIP-1 is the region responsible for binding Ins(1,4,5)P3. In this study we aimed to clarify the role of PRIP-1 at the physiological concentration in Ins(1,4,5)P3-mediated Ca2+ signaling, as we had previously used COS-1 cells overexpressing PRIP-1 (Takeuchi et al., 2000, Biochem J 349:357-368). For this purpose we employed PRIP-1 knock out (PRIP-1-/-) mice generated previously (Kanematsu et al., 2002, EMBO J 21:1004-1011). The increase in free Ca2+ concentration in response to purinergic receptor stimulation was lower in primary cultured cortical neurons prepared from PRIP-1-/- mice than in those from wild type mice. The relative amounts of [3H]Ins(1,4,5)P3 measured in neurons labeled with [3H]inositol was also lower in cells from PRIP-1-/- mice. In contrast, PLC activities in brain cortex samples from PRIP-1-/- mice were not different from those in the wild type mice, indicating that the hydrolysis of Ins(1,4,5)P3 is enhanced in cells from PRIP-1-/- mice. In vitro analyses revealed that type1 inositol polyphosphate 5-phosphatase physically interacted with a PH domain of PRIP-1 (PRIP-1PH) and its enzyme activity was inhibited by PRIP-1PH. However, physical interaction with these two proteins did not appear to be the reason for the inhibition of enzyme activity, indicating that binding of Ins(1,4,5)P3 to the PH domain prevented its hydrolyzation. Together, these results indicate that PRIP-1 plays an important role in regulating the Ins(1,4,5)P3-mediated Ca2+ signaling by modulating type1 inositol polyphosphate 5-phosphatase activity through binding to Ins(1,4,5)P3.

Family, twin, and adoption studies show that psychiatric diseases including bipolar disorder (BP) and unipolar disorder (UP) have a substantial genetic component. For these illnesses, both positive and negative associations have been reported for two polymorphisms located in the serotonin transporter gene (5-HTT) on chromosome 17: a 17-base-pair (bp) variable-number tandem-repeat (VNTR) in intron 2 and a 44-bp insertion/deletion in the promoter region. Thus, associations between these 5-HTT polymorphisms and affective disorders remain unclear. The present work investigates these potential associations in meta-analyzes that maximize the power to find associations between each disease and the two 5-HTT polymorphisms. We applied meta-analysis techniques to case-control studies of two 5-HTT polymorphisms and two affective disorders (BP and UP), resulting in four meta-analyzes. For each polymorphism, we assessed the evidence for allelic associations, heterogeneity among studies, the influence of individual studies, and the potential for publication bias. The short allele(s) of the 44-bp insertion/deletion polymorphism showed a significant association for BP (odds ratio (OR) = 1.13, P = 0.001) but not UP. For the 17-bp VNTR, an increase in the number of tandem repeats had no significant association with any of the disorders. The small but significant effects of the 44-bp insertion/deletion polymorphism for BP is consistent with being one of many genes that contributes to the multi-factorial nature of these psychiatric disorders.

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are inflammatory diseases of the central nervous system (CNS). Activated coagulation factors are associated with inflammation and are elevated in the plasma of animals with EAE. Thrombin is a key coagulation factor and its major endogenous inhibitors are antithrombin III (ATIII) in the plasma and protease nexin 1 (PN-1) in the brain. We measured the capacity of brain homogenates to inhibit exogenous thrombin and the CNS levels of ATIII and PN-1 during the course of EAE. Acute EAE was induced in SJL/J mice by immunization with mouse spinal cord homogenates. On Days 8, 13, and 22 post-immunization, inhibition of exogenous thrombin activity was measured by a recently developed fluorimetric assay. PN-1 and ATIII were assayed both by immunohistochemistry and by immunoblots in the brain and spinal cord. Total brain thrombin inhibitory activity increased (32%) in EAE mice at the peak of clinical disease (Day 13, P=0.04 compared to controls). Brain ATIII also increased at the peak of disease (2.5-fold higher than controls, P=0.0001), and correlated significantly with clinical scores at all stages of disease (r=0.72, P=0.0068). In contrast, PN-1 elevations were more pronounced at the preclinical stage on Day 8 (3-fold higher than controls, P=0.01) than on Day 13 (1.4-fold higher, P=0.005). Increased brain thrombin inhibition at the clinical peak of EAE probably reflects increased influx of plasma thrombin inhibitors. Early PN-1 changes represent a potential target for thrombin modulating drugs in EAE and MS.

The neurotransmitter serotonin (5-HT) has been shown to regulate alcohol consumption in both animals and humans. Since activity of the 5-HT transporter protein (5-HTT) regulates 5-HT levels, the gene encoding this protein may contribute to the risk of alcohol dependence (AD). Studies of the association to AD of a functional insertion-deletion polymorphism in the 5-HTT-linked promoter region (5-HTTLPR) have yielded inconsistent results. We conducted a meta-analysis of data from 17 published studies (including 3,489 alcoholics and 2,325 controls) investigating the association between 5-HTTLPR alleles and AD. The frequency of the short (S) allele at 5-HTTLPR was significantly associated with AD [odds ratio (OR) = 1.18, 95% CI = 1.03-1.33). Moreover, a greater association with the S allele was seen among individuals with AD complicated by either a co-morbid psychiatric condition or an early-onset or more severe AD subtype [OR = 1.34 (95% CI = 1.11-1.63)]. Allelic variation at 5-HTTLPR contributes to risk for AD, with the greatest effect observed among individuals with a co-occurring clinical feature.

Two putatively functional polymorphisms of the serotonin transporter gene (HTT, SLC6A4) were examined for associations with risk for pervasive developmental disorders (PDDs) and specific autism phenotypes. Dutch patients diagnosed with PDD (N = 125, age range 5-20 years, DSM-IV-TR based criteria, ADI-R and ADOS behavioral assessments) and their parents (N = 230) were genotyped for promoter ins/del (5-HTTLPR) and intron 2 variable number of tandem repeats (VNTR) alleles. Using the transmission disequilibrium test (TDT), no disorder-specific preferential transmission of promoter (long and short) or intron 2 (10- and 12-repeat) alleles was observed. However, multivariate analysis of continuous autism-related behavioral measures revealed that subjects with intron 2 12/12 genotype were significantly more impaired in the rigid-compulsive domain (P = 0.008). Quantitative TDT (QTDT) analysis also showed significant association of the intron 2 VNTR 12-repeat allele with rigid-compulsive behavior (P = 0.015). These results suggest that intron 2 VNTR alleles or nearby polymorphisms in linkage disequilibrium may play a role in specific aspects of the behavioral phenotype of autism.

Expression profiling analysis revealed ectopic high expression of mouse TFF3 in non-tumor liver tissues from the hepatocellular carcinoma (HCC) susceptible PWK/Rbrc strain. TFF3 is a member of the trefoil factor family peptides, which are small secreted proteins regulating mucosal regeneration and repair, and which are overexpressed during inflammatory processes and cancer progression. We, therefore, analyzed the TFF3 expression extensively in mouse and human HCCs. Expression of the mouse TFF3 gene was significantly increased in 6 out of 7 HCCs from a PWK spontaneous tumor model and in all 7 HCCs from an SV40T antigen-induced transgenic MT-D2C57BL/6 model. In humans, 8 of 20 HCCs (40%) had overexpression of TFF3 in both mRNA level and protein level. We then analyzed DNA methylation patterns of the TFF3 promoter region to evaluate expression regulation of promoter methylation. In mouse HCCs, we demonstrated that two CpGs, at positions -992 and +109, were hypomethylated in 13 of 14 mouse HCCs. In human HCCs, hypomethylation at CpG -260 was associated with TFF3 overexpression (p=0.04). These results indicate that TFF3 overexpression may be a critical process in mouse and human hepatocellular carcinogenesis, and the specific promoter CpG hypomethylation may be one of the regulation mechanisms of TFF3 overexpression in HCCs.

Minocycline has been shown to be neuroprotective in various models of neurodegenerative diseases. However, its potential in Huntington's disease (HD) models characterized by calpain-dependent degeneration and inflammation has not been investigated. Here, we have tested minocycline in phenotypic models of HD using 3-nitropropionic acid (3NP) intoxication and quinolinic acid (QA) injections. In the 3NP rat model, where the development of striatal lesions involves calpain, we found that minocycline was not protective, although it attenuated the development of inflammation induced after the onset of striatal degeneration. The lack of minocycline activity on calpain-dependent cell death was also confirmed in vitro using primary striatal cells. Conversely, we found that minocycline reduced lesions and inflammation induced by QA. In cultured cells, minocycline protected against mutated huntingtin and staurosporine, stimulations known to promote caspase-dependent cell death. Altogether, these data suggested that, in HD, minocycline may counteract the development of caspase-dependent neurodegeneration, inflammation, but not calpain-dependent neuronal death.

We previously reported evidence that patients with periodontitis have serum antibodies to oral Gram positive bacteria that are cross-reactive with epithelial antigens. In the present report cross-reactive epithelial antigens including CD24, lactate dehydrogenase A [LDM-A], antioxidant protein 2 [AOP 2] and nuclear factor of activated T cells 5 [NFAT 5], were identified by screening a cDNA expression library with pooled patient sera. Titres of antibodies to CD24 peptide correlated negatively with indices of periodontal disease severity. Strong expression of CD24 in the reactive periodontal epithelium and inflamed gingival attachment contrasted with low to undetectable expression in the external gingival epithelium. In periodontitis, a local action of these auto-reactive antibodies could modulate the regulatory potential associated with expression of CD24 in this epithelium.

<h4>Aim</h4>To determine the correlation between portal hemodynamics and spleen function among different grades of cirrhosis and verify its significance in cirrhosis staging.<h4>Methods</h4>The portal and splenic vein hemodynamics and spleen size were investigated by ultrasonography in consecutive 38 cirrhotic patients with cirrhosis (Child's grades A to C) and 20 normal controls. The differences were compared in portal vein diameter and flow velocity between patients with and without ascites and between patients with mild and severe esophageal varices. The correlation between peripheral blood cell counts and Child's grades was also determined.<h4>Results</h4>The portal flow velocity and volume were significantly lower in patients with Child's C (12.25+/-1.67 cm/s vs 788.59+/-234 mm/min, respectively) cirrhosis compared to controls (19.55+/-3.28 cm/s vs 1254.03+/-410 mm/min, respectively) and those with Child's A (18.5+/-3.02 cm/s vs 1358.48+/-384 mm/min, respectively) and Child's B (16.0+/-3.89 cm/s vs 1142.23+/-390 mm/min, respectively) cirrhosis. Patients with ascites had much lower portal flow velocity and volume (13.0+/-1.72 cm/s vs 1078+/-533 mm/min) than those without ascites (18.6+/-2.60 cm/s vs 1394+/-354 mm/min). There was no statistical difference between patients with mild and severe esophageal varices. The portal vein diameter was not significantly different among the above groups. There were significant differences in splenic vein diameter, flow velocity and white blood cell count, but not in spleen size, red blood cell and platelet counts among the various grades of cirrhosis. The spleen size was negatively correlated with red blood cell and platelet counts (r = -0.620 and r = -0.8.34, respectively).<h4>Conclusion</h4>An optimal system that includes parameters representing the portal hemodynamics and spleen function should be proposed for cirrhosis staging.

The kinetochore is a proteinaceous structure that assembles onto centromeric DNA and mediates chromosome attachment to microtubules during mitosis. This description is deceivingly simple: recent proteomic studies suggest that the diminutive kinetochores of Saccharomyces cerevisiae are comprised of at least 60 proteins organized into as many as 14 different subcomplexes. Many of these proteins, such as the centromeric histone variant CENP-A, and entire subcomplexes, such as the Ndc80(Hec1) complex, are conserved from yeast to humans despite the diverse nature of the DNA sequences on which they assemble. There have recently been advances in our understanding of the molecular basis of how kinetochores establish dynamic attachments to spindle microtubules, and how these attachments are correctly oriented to ensure segregation of sister chromatids to daughter cells.

In this study we examined the role and regulation of OX40 signals during CD4 T cell priming on dendritic cells (DCs). Contrary to expectation, OX40-deficient cells proliferated more rapidly than their normal counterparts, particularly when stimulated with peptide in the absence of added cytokines. This proliferative advantage was not apparent for Th2-differentiated cells. When the reasons for this were investigated, we found that the cytokine IL-4 specifically down-regulated expression of OX40 ligand on T, B, and DCs, but not on the CD4(+)CD3(-) cells linked with selection of Th2 cells into the memory compartment. OX40 ligand expression was also down-regulated on rapidly proliferating Th1 effectors. These data are compatible with OX40 signals acting during priming as a check on naive T cell proliferation while T cells integrate additional DC signals. This would serve to limit inappropriate T cell responses. In contrast, OX40 signals from CD4(+)CD3(-) cells located in the outer T zone select proliferating Th2 effectors into the memory T cell pool.

<h4>Background</h4>The prevalence of iron overload and the influence of mutations in the HFE and TRF2 gene on biochemical markers of iron overload among renal transplant patients is unknown.<h4>Methods</h4>Serum iron, ferritin, transferrin saturation (TSAT), and liver function parameters were analyzed in a cohort of 438 renal transplants. In patients with iron overload, the time course of biochemical markers of iron status as well as the influence of iron loading mutations was investigated during a time period of 5 years.<h4>Results</h4>Of 438 renal transplant patients 41 (9.4%) presented with an iron loading phenotype (TSAT above 40% and/or ferritin above 800 ng/mL). Mutations in the HFE gene were present in 12 of 33 (36.3%) patients with iron overload. Among these one patient was homozygous for HFE C282Y, and two patients were compound heterozygous for HFE C282Y/H63D. No individual tested positive for nine other mutations in HFE as well as theTRF2 Y250X mutation. Over time we observed a decrease of mean iron and ferritin levels, and of mean TSAT in our study sample. In patients with mutations in HFE this decrease was less pronounced as compared to patients without mutations. We found an independent positive association between the presence of mutations in HFE and serum alanine-aminotransferase levels at follow-up (P= 0.003).<h4>Conclusion</h4>Our study demonstrates that iron overload is frequently present in renal transplant patients and shows a continuous decrease over time. This decrease is possibly impaired by the HFE C282Y and HFE H63D mutations. Furthermore, mutations in HFE may influence liver function as reflected by increased alanine-aminotransferase concentrations.

Renal stones have afflicted humans for millennia but there is still no solution to this problem. This review discusses the laboratory and metabolic aspects of the clinical management of patients with renal stones, both primary and secondary in origin. First, non-pharmacological interventions such as increased fluid intake, decreased protein consumption, dietary changes in sodium, calcium, oxalate, potassium, purine, vitamins, and essential fatty acids are considered. Then specific pharmacological treatment to modify urine calcium, oxalate, urate, citrate, and acidity are considered. Finally, more unusual types of stone are examined.

Arterial tortuosity associated with hyperextensible skin and hypermobility of joints, features that are characteristics of Ehlers-Danlos syndrome (EDS), has been described in several families. An arterial tortuosity locus has recently been mapped to chromosome 20q13. Here, we report a consanguineous Kurdish family in which an affected child manifested elongation and severe tortuosity of the aorta, carotid, and other arteries. Additional clinical symptoms include loose skin, hypermobile joints, hernias, and facial features that resemble EDS individuals. To examine whether the arterial tortuosity locus was involved in this child, homozygosity analysis was performed using microsatellite markers on 20q13. The affected child was found homozygous, whereas the unaffected parents and three siblings were heterozygous. Additional typing defined the genomic interval to a 37-cm region within which the arterial tortuosity locus is located. Three functional candidate genes (B4GALT5, KCNB1, and PTGIS) were sequenced. No mutations were discovered in the coding regions of these three genes and the promoter regions of B4GALT5 and KCNB1 genes. Moreover, the B4GALT5 mRNA expression was unaltered in patient-derived lymphoblastoid cells. In the PTGIS gene promoter, the affected child was homozygous for eight variable number of tandem repeats, while parents and unaffected siblings carried six repeats.

It is now widely recognized that the packaging of genomic DNA, together with core histones, linker histones, and other functional proteins into chromatin profoundly influences nuclear processes such as transcription, replication, DNA repair, and recombination. Whereas earlier structural studies portrayed nucleosomes (the basic repeating unit of chromatin) as monolithic and static macromolecular assemblies, we now know that they are highly dynamic and capable of extensive crosstalk with the cellular machinery. Histone variants have evolved to locally alter chromatin structure, whereas histone chaperones and other cellular factors promote histone exchange and chromatin fluidity. Both of these phenomena likely facilitate interconversion between different chromatin states that show varying degrees of transcriptional activity.

<h4>Aim</h4>Colorectal cancers result from the accumulation of several distinct genetic alterations. This study was to investigate the frequency and prognostic value of loss of heterozygosity (LOH) and microsatellite instability (MSI) at 14 genetic loci located near or within regions containing important genes implicated in colorectal tumorigenesis.<h4>Methods</h4>We studied colorectal cancers with corresponding normal mucosae in 207 patients (139 males and 68 females, mean age at the time of tumor resection 66.2+/-12.4 years, range 22-88 years). There were 37 right-sided colonic tumors, 85 left-sided colonic tumors and 85 rectal tumors. The distribution of tumor staging was stage I in 25, stage II in 73, stage III in 68, and stage IV in 41. We analyzed the LOH and MSI of HPC1, hMSH2, hMLH1, APC, MET, P53, NH23-H1, DCC, BAT25, BAT26, D17S250, MYCL1 and D8S254 with fluorescent polymerase chain reaction and denatured gel electrophoresis. High-frequency LOH was determined to be greater than three, or more than 50% of the informative marker with LOH. High-frequency MSI (MSI-H) was determined as more than four markers with instability (>30%). Correlations of LOH and MSI with clinical outcomes and pathological features were analyzed and compared.<h4>Results</h4>The occurrence of MSI-H was 7.25%, located predominantly in the right colons (7/15) and had a higher frequency of poor differentiation (6/15) and mucin production (7/15). LOH in at least one genetic locus occurred in 78.7% of the tumors and was significantly associated with disease progression. Of the 166 potentially cured patients, 45 developed tumor recurrence within 36 mo of follow-up. Clinicopathological factors affecting 3-year disease-free survival (DFS) were TNM staging, grade of differentiation, preoperative CEA level, and high LOH status. Patients with high LOH tumors had a significantly lower DFS (50%) compared with patients with low LOH tumors (84%). Of the patients developing subsequent tumor recurrence, the number and percentage of LOH were 2.97 and 46.8% respectively, similar to the stage IV disease patients. TNM staging had the most significant impact on DFS, followed by high LOH status.<h4>Conclusion</h4>Clinical manifestations of LOH and MSI are different in colorectal cancer patients. High-frequency LOH is associated with high metastatic potential of colorectal cancers.

<h4>Aim</h4>Cysteine peptidase (CP) and its inhibitor (CPI) are a matrix protease that may be associated with colorectal carcinoma invasion and progression, and vitamin E is also a stimulator of the immunological system. Our purpose was to determine the correlation between the expression of cysteine peptidases and their endogenous inhibitors, and the level of vitamin E in sera of patients with colorectal cancer in comparison with healthy individuals.<h4>Methods</h4>The levels of cysteine peptidases and their inhibitors were determined in the sera of patients with primary and metastatic colorectal carcinoma and healthy individuals using fluorogenic substrate, and the level of vitamin E was determined by HPLC.<h4>Results</h4>The levels of cysteine peptidases and their inhibitors were significantly higher in the metastatic colorectal cancer patients than that in the healthy controls (P<0.05). The activity of CP increased 2.2-fold, CPI 2.8-fold and vitamin E decreased 3.4-fold in sera of patients with metastasis in comparison with controls. The level of vitamin E in healthy individuals was higher, whereas the activity of cysteine peptidases and their inhibitors associated with complexes was lower than that in patients with cancer of the digestive tract.<h4>Conclusion</h4>These results suggest that the serum levels of CP and their inhibitors could be an indicator of the prognosis for patients with metastatic colorectal cancer. Vitamin E can be administered prophylactically to prevent digestive tract neoplasmas.

Isolated alterations of biochemical markers of liver damage in a seemingly healthy patient can present a challenge for the clinician. In this review we provide a guide to interpreting alterations to liver enzyme levels. The functional anatomy of the liver and pathophysiology of liver enzyme alteration are briefly reviewed. Using a schematic approach that classifies enzyme alterations as predominantly hepatocellular or predominantly cholestatic, we review abnormal enzymatic activity within the 2 subgroups, the most common causes of enzyme alteration and suggested initial investigations.

<h4>Background and aims</h4>Juvenile hemochromatosis is a severe form of hereditary iron overload that has thus far been linked to pathogenic mutations of the gene coding for hemojuvelin (HJV), on chromosome 1, or, more rarely, that coding for hepcidin ( HAMP ), on chromosome 19. A milder adult-onset form is due to pathogenic mutations of HFE or, rarely, serum transferrin receptor 2.<h4>Methods</h4>We studied a pedigree with siblings affected by both juvenile and adult-onset hereditary hemochromatosis. Affected subjects underwent full clinical evaluation, as well as microsatellite and gene sequencing analysis.<h4>Results</h4>Two siblings (male and female, aged 24 and 25 years, respectively) were hospitalized for severe endocrinopathy and cardiomyopathy. At age 18 and 17 years, they had presented with impotence and amenorrhea, respectively, and increased serum iron levels. Hypogonadotropic hypogonadism was confirmed in both, and liver biopsy showed marked hepatic iron accumulation and micronodular cirrhosis. Iron levels were normalized after 24 months (female) and 36 months (male) of weekly phlebotomies. Microsatellite analysis showed no linkage with chromosome 1 and 19, and gene sequencing showed no hemojuvelin or hepcidin gene mutations. Instead, combined mutations of HFE (C282Y/H63D compound heterozygosity) and serum transferrin receptor 2 (Q317X homozygosity) were found. A 21-year-old brother with a milder phenotype resembling classic adult-onset hereditary hemochromatosis carried only the Q317X serum transferrin receptor 2 homozygote mutation.<h4>Conclusions</h4>Juvenile hereditary hemochromatosis is not a distinct monogenic disorder invariably due to hemojuvelin or hepcidin mutations: it may be genetically linked to the adult-onset form of hereditary hemochromatosis.

Liver fibrosis is the excessive accumulation of extracellular matrix proteins including collagen that occurs in most types of chronic liver diseases. Advanced liver fibrosis results in cirrhosis, liver failure, and portal hypertension and often requires liver transplantation. Our knowledge of the cellular and molecular mechanisms of liver fibrosis has greatly advanced. Activated hepatic stellate cells, portal fibroblasts, and myofibroblasts of bone marrow origin have been identified as major collagen-producing cells in the injured liver. These cells are activated by fibrogenic cytokines such as TGF-beta1, angiotensin II, and leptin. Reversibility of advanced liver fibrosis in patients has been recently documented, which has stimulated researchers to develop antifibrotic drugs. Emerging antifibrotic therapies are aimed at inhibiting the accumulation of fibrogenic cells and/or preventing the deposition of extracellular matrix proteins. Although many therapeutic interventions are effective in experimental models of liver fibrosis, their efficacy and safety in humans is unknown. This review summarizes recent progress in the study of the pathogenesis and diagnosis of liver fibrosis and discusses current antifibrotic strategies.

Chondrocalcinosis, chronic pseudo-osteoarthritis arthropathy, and osteoporosis are classic osteoarticular complications of hemochromatosis (HC). Within HC, femoral head aseptic osteonecrosis (FHAO) is not notified in textbooks. We describe 3 cases of FHAO occurring in this setting in 3 patients homozygous for the C282Y mutation on HFE gene who had no other risk factors for FHAO. FHAO was diagnosed 9 years before (Case 1), concomitantly with (Case 3), or 9 years after HC (Case 2). In one case, FHAO occurred although phlebotomies were regularly carried out. There are scarce data available in the literature on HC and FHAO. Our observations suggest FHAO may be an indicator for HC, and iron balance should be determined before considering FHAO as idiopathic. Thus phlebotomy may not be protective against the occurrence of FHAO. Studies are needed to determine the prevalence of HC in consecutive patients with FHAO.

<h4>Background</h4>Severe, persistent asthma is characterized by airway smooth muscle hyperplasia, inflammatory cell infiltration into the smooth muscle, and increased expression of many cytokines, including IL-4, IL-13, IL-1beta, and TNF-alpha. These cytokines have the potential to alter the expression of surface receptors such as CD40 and OX40 ligand on the airway smooth muscle cell.<h4>Objective</h4>To examine whether cytokines alter expression of CD40 and OX40 ligand on airway smooth muscle cells and identify any differences in response between asthmatic and nonasthmatic airway smooth muscle cells.<h4>Methods</h4>We used flow cytometry and immunohistochemistry to detect CD40 and OX40 ligand on airway smooth muscle cells cultured in the presence of TNF-alpha, IL-1beta, IL-4, or IL-13. Prostaglandin E 2 levels were assessed by ELISA.<h4>Results</h4>TNF-alpha increased expression of both CD40 and OX40 ligand on both asthmatic and nonasthmatic airway smooth muscle cells. The level of expression was significantly greater on the asthmatic cells. IL-1beta alone had no effect, but it attenuated the TNF-induced expression of both CD40 and OX40 ligand. The mechanism of inhibition was COX-dependent for CD40 and was COX-independent but cyclic AMP-dependent for OX40 ligand. IL-4 and IL-13 had no effect.<h4>Conclusion</h4>Our study has demonstrated that TNF-alpha and IL-1beta have the potential to modulate differentially the interactions between cells present in the inflamed airways of a patient with asthma and therefore to contribute to the regulation of airway inflammation and remodeling.

MTJ-1 associates with a glucose-regulated protein of Mr approximately 78,000(GRP78) in the endoplasmic reticulum and modulates GRP78 activity as a chaperone. GRP78 also exists on the cell surface membrane, where it is associated with a number of functions. MHC class I Ags on the cell surface are complexed to GRP78. GRP78 also serves as the receptor for alpha2-macroglobulin-dependent signaling and for uptake of certain pathogenic viruses. The means by which GRP78, lacking a transmembrane domain, can fulfill such functions is unclear. In this study we have examined the question of whether MTJ-1, a transmembrane protein, is involved in the translocation of GRP78 to the cell surface. MTJ-1 and GRP78 coimmunoprecipitated from macrophage plasma membrane lysates. Silencing of MTJ-1 gene expression greatly reduced MTJ-1 mRNA and protein levels, but also abolished cell surface localization of GRP78. Consequently, binding of the activated and receptor-recognized form of alpha2-macroglobulin to macrophages was greatly reduced, and activated and receptor-recognized form of alpha2-macroglobulin-induced calcium signaling was abolished in these cells. In conclusion, we show that in addition to assisting the chaperone GRP78 in protein quality control in the endoplasmic reticulum, MTJ-1 is essential for transport of GRP78 to the cell surface, which serves a number of functions in immune regulation and signal transduction.

Recent advances in our understanding of iron metabolism and the epidemiology of iron overload disorders have shown that hereditary forms of hemochromatosis can result from mutations in several iron metabolism genes other than HFE, including Hamp, HJV, TFR2, and SCL40A. These "non-HFE" forms of hemochromatosis are much rarer than HFE-related hemochromatosis but exhibit a similar phenotype, and with the exception of ferroportin disease, a similar pattern of inheritance and parenchymal iron accumulation. Therefore, these diseases can be thought of as variant forms of a primary hepatic iron overload syndrome; thus, a unified approach can be used for evaluation and diagnosis. Management generally consists of periodic phlebotomies until iron is depleted.

Presenilin (PS) plays an essential role in intramembranous gamma-secretase processing of amyloid precursor protein (APP) and several membrane-bound proteins. Here we report that selective accumulation of a membrane-tethered deleted in colorectal cancer (DCC) derivative (DCC-alpha) correlates with extensive neurite outgrowth in transfected neuroblastoma cells and axodendritic connectivity associated with increased spine density in cortical neurons derived from PS1(-/-) embryos, as well as wild-type neurons treated with gamma-secretase inhibitors. cAMP-dependent signaling was also increased in both the neuroblastoma and cortical neuron systems. As a physiological consequence of increases in axodendritic connectivity and in the magnitude of cAMP-dependent signaling, short- and long-term glutamatergic synaptic transmission was enhanced in PS-deficient neurons. Together, these results demonstrate for the first time that PS-mediated gamma-secretase activity attenuates receptor-mediated intracellular signaling pathways that are critical in regulating glutamatergic synaptic transmission and memory processes.

Isokinetic dynamometers have been measured for torque and force reliability in the past, but little research has been performed on rate of velocity development (RVD) measures. The purpose of this study was to determine the reliability of RVD measures on an isokinetic device at slow and fast speeds. Twenty volunteers performed 5 repetitions of concentric knee extension at 1.04 and 4.18 rad.s(-1) on a Kin-Com isokinetic dynamometer. Each subject was identically posttested 7 days later. Data were separated into 3 velocity range-of-motion (ROM) phases of RVD, load range (LR), and deceleration (DCCROM). Analyses of variance (ANOVAs) were performed to analyze the mean data between day 1 and day 2, while intraclass correlation coefficients (ICCs) were performed for reliability. Results at 1.04 rad.s(-1) demonstrated a low but significant (p < 0.05) ICC value (0.58) only for LR, while at 4.18 rad.s(-1) RVD (0.87), LR (0.83), and DCC (0.55) all exhibited significant ICC values. Percent error for high-speed testing ranged from 1.4-3.19%. No variable exhibited a significant mean difference between testing days. These results collectively point to moderate to high phase reliability for RVD measures at fast speeds of testing, while the slow speed showed very low reliability. Therefore, care should be exercised at slow speeds when comparing RVD measures from test to test.

Low dietary folate intake is associated with an increased risk for colon cancer; however, relevant genetic animal models are lacking. We therefore investigated the effect of targeted ablation of two folate transport genes, folate binding protein 1 (Folbp1) and reduced folate carrier 1 (RFC1), on folate homeostasis to elucidate the molecular mechanisms of folate action on colonocyte cell proliferation, gene expression, and colon carcinogenesis. Targeted deletion of Folbp1 (Folbp1(+/-) and Folbp1(-/-)) significantly reduced (P < 0.05) colonic Folbp1 mRNA, colonic mucosa, and plasma folate concentration. In contrast, subtle changes in folate homeostasis resulted from targeted deletion of RFC1 (RFC1(+/-)). These animals had reduced (P < 0.05) colonic RFC1 mRNA and exhibited a 2-fold reduction in the plasma S-adenosylmethionine/S-adenosylhomocysteine. Folbp1(+/-) and Folbp1(-/-) mice had larger crypts expressed as greater (P < 0.05) numbers of cells per crypt column relative to Folbp1(+/+) mice. Colonic cell proliferation was increased in RFC1(+/-) mice relative to RFC1(+/+) mice. Microarray analysis of colonic mucosa showed distinct changes in gene expression specific to Folbp1 or RFC1 ablation. The effect of folate transporter gene ablation on colon carcinogenesis was evaluated 8 and 38 weeks post-azoxymethane injection in wild-type and heterozygous mice. Relative to RFC1(+/+) mice, RFC1(+/-) mice developed increased (P < 0.05) numbers of aberrant crypt foci at 8 weeks. At 38 weeks, RFC1(+/-) mice developed local inflammatory lesions with or without epithelial dysplasia as well as adenocarcinomas, which were larger relative to RFC1(+/+) mice. In contrast, Folbp1(+/-) mice developed 4-fold (P < 0.05) more lesions relative to Folbp1(+/+) mice. In conclusion, Folbp1 and RFC1 genetically modified mice exhibit distinct changes in colonocyte phenotype and therefore have utility as models to examine the role of folate homeostasis in colon cancer development.

Two isoforms of rat ether-a-go-go (Eag) K channels, rEag1 and rEag2, are widely expressed in many regions of the brain. The neurophysiological roles of these channels, however, are unclear. We addressed this issue by studying their subcellular localizations in hippocampal neurons. Immunofluorescence studies using markers for different compartments of neurons demonstrated a differential expression pattern of rEag1 and rEag2 K channels in the somatodendritic region. Furthermore, rEag1 K channels were in close proximity to synaptophysin and densin-180, but not GAD65. Our data suggest that both rEag1 and rEag2 K channels may play a pivotal role in the regulation of the excitability of dendrites and somas, and that rEag1 K channels may modulate the postsynaptic signaling of glutamatergic synapses.

<h4>Background</h4>Thrombophilia refers to series of acquired and inherited conditions that confer a tendency to thrombus formation. The exact relationship between thrombophilia and MI is not well established.<h4>Objectives</h4>To determine the prevalence of thrombophilia in young patients with their first MI and few conventional risk factors.<h4>Methods</h4>We evaluated the baseline characteristics and the thrombophilia profile, including anti-cardiolipin antibodies, activated protein C resistance (APCR) with the factor V Leiden mutation, prothrombin G20210A mutation, protein C, protein S, and antithrombin-III levels, among 85 consecutive patients (<50 year old) who were admitted to CCU with their first MI. Patients were divided into two groups: group A-patients with < or =1 risk factor and group B-patients with > or =2 risk factors.<h4>Results</h4>92% were male and 55% with anterior wall MI. Overall, the risk factor profile was: smoking in 60%, hyperlipidemia in 42%, positive family history in 29%, hypertension in 18%, diabetes mellitus in 13%, and obesity in 8%. Forty-seven percent of patients had < or =1 risk factor (n=40, group A) and 53% had > or =2 risk factors (n=45, group B). The prevalence of the prothrombin mutation was 15% in group A compared to 7% in group B (p=0.12). APCR secondary to a heterozygous genotype of factor V Leiden mutation was found in 20% in group A compared to 2% in group B (p<0.01). Anti-cardiolipin antibodies were found in 16% in group A compared to 22% in group B (p=ns). Finally, we have found that the likelihood of identifying at least one thrombophilia marker was 50% in group A compared to 29% in group B (p=0.046).<h4>Conclusions</h4>The likelihood to detect at least one thrombophilia marker in young patients with MI and few conventional risk factors is significantly high. Thrombophilia may contribute to the development of MI in this specific group of young patients.

<h4>Background and objectives</h4>The HFE protein interacts with the transferrin receptor (TfR) to regulate cellular iron uptake. Nucleated erythroid cells have the highest number of TfR and the greatest iron uptake. The aim of this study was to investigate whether erythroid iron uptake is directly affected by HFE mutations.<h4>Design and methods</h4>Iron status and erythropoiesis was investigated in sixty, asymptomatic HFE C282Y homozygotes. Reverse transcription-polymerase chain reaction, flow cytometry and immunocytochemistry were employed to investigate the HFE expression profile of normal peripheral blood, nucleated erythroid cells and several cultured cell lines.<h4>Results</h4>The HFE C282Y homozygous subjects showed subtle erythropoietic changes with raised transferrin saturation and reticulocyte counts and low-normal serum transferrin receptor levels, but normal erythrocyte count and mean cell volume. HFE mRNA was detected in macrophages and monocytes and HFE protein was detected in granulocytes and at low levels in monocytes. Cultured primary human erythroid colonies did not express HFE mRNA or protein.<h4>Interpretation and conclusions</h4>There is evidence that HFE C282Y homozygotes display increased plasma iron turnover and increased erythropoiesis, despite there being no evidence that HFE is expressed in erythroid colonies with a normal HFE genotype. It is likely that HFE mutations do not directly alter erythroid iron handling, but alter the supply of iron to the erythroid tissues.

Juvenile hemochromatosis is a severe form of hereditary iron overload. It can be caused by mutations in either hepcidin or hemojuvelin genes. In this study we identified the molecular basis of juvenile hemochromatosis in three Australian families and assessed the role of potential modifying genes in individuals carrying HFE mutations.

Chemokines are a large family of secreted proteins that play an important role in the migration of leukocytes during hematopoiesis and inflammation. Chemokines and their receptors are also widely distributed in the CNS. Although recent investigations are beginning to elucidate chemokine function within the CNS, relatively little is known about the CNS function of this important class of molecules. To better appreciate the CNS function of chemokines, the role of signaling by stromal cell-derived factor-1 (SDF-1) through its receptor, chemokine (CXC motif) receptor 4 (CXCR4), was analyzed in zebrafish embryos. The SDF-1/CXCR4 expression pattern suggested that SDF-1/CXCR4 signaling was important for guiding retinal ganglion cell axons within the retina to the optic stalk to exit the retina. Antisense knockdown of the ligand and/or receptor and a genetic CXCR4 mutation both induced retinal axons to follow aberrant pathways within the retina. Furthermore, retinal axons deviated from their normal pathway and extended to cells ectopically expressing SDF-1 within the retina. These data suggest that chemokine signaling is both necessary and sufficient for directing retinal growth cones within the retina.

Huntington's disease (HD) is an incurable, adult-onset, dominantly inherited neurodegenerative disease, caused by a CAG expansion in the 5' coding region of the gene HD [encoding huntingtin (htt), which is ubiquitously expressed in all tissues]. The disease progresses inexorably with devastating clinical effects on motor, cognitive and psychological functions; death occurring approximately 18 years from the time of onset. These clinical symptoms primarily relate to the progressive death of medium-spiny GABA-ergic neurons of the striatum and in the deep layers of the cortex; during the later stages of the disease, the degeneration extends to a variety of brain regions, including the hypothalamus and hippocampus. The mechanism by which mutant htt leads to neuronal cell death and the question of why striatal neurons are targeted both remain to be further investigated. Certainly htt is required for cell survival and impairment of wild-type htt function can be involved in neurodegeneration, but considerable evidence also shows that trinucleotide repeat expansion into glutamine (polyQ domain) endows the protein with a newly acquired toxic activity. The increasing availability of HD animal models have allowed not only to investigate the function of htt, but also to screen and test potential therapeutic drugs in the promising area of neurotherapeutics. So, thorough analysis of these molecular and biochemical events, assessing the validity of candidate mechanisms, provides a means to identify effective therapeutic strategies for cellular repair. Here, the rationale and efficacy of different therapies are compared and alternative therapies are reviewed including intrastriatal transplantation of human fetal striatal tissue to support the cell replacement strategy in HD. Since functional restoration through neuronal replacement probably could be combined with neuroprotective strategies for optimum clinical benefit, in vivo and ex vivo gene therapy for delivery of neuroprotective growth factor molecules are also considered.

The anti-Hu syndrome is the most common paraneoplastic neurologic syndrome but the exact mechanism of immune mediated neuronal injury remains unknown. Anti-Hu antibodies do not appear to play a pivotal role in the pathogenesis of the disease. To assess cell-mediated immunity, we selected 51 peptides from the Hu-D sequence and tested their ability to bind to six common HLA class I molecules. Stable complexes with purified HLA molecules were obtained with 19/51 (37%) selected peptides. Subsequently, the ability of the 19 HLA-binding peptides to stimulate T cells from 10 patients and 10 control subjects was evaluated by detecting IFN-gamma secretion. An anti-peptide T-cell response was observed in 7/10 Hu-positive patients but also in 3/10 control subjects. Overall, a significant T-cell activation occurred in response to 74% (14 out of 19) of the selected peptides in the Hu-positive patients vs. 16% (3 out of 19) in the control group (p < 0.001). In addition, T cells of patients tested within 3 months of the onset of anti-Hu syndrome responded to 82% (14 out of 17) of assessed Hu-D peptides vs. 37% (7 out of 19) in patients tested 1 year or more after developing the syndrome (p < 0.01). Thus, the present study suggests a role of cellular immunity during the course of anti-Hu syndrome.

The availability of serum blood chemistries for screening both symptomatic and asymptomatic patients has resulted in a marked increase in the number of abnormal liver chemistry tests that must be interpreted by physicians. Usually the first step in the evaluation of a patient with elevated liver enzymes is to repeat the test to confirm the result. If the result is still abnormal, it seems wise to differentiate between a predominant "necrotic pattern" of liver chemistry, as indicated by an elevation of ALT- or AST-activity or a predominant "cholestatic pattern", as indicated by elevated activities of g-GT and alkaline phosphatase. In patients with elevated serum amino transferases hepatic diseases should be excluded primarily with non-invasive serologic tests. The most common causes of elevated amino transferase levels are chronic hepatitis B and C, autoimmunhepatitis, non-alcoholic steatohepatitis, hemochromatosis, Wilson-disease and (only recently recognized) celiac sprue. In the case of a dominant "cholestatic pattern", primary biliary cirrhosis, primary sclerosing cholangitis, but also drugs and granulomatose hepatitis must be excluded. If non-invasive serologic studies remain inconclusive, ultrasound, mini-laparoscopy and liver biopsy will help to establish the final diagnoses.

Nonspherocytic hereditary anemias are occasionally accompanied by significant iron overload but the significance for the development of chronic liver disease is not clear. We described two cases of patients with chronic liver disease and severe iron overload due to chronic hereditary hemolysis. Both patients have had signs of liver cirrhosis and severe hemolysis since childhood. A hereditary pyruvate kinase deficiency (PKD) was discovered as the underlying reason for the hemolysis. Sequencing of the pyruvate kinase gene showed a mutation within exon 11. Liver histology in both patients revealed cirrhosis and a severe iron overload but primary hemochromatosis was excluded by HFE-gene analysis. An iron reduction therapy with desferrioxamine led to significant decrease of serum ferritin and sustained clinical improvement. PKD-induced hemolysis may cause severe iron overload even in the absence of HFE-genotype abnormalities. This secondary iron overload can lead to chronic liver disease and cirrhosis. Therefore, the iron metabolism of PKD patients has to be closely monitored and iron overload should be consequently treated.

This study was conducted to determine if an oral squamous cell carcinoma alters mRNA expression of serotonin transporter (5-HTT) in the central nervous system. KB cell line derived from a human oral squamous cell carcinoma was inoculated into nude mice, and mRNA expression level of 5-HTT in the dorsal raphe nucleus (DRN) was examined by in situ hybridization when the tumor mass reached to -10% of total body weight. Plasma leptin levels were determined by radioimmunoassay method using a commercial kit. 5-HTT mRNA level was significantly decreased in the DRN of tumor bearing mice, compared to the age-matching non-tumor control. Plasma leptin level decreased concomitantly in tumor bearing mice. These results suggest that oral carcinoma may suppress 5-HTT gene expression in the central nervous system, perhaps in relation with decreased plasma leptin level.

Studies of tyrosine phosphorylation in the human duodenum have indicated that proliferating cells in the middle portion of the duodenal crypt were devoid of this feature, suggesting that tyrosine kinase activation is not a dominant factor in crypt cell proliferation, and that consequently tyrosine phosphatase activity may be a more critical factor in crypt cell development. We investigated the expression of the leukocyte common antigen-related receptor (LAR) family of tyrosine phosphatases. A flow cytometry system was used to examine cells from the surface, mid-portion, and lower part of the crypt. Individual cell populations were immunostained with anti-LAR antibodies using phycoerythrin-conjugated anti-CD3 to discriminate between epithelial cells (CD3-) and intraepithelial lymphocytes (CD3+). Epithelial cells expressed LAR throughout the crypt. Expression of LAR was maximal in the mid-portion of the crypt with lower expression at the top of the villi. Intraepithelial lymphocytes expressed low levels of LAR at the tips of the villi with stronger expression extending towards the base of the crypt. These findings were confirmed by immunohistochemistry on paraffin-fixed sections. Of note, peripheral blood lymphocytes expressed less LAR than IEL. These observations suggest the possibility that tyrosine phosphatase LAR may be of importance in the regulation of crypt cell proliferation. Moreover, as the extracellular domain of LAR has homology with adhesion molecules, the finding of this molecule on IEL could suggest a possible functional role in homing of this unique lymphocyte.

The retinoblastoma tumor suppressor (pRb) has traditionally been studied as a negative regulator of cell cycle progression through its interactions with the E2F family of transcription factors. Utilizing prostate epithelial cell lines established from Rb+/+ and Rb-/- prostate tissues, we previously demonstrated that Rb-/- epithelial cells were not transformed and retained the ability to differentiate in vivo despite the lack of pRb. To further study the effects of pRb loss in an epithelial cell population, we utilized oligonucleotide microarrays to identify any pRb-dependent transcriptional regulation during serum depletion-induced growth arrest. These studies identified 120 unique transcripts regulated by growth arrest in Rb+/+ cells. In these wild-type cells, the majority (80%) of altered transcripts were downregulated, including 40 previously identified E2F target genes. Although the transcriptional repression of E2F target genes is characteristic of pRb pocket protein family activity, further analysis revealed that, compared to Rb+/+ cells, Rb-/- cells exhibited a nearly identical response for all transcripts including those of E2F target genes. These findings demonstrate that pRb is not strictly required for the vast majority of transcriptional alterations associated with growth arrest.

<h4>Objective</h4>To investigate the role of loss of heterozygosity (LOH) in tumor suppressor genes (TSG) and microsatellite instability (MSI) in hepatocarcinogenesis, as well as their correlation with clinicopathologic features.<h4>Methods</h4>LOH in 6 TSG (APC, DCC, MCC, OGG1, p53 and RB1) was detected in 36 informative cases of hepatocellular carcinoma (HCC), among 92 surgically resected HCC. Thirteen polymorphic microsatellite markers were also studied in 15 of these cases by microdissection-based PCR amplification and direct DNA sequencing. The correlation between genetic alterations and clinicopathologic features was analyzed.<h4>Results</h4>The overall incidence of LOH in HCC was 41.7% (15/36). There was no LOH in MCC gene. 46.2% (6/13) microsatellites showed LOH in 9 of the 15 cases of HCC (60%). Certain clinicopathologic differences were observed between cases (number = 7) with LOH in APC, OGG1 and DCC ("type I") and cases (number = 8) with LOH in p53 and RB1 ("type II"). The mean tumor size of these two types was 2.9 (+/- 1.7) cm and 7.2 (+/- 3.4) cm, respectively (P < 0.01); and the mean survival was 72.0 (+/- 38.6) months, and 51.0 (+/- 30.4) months, respectively (P < 0.05).<h4>Conclusions</h4>Compared with MSI pathway, LOH pathway plays a more important role in the development of HCC. A multistep hepatocarcinogenesis is likely, with LOH in APC, OGG1 and DCC ("type I") being an early event and LOH in p53 and RB1 ("type II") being a late event. On the other hand, MCC gene seems to play no role in the whole process.

PRDX6 is a cytosolic member of the peroxiredoxin family of antioxidant proteins, which protect cells from oxidative damage by reducing cellular peroxides. Knockout studies and transgenic overexpression of Prdx6 in mice have demonstrated an important role for this protein in the defense against oxidative stress. Using Northern blotting with various Prdx6 probes, we have revealed the existence of multiple transcripts with distinct tissue distributions and regulation, including the major 1.4-kb transcript highly expressed in liver and lung, and two additional transcripts expressed primarily in liver. We hypothesized that these additional transcripts correspond either to alternative Prdx6 mRNAs or to highly related genes such as the intronless genes Aop2-rs1 and Aop2-rs2. A combination of Northern blotting, RACE, and EST and genomic sequence analysis has determined that all three liver transcripts are derived from the Prdx6 gene, as they are absent in Prdx6-null mice and differ in their 3' UTRs, suggesting the utilization of different transcription termination signal sequences which we have identified by sequence analysis. We found the Aop2-rs1 gene to be exclusively expressed in testis as a 1.2-kb transcript and have identified putative regulatory elements in its promoter. In contrast, Aop2-rs2 appears not to be expressed in any tissues, although we have evidence for the existence of other related genes that are expressed in a tissue-specific manner. Since the Prdx6 transcripts exhibit differential regulation in response to growth and oxidative stress, further investigation may reveal their distinct roles in the cell and mechanism of regulation.

It is as futile to ask how much of the phenotype of an organism is due to nature and how much to its nurture as it is to determine how much of the area of a rectangle is due to its length and how much to its height. Phenotype and area are joint products. The spectacular success of genomics, unfortunately, threatens to re-awaken belief in genes as the principal determinants of human behavior. This paper develops the thesis that gene expression is modified by environmental inputs and that the impact of the environment on a given organism is modified by its genome. Genes set the boundaries of the possible; environments parse out the actual.