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The expression of DCC (deleted in colorectal cancer) is often markedly reduced in colorectal and other cancers. However, the rarity of point mutations identified in DCC coding sequences and the lack of a tumor predisposition phenotype in DCC hemizygous mice have raised questions about its role as a tumor suppressor. DCC also mediates axon guidance and functions as a dependence receptor; such receptors create cellular states of dependence on their respective ligands by inducing apoptosis when unoccupied by ligand. We now show that DCC drives cell death independently of both the mitochondria-dependent pathway and the death receptor/caspase-8 pathway. Moreover, we demonstrate that DCC interacts with both caspase-3 and caspase-9 and drives the activation of caspase-3 through caspase-9 without a requirement for cytochrome c or Apaf-1. Hence, DCC defines an additional pathway for the apoptosome-independent caspase activation.

Kartagener syndrome (KS) is a trilogy of symptoms (nasal polyps, bronchiectasis, and situs inversus totalis) that is associated with ultrastructural anomalies of cilia of epithelial cells covering the upper and lower respiratory tracts and spermatozoa flagellae. The axonemal dynein intermediate-chain gene 1 (DNAI1), which has been demonstrated to be responsible for a case of primary ciliary dyskinesia (PCD) without situs inversus, was screened for mutation in a series of 34 patients with KS. We identified compound heterozygous DNAI1 gene defects in three independent patients and in two of their siblings who presented with PCD and situs solitus (i.e., normal position of inner organs). Strikingly, these five patients share one mutant allele (splice defect), which is identical to one of the mutant DNAI1 alleles found in the patient with PCD, reported elsewhere. Finally, this study demonstrates a link between ciliary function and situs determination, since compound mutation heterozygosity in DNAI1 results in PCD with situs solitus or situs inversus (KS).

Axonal growth cones that cross the nervous system midline change their responsiveness to midline guidance cues: They become repelled by the repellent Slit and simultaneously lose responsiveness to the attractant netrin. These mutually reinforcing changes help to expel growth cones from the midline by making a once-attractive environment appear repulsive. Here, we provide evidence that these two changes are causally linked: In the growth cones of embryonic Xenopus spinal axons, activation of the Slit receptor Roundabout (Robo) silences the attractive effect of netrin-1, but not its growth-stimulatory effect, through direct binding of the cytoplasmic domain of Robo to that of the netrin receptor DCC. Biologically, this hierarchical silencing mechanism helps to prevent a tug-of-war between attractive and repulsive signals in the growth cone that might cause confusion. Molecularly, silencing is enabled by a modular and interlocking design of the cytoplasmic domains of these potentially antagonistic receptors that predetermines the outcome of their simultaneous activation.

Bothrops lanceolatus venom contains caseinolytic, phospholipase, esterase and haemorrhagic activities. We have investigated the coagulant and anticoagulant actions of B. lanceolatus venom on human citrated plasma and on purified plasma components. Although B. lanceolatus venom up to 50 microg/ml was unable to clot citrated plasma, at concentrations > or = 5 microg/ml the venom dose-dependently clotted purified human fibrinogen, indicating the presence of a thrombin-like enzyme. Human plasma (final concentration > or = 12.5%) dose-dependently inhibited the venom-induced fibrinogen clotting. This finding suggested that endogenous plasma protease inhibitors can affect the venom's action on fibrinogen. To investigate this possibility, B. lanceolatus venom was incubated with different plasma protease inhibitors and the activity on fibrinogen tested. alpha(2)-Macroglobulin and alpha(1)-antitrypsin did not interfere with the coagulant activity of the venom whereas the antithrombin-III/heparin complex partially inhibited this activity. A non-toxic, acidic phospholipase A(2) purified from B. lanceolatus venom prolonged the activated partial thromboplastin time in human plasma from 39.7+/-0.5 s (control with saline) to 60.2+/-0.9 s with 50 microg of PLA(2) (p<0.001), suggesting an anticoagulant activity associated with this enzyme. This anticoagulant activity may account for some of the effects of the venom on blood coagulation.

The interstitial insertion of genetic material from one chromosome into another can achieve the type of gene-gene fusions more usually associated with chromosome translocations. An example of such an interstitial insertion, which has created an MLL-AF10 fusion in an acute myeloid leukaemia, has been analysed at the genomic level. The genomic fusion, which resulted in the juxtaposition of 3' AF10 sequence to 5' MLL sequence, was identified within MLL and AF10 intronic sequences. It was further established that the remaining 3' MLL sequence, from exon 6 onwards, was fused to novel sequence of unknown origin (named FM3 for fused to MLL 3'). The points of fusion of these 5' and 3' portions of MLL matched to adjacent nucleotides and lay between exons 5 and 6. The FM3 sequence was shown to be from chromosome arm 10p and located close to AF10 in a proximal position. It was subsequently demonstrated that in the leukaemia a third fusion existed between 5' AF10 and the FM3 sequence at a point immediately downstream from its fusion to MLL. It was therefore concluded that the MLL-AF10 gene fusion is the result of a simultaneous transposition of genetic material into the MLL gene and the joining of the remaining free ends on chromosome 10. This kind of event, characterised completely here for the first time, is a means to achieve a fusion when the genes involved lie in opposite orientations and results in three genomic junctions.

Deficiency in the monoamine degradation enzyme monoamine oxidase A (MAOA) or prenatal exposure to the monoamine uptake inhibitor cocaine alters behavior in humans and rodents, but the mechanisms are unclear. In MAOA knock-out mice, inhibiting serotonin synthesis during development can prevent abnormal segregation of axons in the retinogeniculate and somatosensory thalamocortical systems. To investigate this effect, we crossed MAOA knock-outs with mice lacking the serotonin transporter 5-HTT or the 5-HT1B receptor, two molecules present in developing sensory projections. Segregation was abnormal in 5-HTT knock-outs and MAOA/5-HTT double knock-outs but was normalized in MAOA/5-HT1B double knock-outs and MAOA/5-HTT/5-HT1B triple knock-outs. This demonstrates that the 5-HT1B receptor is a key factor in abnormal segregation of sensory projections and suggests that serotonergic drugs represent a risk for the development of these projections. We also found that the 5-HT1B receptor has an adverse developmental impact on beam-walking behavior in MAOA knock-outs. Finally, because the 5-HT1B receptor inhibits glutamate release, our results suggest that visual and somatosensory projections must release glutamate for proper segregation.

Luteinizing hormone-releasing hormone (LHRH) neurons migrate from the vomeronasal organ (VNO) to the forebrain in all mammals studied. In mice, most LHRH neuron migration is dependent on axons that originate in the VNO but bypass the olfactory bulb and project into the basal forebrain. Thus, cues that regulate the trajectories of these vomeronasal axons are candidates for determining the destination of LHRH neurons. Using in situ hybridization techniques, we examined the expression of Deleted in colorectal cancer (DCC), a vertebrate receptor for the guidance molecule netrin-1, during development of the olfactory system. DCC is expressed by cells in the olfactory epithelium (OE) and VNO, and in cells migrating from the OE and VNO from embryonic day 11 (E11) to E14. Some DCC(+) cells on vomeronasal axons in the nose also express LHRH. However, DCC expression is downregulated beginning at E12, so few if any LHRH neurons in the forebrain also express DCC. In rat, DCC is expressed on TAG-1(+) axons that guide migrating LHRH neurons. We therefore examined LHRH neuron migration in DCC(-/-) mice and found that trajectories of the caudal vomeronasal nerve and positions of LHRH neurons are abnormal. Fewer than the normal number of LHRH neurons are found in the basal forebrain, and many LHRH neurons are displaced into the cerebral cortex of DCC(-/-) mice. These results are consistent with the idea that DCC regulates the trajectories of a subset of vomeronasal axons that guide the migration of LHRH neurons. Loss of DCC function results in the migration of many LHRH neurons to inappropriate destinations.

<h4>Objectives</h4>To identify the causes of mortality in children with vertically transmitted human immunodeficiency virus (HIV) infection and to study age-related mortality trends.<h4>Methods</h4>In the multicenter P(2)C(2) HIV Study, 816 children born to HIV-infected mothers were followed for a median of 3.6 years. Two hundred five study participants with HIV infection were enrolled at a median age of 23 months; 611 were enrolled either prenatally or in the neonatal period before their HIV infection status was known. There were 121 deaths in study patients. The cause of death for all patients, its relationship to HIV infection, and pulmonary or cardiac involvement were determined. Age trends in disease-specific mortality were summarized for the HIV-related deaths.<h4>Results</h4>Ninety-three children died of HIV-related conditions. Infection was the most prevalent cause of death for children under 6 years of age with 32.3% caused by pulmonary infection and another 16.9% caused by nonpulmonary infection. The frequency of pulmonary disease as the underlying cause of death decreased significantly with increasing age: 5/9 (55.6%) by age 1, 1/12 (8.3%) after age 10 years. The frequency of chronic cardiac disease as the underlying cause increased with age-0% by age 1 year, 3/12 (25.0%) after age 10 years, as did the frequency of wasting syndrome with disseminated Mycobacterium avium complex-0% by age 1 year, 6/12 (50.0%) after age 10 years.<h4>Conclusions</h4>Children with HIV who survive longer are less likely to die of pulmonary disease or infection and more likely to die of cardiac causes or with wasting syndrome.pediatric acquired immunodeficiency syndrome, mortality, human immunodeficiency virus.

Iron overload is highly prevalent, but its molecular pathogenesis is poorly understood. Recently, DMT1 was shown to be a major apical iron transporter in absorptive cells of the duodenum. In vivo, it is the only transporter known to be important for the uptake of dietary non-heme iron from the gut lumen. The expression and subcellular localization of DMT1 protein in 3 mouse models of iron overload were examined: hypotransferrinemic (Trf(hpx)) mice, Hfe knockout mice, and B2m knockout mice. Interestingly, in Trf(hpx) homozygotes, DMT1 expression was strongly induced in the villus brush border when compared to control animals. This suggests that DMT1 expression is increased in response to iron deficiency in the erythron, even in the setting of systemic iron overload. In contrast, no increase was seen in DMT1 expression in animals with iron overload resembling human hemochromatosis. Therefore, it does not appear that changes in DMT1 levels are primarily responsible for iron loading in hemochromatosis.

<h4>Background</h4>Heterozygotes for the C282Y mutation of the HFE gene may have altered hematology indices and higher iron stores than wild-type subjects.<h4>Methods</h4>We performed a cross-sectional analysis of 1488 females and 1522 males 20-79 years of age drawn from the Busselton (Australia) population study to assess the effects of HFE genotype, age, gender, and lifestyle on serum iron and hematology indices.<h4>Results</h4>Male C282Y heterozygotes had increased transferrin saturation compared with the wild-type genotype. Neither male nor female heterozygotes had significantly increased ferritin values compared with the wild-type genotype. Younger (20-29 years) wild-type males, but not heterozygous males, had significantly lower ferritin values than wild-type males in the older age groups. Compound heterozygous subjects had increased means for serum iron, transferrin saturation, corpuscular volume, and corpuscular hemoglobin compared with the wild-type genotype, and the males also had increased ferritin values (medians 323 vs 177 microg/L; P = 0.003). In both male and female wild-type subjects, an increased body mass index was associated with decreased serum iron and transferrin saturation and increased ferritin values. There was a significant increase in ferritin concentrations in both genders with increasing frequency of red meat consumption above a baseline of 1-2 times per week and alcohol intakes >10 g/day.<h4>Conclusions</h4>Male C282Y heterozygotes had significantly increased transferrin saturation values. Compound heterozygous (C282Y/H63D) subjects formed a separate category of C282Y heterozygotes in whom both iron and red cell indices were significantly increased compared with the wild-type genotype.

OX40 (CD134) and its ligand (OX40L) have been implicated in T cell activation and migration. In this study, we examined the contribution of these molecules to the pathogenesis of experimental autoimmune encephalomyelitis (EAE) by administering a neutralizing mAb against murine OX40L (RM134L) to proteolipid protein (139-151) peptide-induced EAE in SJL mice. Administration of RM134L effectively ameliorated the disease in both actively induced and adoptively transferred EAE models. Histological examination showed that the RM134L treatment greatly reduced mononuclear cell infiltration into the spinal cord. The RM134L treatment did not inhibit the development of pathogenic T cells, given that proliferative response and IFN-gamma production by draining lymph node cells were not reduced or rather enhanced upon restimulation with proteolipid protein (139-151) in vitro, and these cells effectively transferred EAE to naive SJL mice. Flow cytometric analyses showed that the RM134L treatment inhibited the accumulation of OX40-expressing CD4(+) T cells and the migration of adoptively transferred CD4(+) T cells in the spinal cord. Immunohistochemical staining showed that OX40L was most prominently expressed on endothelial cells in the inflamed spinal cord. These results suggest that the OX40/OX40L interaction plays a critical role for the migration of pathogenic T cells into the CNS in the pathogenesis of EAE.

Conditional gene repair mutations in the mouse can assist in cell lineage analyses and provide a valuable complement to conditional gene inactivation strategies. We present a method for the generation of conditional gene repair mutations that employs a loxP-flanked (floxed) selectable marker and transcriptional/translational stop cassette (neostop) located within the first intron of a target gene. In the absence of Cre recombinase, expression of the targeted allele is suppressed generating a null allele, while in the presence of Cre, excision of neostop restores expression to wild-type levels. To test this strategy, we have generated a conditional gene repair allele of the mouse Huntington's disease gene homolog (HDH:). Insertion of neostop within the HDH: intron 1 generated a null allele and mice homozygous for this allele resembled nullizygous HDH: mutants and died after embryonic day 8.5. In the presence of a cre transgene expressed ubiquitously early in development, excision of neostop restored HDH: expression and rescued the early embryonic lethality. A simple modification of this strategy that permits the generation of conventional gene knockout, conditional gene knockout and conditional gene repair alleles using one targeting construct is discussed.

The molecular events that drive the initiation and progression of ovarian adenocarcinoma are not well defined. We have investigated changes in gene expression in ovarian cancer cell lines compared to an immortalized human ovarian surface epithelial cell line (HOSE) using a cDNA array. We identified 17 genes that were under-expressed and 10 genes that were over-expressed in the cell lines compared to the HOSE cells. One of the genes under-expressed in the ovarian cancer cell lines, Id3, a transcriptional inactivator, was selected for further investigation. Id3 mRNA was expressed at reduced levels in 6 out of 9 ovarian cancer cell lines compared to the HOSE cells while at the protein level, all 7 ovarian cancer cell lines examined expressed the Id3 protein at greatly reduced levels. Expression of Id3 mRNA was also examined in primary ovarian tumours and was found in only 12/38 (32%) cases. A search was conducted for mutations of Id3 in primary ovarian cancers using single stranded conformation polymorphism (SSCP) analysis. Only one nucleotide substitution, present also in the corresponding constitutional DNA, was found in 94 ovarian tumours. Furthermore no association was found between LOH at 1p36 and lack of expression of Id3. These data suggest that Id3 is not the target of LOH at 1p36.

Sibling neurons in the embryonic central nervous system (CNS) of Drosophila can adopt distinct states as judged by gene expression and axon projection. In the NB4-2 lineage, two even-skipped (eve)-expressing sibling neuronal cells, RP2 and RP2sib, are formed in each hemineuromere. Throughout embryogenesis, only RP2, but not RP2sib, maintains eve expression. In this report, we describe a P-element induced mutation that alters the expression pattern of EVE in RP2 motoneurons in the Drosophila embryonic CNS. The mutation was mapped to a Drosophila homolog of human AF10/AF17 leukemia fusion genes (alf), and therefore named Dalf. Like its human counterparts, Dalf encodes a zinc finger/leucine zipper nuclear protein that is widely expressed in embryonic and larval tissues including neurons and glia. In Dalf mutant embryos, the RP2 motoneuron no longer maintains EVE expression. The effect of the Dalf mutation on EVE expression is RP2-specific and does not affect other characteristics of the RP2 motoneuron. In addition to the embryonic phenotype, Dalf mutant larvae are retarded in their growth and this defect can be rescued by the ectopic expression of a Dalf transgene under the control of a neuronal GAL4 driver. This indicates a requirement for Dalf function in the nervous system for maintaining gene expression and the facilitation of normal growth.

Screening a human small intestinal library with human serum yielded a clone which encoded a protein res4-22 the gene of which was highly homologous to a recently described gene located in the Huntington's disease locus. Autoantibodies against res4-22 (anti-res4-22), mainly of the immunoglobulin (Ig)A type, were detected in patients with neurological disorders at a higher frequency (18.4%) than in healthy blood donors (8.0%). In neurological patients with cerebral ischaemia anti-res4-22 was found significantly more often (47.4%) than in the total group of neurological patients. Anti-res4-22 positive sera showed significantly more frequently myelin staining in cerebellum and nerve sections than anti-res4-22 negative sera. Our findings demonstrate a new species of human autoantibodies against a newly described protein the function of which is still unknown.

The cell wall imparts structural strength and shape to bacteria. It is made up of polymeric glycan chains with peptide branches that are cross-linked to form the cell wall. The cross-linking reaction, catalyzed by transpeptidases, is the last step in cell wall biosynthesis. These enzymes are members of the family of penicillin-binding proteins, the targets of beta-lactam antibiotics. We report herein the structure of a penicillin-binding protein complexed with a cephalosporin designed to probe the mechanism of the cross-linking reaction catalyzed by transpeptidases. The 1.2-A resolution x-ray structure of this cephalosporin bound to the active site of the bifunctional serine type D-alanyl-D-alanine carboxypeptidase/transpeptidase (EC ) from Streptomyces sp. strain R61 reveals how the two peptide strands from the polymeric substrates are sequestered in the active site of a transpeptidase. The structure of this complex provides a snapshot of the enzyme and the bound cell wall components poised for the final and critical cross-linking step of cell wall biosynthesis.

Porphyria cutanea tarda (PCT), a liver disease with skin lesions caused by excess liver production of uroporphyrin (URO), is associated with consumption of alcoholic beverages or estrogens, and moderate iron overload. Recently, it has been shown that many PCT patients carry mutations in the HFE gene, which is responsible for hereditary hemochromatosis. Mice homozygous for either the null mutation in the Hfe gene or the C282Y missense mutation rapidly accumulate hepatic parenchymal iron similar to patients with hemochromatosis. Here we investigated whether disruption of the murine Hfe gene would result in hepatic uroporphyria. Mice homozygous for the Hfe-null mutation accumulated high levels of hepatic URO when fed 5-aminolevulinate (ALA). Hfe (+/-) mice also accumulated hepatic URO when fed ALA, but at a much slower rate. The amount of accumulated URO in the null mutant mice was similar to that in wild-type mice treated with iron carbonyl in the diet, or injected with iron dextran. Iron in both wild-type and Hfe (+/-) mice was mostly in Kupffer cells. In contrast, Hfe (-/-) mice had considerable parenchymal iron deposition as well, in a pattern similar to that observed in wild-type mice treated with iron carbonyl. URO accumulation was accompanied by 84% and 33% decreases in hepatic uroporphyrinogen decarboxylase activities in Hfe (-/-) and Hfe (+/-) mice, respectively. No increases in CYP1A2 or other cytochrome P450s were detected in the Hfe-null mutant mice. We conclude that this experimental model of uroporphyria is a valid model for further investigations into the mechanism of PCT.

Chronic lymphocytic leukemia (CLL) is associated with impaired immunoglobulin (Ig) class-switching from IgM to IgG and IgA, a defect that leads to recurrent infections. When activated in the presence of leukemic CLL B cells, T cells rapidly up-regulate CD30 through an OX40 ligand and interleukin 4 (IL-4)-dependent mechanism. These leukemia-induced CD30+ T cells inhibit CD40 ligand (CD40L)-mediated S mu-->S gamma and S mu-->S alpha class-switch DNA recombination (CSR) by engaging CD30 ligand (CD30L), a molecule that interferes with the assembly of the CD40-tumor necrosis factor receptor-associated factor (TRAF) complex in nonmalignant IgD+ B cells. In addition, engagement of T cell CD30 by CD30L on neoplastic CLL B cells down-regulates the CD3-induced expression of CD40L. These findings indicate that, in CLL, abnormal CD30-CD30L interaction impairs IgG and IgA production by interfering with the CD40-mediated differentiation of nonmalignant B cells.

HH should be distinguished from the other syndromes of iron overload. Many patients with HH have abnormal serum iron values before the development of any significant symptoms or clinical findings, and liver biopsy is less important in these patients. HFE mutation analysis has strengthened our ability to diagnose HH accurately and is useful in family studies. HFE mutations may play a contributory role in some patients with PCT, NASH, or chronic HCV. Generalized population screening for HH may someday become a reality and lead to the identification and treatment of more patients before they have tissue damage or increased morbidity. With the identification of the HFE gene, we are beginning to unravel many of the mysteries of both normal iron absorption and the disorder of iron metabolism found in patients with HH.

Among voltage-gated Ca2+ channels the non-dihydropyridine-sensitive alpha1E subunit is functionally less well characterized than the structurally related alpha1A (omega-agatoxin-IVA sensitive, P- /Q-type) and alpha1B (omega-conotoxin-GVIA sensitive, N-type) subunits. In the rat insulinoma cell line, INS-1, a tissue-specific splice variant of alpha1E (alpha1Ee) has been characterized at the mRNA and protein levels, suggesting that INS-1 cells are a suitable model for investigating the function of alpha1Ee. In alpha1E-transfected human embryonic kidney (HEK-293) cells the alpha1E-selective peptide antagonist SNX-482 (100 nM) reduces alpha1Ed- and alpha1Ee-induced Ba2+ inward currents in the absence and presence of the auxiliary subunits beta3 and alpha2delta-2 by more than 80%. The inhibition is fast and only partially reversible. No effect of SNX-482 was detected on the recombinant T-type Ca2+ channel subunits alpha1G, alpha1H, and alpha1I showing that the toxin from the venom of Hysterocrates gigas is useful as an alpha1E-selective antagonist. After blocking known components of Ca2+ channel inward current in INS-1 cells by 2 microM (+/-)-isradipine plus 0.5 microM omega-conotoxin-MVIIC, the remaining current is reduced by 100 nM SNX-482 from -12.4 +/- 1.2 pA/pF to -7.6 +/- 0.5 pA/pF (n = 9). Furthermore, in INS-1 cells, glucose- and KCl-induced insulin release are reduced by SNX-482 in a dose-dependent manner leading to the conclusion that alpha1E, in addition to L-type and non-L-type (alpha1A-mediated) Ca2+ currents, is involved in Ca2+ dependent insulin secretion of INS-1 cells.

This study utilized a multidisciplinary approach to examine injury-induced compensatory responses in the aging hippocampal serotonin transporter (5-HTT), a membrane protein implicated in a variety of neurodegenerative disorders. Age-dependent cellular, anatomical, and physiological changes of the 5-HTT were evaluated in female Fischer 344 rats (2 and 17 months) following denervation of the serotonergic afferents (fimbria-fornix and cingulum bundle) to the dorsal hippocampus using the neurotoxicant 5,7-dihydroxytryptamine (5,7-DHT). Seven days following 5,7-DHT administration, a uniform loss of the hippocampal 5-HTT immunoreactivity was observed in both age groups. However, at 21 days 5-HTT immunoreactivity in young 5,7-DHT-treated animals was similar to control levels, indicative of recovery, while older animals exposed to 5,7-DHT did not show recovery of hippocampal 5-HTT expression. 5-HTT binding site density, as determined by quantitative autoradiography ([3H]citalopram), supported the immunohistochemical results by demonstrating a recovery of 5-HTT binding sites in young, but not old animals, at 21 days following the lesion (P < 0.001). Furthermore, cellular electrophysiological function of hippocampal CA1 pyramidal neurons in 3- and 18-month-old F344 rats at 21 days following 5,7-DHT or vehicle treatment were assessed using in vivo microiontophoretic application of serotonin (5-HT). Independent of changes in sensitivity to the inhibitory effects of 5-HT application, the time to recovery of cell firing following application of 5-HT was significantly increased in the 18-month 5,7-DHT group compared to the 18-month vehicle and 3-month 5,7-DHT groups (60 and 59% increases, respectively; P < 0.05). Overall, these series of studies comprise a model which can be used to identify cellular events underlying both the formation of injury-induced compensatory processes in younger animals and the lack thereof with advancing age.

The aim of this study was to investigate the regulation of Rb protein expression in relation to increased differentiation and induction of apoptosis in colonic epithelial cells. In vivo, Rb protein expression was found to be down-regulated towards the top of the normal colonic crypt, coincident with the region of differentiation and apoptosis, but highly expressed in colonic carcinoma tissue. Using in vitro models to study the regulation of Rb expression in pre-malignant colonic epithelial cells, we have been able to show for the first time that Rb protein expression is transcriptionally down-regulated in differentiated pre-malignant cells (in post-confluent cultures) but not in malignant colorectal epithelial cells. Furthermore, suppression of rb protein function by the HPV-E7 viral oncoprotein increased both spontaneous and DNA damage-induced apoptosis. These results suggest that Rb is able to act as a survival factor in colonic epithelial cells by suppressing apoptosis, and that over-expression of pRb in colorectal tumour cells can cause a loss of sensitivity to apoptotic signalling, resulting in aberrant cell survival and resistance to therapy.

We assessed the relative expression of oestrogen receptor (ER)alpha and oestrogen receptor (ER)beta mRNAs in 36 human endometrial cancers using a multiplex polymerase chain reaction (PCR). To determine whether or not the expression of ER subtypes in endometrial cancers is associated with clinicopathological parameters, we examined correlations between ER subtypes and age, tumour grade and depth of myometrial invasion. Using multiple regression analysis, myometrial invasion showed a significant correlation with ER-beta: ER-alpha ratio (r = 0.54, P = 0.0007). The ER-beta:ER-alpha ratio was high in advanced invasive carcinoma. Western blotting analysis showed that ER-beta proteins were highly expressed in comparison with ER-alpha proteins in endometrial cancer with severe myometrial invasion. Our results suggest that ER-beta is important in the progression of myometrial invasion.

The doxorubicin (DOX) prodrug N-[4-doxorubicin-N-carbonyl (oxymethyl) phenyl] O-beta-glucuronyl carbamate (DOX-GA3) was synthesised for specific activation by human beta-glucuronidase, which is released in necrotic areas of tumour lesions. This novel prodrug was completely activated to the parent drug by human beta-glucuronidase with V(max)= 25.0 micromol x min(-1) x mg(-1) and K(m) = 1100 microM. The pharmacokinetics and distribution of DOX-GA3 in nude mice bearing human ovarian cancer xenografts (OVCAR-3) were determined and compared with DOX. Administration of DOX at 8 mg x kg(-1) i.v. (maximum tolerated dose, MTD) to OVCAR-3-bearing mice resulted in a peak plasma concentration of the drug of 16.4 microM (t = 1 min). A 7.6-times lower peak plasma concentration of DOX was measured after injection of DOX-GA3 at 250 mg x kg(-1) i.v. (50% of MTD). In normal tissues the prodrug showed peak DOX concentrations that were up to 5-fold (heart) lower than those found after DOX administration. DOX-GA3 activation by beta-glucuronidase in the tumour yielded an almost 5-fold higher DOX peak concentration of 9.57 nmol x g(-1) (P< 0.05) than the peak concentration of only 2.14 nmol x g(-1) observed after DOX. As a consequence, the area under the curve of DOX calculated in tumour tissue after DOX-GA3 (13.1 micromol x min(-1) x g(-1)) was 10-fold higher than after DOX (1.31 micromol x min(-1) x g(-1)). The anti-tumour effects of DOX-GA3 and DOX were compared at equitoxic doses in OVCAR-3 xenografts at a mean tumour size of 125 mm(3). The prodrug given i.v. at 500 mg x kg(-1) weekly x 2 resulted in a maximum tumour growth inhibition of 87%, while the standard treatment with DOX at a dose of 8 mg x kg(-1) i.v. weekly x 2 resulted in a maximum tumour growth inhibition of only 56%. Treatment with DOX-GA3 was also given to mice with larger tumours containing more necrosis. For tumours with a mean size of 400 mm(3) the specific growth delay by DOX-GA3 increased from 2.7 to 3.9. Our data indicate that DOX-GA3 is more effective than DOX and suggest that the prodrug will be specifically advantageous for treatment of advanced disease.

As well as being a passive support, the extracellular matrix also regulates key biological processes such as invasion, differentiation and angiogenesis. We have therefore developed an in vitro model of bladder cancer invasion using de-epithelialized rat bladder to allow for tumour cell-extracellular matrix interactions. Onto this we have seeded a panel of human bladder cancer cell lines (RT4, RT112, 253J and EJ28 (T24)) representing progression from well to poorly differentiated phenotypes and used as models of superficial to invasive bladder cancer. The better differentiated cell lines RT4 and RT112 reproducibly grew as stratified epithelium, whereas poorly differentiated EJ28 cells invaded across a broad front. Invasion was not simply related to proliferation rate, measured either as doubling time on plastic (non-invasive 253J and invasive EJ28 having the same doubling time) or by Ki-67 proliferation index within the model. We used the model to test the ability of 4 compounds that interfere with tumour cell-extracellular matrix interactions (suramin, N-acetylcysteine and the urokinase plasminogen activator pathway antagonists A5 compound and monoclonal antibody Mab 3936) to inhibit invasion. At non-toxic concentrations, all significantly inhibited invasion (P< 0.05), although to varying degrees, suramin and A5 almost completely and N-acetylcysteine the least. In conclusion, this model shows the urokinase system is important for bladder invasion and can be used to investigate other mechanisms of bladder cancer invasion and also for the testing of intravesical drugs.

Severe combined immunodeficient (SCID) mice injected i.v. with the human T-ALL cell line CCRF CEM (SCID-CEM mice) develop within 50 days life-threatening multi-organ growth of leukaemia cells. The development of leukaemia in SCID-CEM mice treated with three 10 microg i.v. doses of the anti-CD7 immunotoxin (IT) HB2-SAPORIN or the anti-CD38 IT OKT10-SAPORIN was significantly delayed compared with PBS sham-treated animals but 90% of animals treated with either IT eventually developed disseminated leukaemia cell growth. In contrast treatment of SCID-CEM mice with a combination of both ITs led not only to a significantly greater delay in time to leukaemia development but also in the numbers of animals remaining leukaemia free (60%). The native HB2 and OKT10 antibodies (both murine IgG1antibodies) exerted significant, though relatively weak therapeutic effects, probably mediated through an antibody-dependent cellular cytotoxicity (ADCC) mechanism. Moreover, there was no in vivo additivity of therapeutic effect when both antibodies were used in combination. Apparent, however, was that the combination of HB2-SAPORIN IT with OKT10 antibody led to an intermediate therapeutic effect that was significantly greater than that obtained when either was used alone but significantly less than that obtained when the two IT combination was utilized. This was similarly the case for the combination of OKT10-SAPORIN IT with HB2 antibody though the effect was less pronounced in this instance. This result suggests that the therapeutic effect of IT + antibody treatment results from an additivity between antibody-mediated delivery of saporin combined with a SCID mouse NK cell-mediated ADCC attack on the target cell directed through target cell bound antibody Fc engagement with FcgammaRIII on the NK cell surface. The combination of both ITs however gave the best therapeutic outcome in SCID-CEM mice probably as the result of (i) delivery of greater amounts of saporin to target CEM cells positive for both CD7 and CD38, (ii) delivery of an effective dose of saporin to CEM cells downregulated or negative for one of the target antigens and (iii) through ADCC mechanisms that interact additively with IT action. We have previously proposed that combination IT therapy would be one means of overcoming the problem of heterogeneity of antigen expression within a global tumour cell population and these additional findings support this and provide a further strengthening of the rationale for employing cocktails of ITs for the treatment of human malignancies.

A marked antitumour efficacy is currently obtained by oxaliplatin (LOHP)-fluorouracil (FU)-folinic acid (FA) combination and by CPT11-FU-FA combination. Logically, the triple association LOHP, CPT11 and FUFA will be soon tested in cancer patients. The aim of the present study was to compare two schedules combining SN38 (the active metabolite of CPT11, irinotecan) with FU-FA and LOHP. The two schedules differed by the SN38 position. The relative contribution of each drug in the resulting global cytotoxicity was evaluated. Two human colon cancer cell lines were used (WIDR and SW620 both p53 mutated). LOHP plus FA were applied for 2 h, just before a 48 h FU exposure. The SN38 sequence was applied for 24 h, starting either 48 h before LOHP-FA (schedule A), or just after LOHP-FA exposure (schedule B). Cytotoxicity was assessed by the 3-(4,5-demethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) test and drug interactions were analysed according to the Chou and Talalay method, based on the computation of a combination index (CI). The SN38 position significantly induces a shift from additivity-antagonism when SN38 was applied after LOHP, towards additivity-synergism when SN38 was applied first (P = 0.03). The relative contribution (RC) of each drug in the overall cytotoxicity of the triple combination was defined as the drug concentration giving 50% cell lethality (IC(50)) of the double association without that drug divided by the IC(50)of the triple association. Whatever the SN38 position, the larger contribution was made by LOHP (median RC = 2.4) and the smaller by SN38 (median RC = 1.1). In addition, the contribution of FUFA was improved when SN38 was applied first (median RC = 2.2) as compared to the opposite schedule (median RC = 1.2). Results were in agreement between the two explored cell lines. The present data should be taken into account when establishing the rationale of future trials combining CPT11, LOHP and FU-FA.

Iron is vital for almost all living organisms by participating in a wide variety of metabolic processes, including oxygen transport, DNA synthesis, and electron transport. However, iron concentrations in body tissues must be tightly regulated because excessive iron leads to tissue damage, as a result of formation of free radicals. Disorders of iron metabolism are among the most common diseases of humans and encompass a broad spectrum of diseases with diverse clinical manifestations, ranging from anemia to iron overload and, possibly, to neurodegenerative diseases. The molecular understanding of iron regulation in the body is critical in identifying the underlying causes for each disease and in providing proper diagnosis and treatments. Recent advances in genetics, molecular biology and biochemistry of iron metabolism have assisted in elucidating the molecular mechanisms of iron homeostasis. The coordinate control of iron uptake and storage is tightly regulated by the feedback system of iron responsive element-containing gene products and iron regulatory proteins that modulate the expression levels of the genes involved in iron metabolism. Recent identification and characterization of the hemochromatosis protein HFE, the iron importer Nramp2, the iron exporter ferroportin1, and the second transferrin-binding and -transport protein transferrin receptor 2, have demonstrated their important roles in maintaining body's iron homeostasis. Functional studies of these gene products have expanded our knowledge at the molecular level about the pathways of iron metabolism and have provided valuable insight into the defects of iron metabolism disorders. In addition, a variety of animal models have implemented the identification of many genetic defects that lead to abnormal iron homeostasis and have provided crucial clinical information about the pathophysiology of iron disorders. In this review, we discuss the latest progress in studies of iron metabolism and our current understanding of the molecular mechanisms of iron absorption, transport, utilization, and storage. Finally, we will discuss the clinical presentations of iron metabolism disorders, including secondary iron disorders that are either associated with or the result of abnormal iron accumulation.

The Escherichia coli SOS-regulated umuDC gene products participate in a DNA damage checkpoint control and in translesion DNA synthesis. Specific interactions involving the UmuD and UmuD' proteins, both encoded by the umuD gene, and components of the replicative DNA polymerase, Pol III, appear to be important for regulating these two biological activities of the umuDC gene products. Here we show that overproduction of the epsilon proofreading subunit of Pol III suppresses the cold sensitivity normally associated with overexpression of the umuDC gene products. Our results suggest that this suppression is attributable to specific interactions between UmuD or UmuD' and the C-terminal domain of epsilon.

Currently, a major focus of human genetics is the utilization of single-nucleotide polymorphisms for clinical diagnostics, whole-genome linkage disequilibrium screens to identify common disease genes such as Alzheimer disease, determination of the recent evolutionary history of a species, and the process of speciation. We have examined single-nucleotide extension coupled with high-performance liquid chromatography as a method to simultaneously genotype two SNPs occurring in the coding region of the HFE gene that produce clinical effects. This assay allows concurrent genotyping of the C282Y and H63D mutations in 11 min and is 100% concordant with current testing methods for both of these mutations.

Thyroid lesions develop in most NOD.H-2h4 mice 6 weeks after they are given 0.05% NaI in drinking water. B cells are required for spontaneous autoimmune thyroiditis (SAT) development, and anti-thyroglobulin autoantibody levels correlate with SAT severity. Immunohistochemical staining of thyroids obtained 2-10 weeks after administration of NaI water suggested that CD4+ T cells initially infiltrated the thryoid, followed by CD8+ T cells and B cells. Intrathyroidal CD4+ T cells are more numerous than CD8+ T cells. CD4+ T cells and B cells form aggregates in the thyroid, while CD8+ T cells are scattered throughout the thyroid. Intrathyroidal germinal centre-like structures could be observed in thyroid lesions with 2-3+ SAT and intrathyroidal B cells co-expressed OX40L. By RT-PCR, intrathyroidal expression of OX40L, OX40, CD40L, IL-2R, CTLA-4 and Igbeta mRNA correlated closely with the SAT severity score. These molecules were not expressed in normal thyroids. In the spleen, OX40L-positive cells were detected at 2 weeks and increased 4-6 weeks after NaI water. OX40, OX40L, CD40L, IL-2R and B7-1 as well as IFN-gamma and IL-4 mRNA were minimally expressed in normal spleens, usually began to be expressed at 2 weeks and increased to maximal level 4-8 weeks after NaI water. These results suggest that in NOD.H-2h4 mice, the OX40L, OX40, CD40L and B7 molecules, which increase in the spleen and thyroid of these mice after receiving NaI water, may play a role in SAT development, implying that one or more of these molecules might be good targets for the prevention or treatment of SAT.

The aim of this study was to elucidate whether or not p53 genetic heterogeneity would occur while colorectal carcinoma was limited to the mucosa. Eight cases of endoscopically resected colorectal intramucosal carcinomas were analyzed to determine the p53 gene sequence (exons 5 to 8). Six out of 8 cases showed p53 gene mutations, and in all of them, the mutational status was heterogeneous. In 4 cases, mutated codons were heterogeneous as well. These data indicate that p53 gene alterations in colorectal carcinomas occur and diverge at the stage of intramucosal carcinoma, supporting our previously proposed hypothesis that colorectal carcinomas can be composed of various subclones as regards p53 gene mutation, while the carcinoma is limited to the mucosa, and one of these subclones commences invasion to the submucosa after clonal selection, thus generating a monoclonal invasive carcinoma.

Hereditary hemochromatosis (HH) is a common disorder of iron metabolism caused by mutation in HFE, a gene encoding an MHC class I-like protein. Clinical studies demonstrate that the severity of iron loading is highly variable among individuals with identical HFE genotypes. To determine whether genetic factors other than Hfe genotype influence the severity of iron loading in the murine model of HH, we bred the disrupted murine Hfe allele onto three different genetically defined mouse strains (AKR, C57BL/6, and C3H), which differ in basal iron status and sensitivity to dietary iron loading. Serum transferrin saturations (percent saturation of serum transferrin with iron), hepatic and splenic iron concentrations, and hepatocellular iron distribution patterns were compared for wild-type (Hfe +/+), heterozygote (Hfe +/-), and knockout (Hfe -/-) mice from each strain. Although the Hfe -/- mice from all three strains demonstrated increased transferrin saturations and liver iron concentrations compared with Hfe +/+ mice, strain differences in severity of iron accumulation were striking. Targeted disruption of the Hfe gene led to hepatic iron levels in Hfe -/- AKR mice that were 2.5 or 3.6 times higher than those of Hfe -/- C3H or Hfe -/- C57BL/6 mice, respectively. The Hfe -/- mice also demonstrated strain-dependent differences in transferrin saturation, with the highest values in AKR mice and the lowest values in C3H mice. These observations demonstrate that heritable factors markedly influence iron homeostasis in response to Hfe disruption. Analysis of mice from crosses between C57BL/6 and AKR mice should allow the mapping and subsequent identification of genes modifying the severity of iron loading in this murine model of HH.

The deleted in colorectal cancer (DCC) protein is important in the pathway guidance of cells and cell processes during neural development, and DCC has also been implicated in the aberrant cellular migrations of neuroblastoma dissemination. We attempted to further define DCC protein function by the overexpression of full-length and truncated DCC constructs in a human neuroblastoma cell line. Overexpression of the truncated DCC protein resulted in a less epithelioid morphology. This was accompanied by decreases in expression of N-cadherin and alpha- and beta-catenin by immunoblot and Northern blot analysis. Levels of desmoglein were relatively less affected, whereas endogenous DCC protein levels were increased in the truncated transfectants. N-cadherin immunofluorescence was consistent with the immunoblot studies and localized the protein to the cytoplasm and sites of cell-cell contact. Cell aggregation studies demonstrated diminished calcium-dependent aggregation in the truncated transfectants. In conclusion, overexpression of a truncated DCC protein in neuroblastoma cells resulted in the loss of an epithelioid morphology, diminished expression of N-cadherin and alpha- and beta-catenin, and diminished calcium-dependent cell adhesion. These studies provide the first evidence of an apparent functional link between DCC and N-cadherin/catenin-dependent cell adhesion.

<h4>Objective</h4>In healthy conditions, factors inducing or inhibiting coagulation and factors inducing or inhibiting fibrinolysis are in balance. In ulcerative colitis, hypercoagulation is presumed, which may explain part of the clinical features of this disease. Therapy strategies affecting hemostasis may improve the course of ulcerative colitis. This study was conducted to evaluate the balance of coagulation and fibrinolysis in the course of treatment of active ulcerative colitis.<h4>Methods</h4>Patients with active ulcerative colitis were studied by serial determination of markers of the coagulation cascade (thrombin-antithrombin complexes and fibrin degradation products [FbDP]) and the fibrinolytic cascade (fibrinogen degradation products [FgDP]). Parameters of inflammation were also measured (C-reactive protein [CRP], erythrocyte sedimentation rate [ESR], albumin, platelet count, and fibrinogen). Disease activity was assessed by endoscopic and histopathological scores. Follow-up measurement was performed in the course of treatment at the third or fourth month after baseline. Measurements were compared with healthy controls.<h4>Results</h4>Thirty-three patients and 22 healthy controls were included. During active ulcerative colitis, inflammatory parameters (CRP, ESR, platelet count) and hemostatic parameters (thrombin-antithrombin complexes, fibrinogen, FgDP, and FbDP) were elevated in comparison with healthy controls. Albumin was decreased and antithrombin-III remained unchanged. During treatment, disease activity decreased significantly endoscopically and histopathologically (p < 0.001). CRP, ESR, platelet count, and fibrinogen also decreased significantly. The hemostatic balance, expressed as the ratio between the plasmin-dependent generation of FgDP and coagulation-dependent generation of FbDP, increased from 0.69 to 1.12 during treatment, mainly because of a decrease of FbDP. In healthy controls, this ratio was<h4>Conclusions</h4>The coagulation and fibrinolytic cascades were activated in active ulcerative colitis, with a hemostatic imbalance in favor of coagulation. This hypercoagulability persisted in 20% (7/33) of patients with ulcerative colitis in remission. The decrease of FbDP and the increase in the FgDP/FbDP ratio during reconvalescence of ulcerative colitis showed that the coagulation cascade was more activated than the fibrinolytic cascade in active disease.

<h4>Objective</h4>The relationship between the degree of iron overload and the presence of cirrhosis has not been clearly established in hemochromatosis. Severe iron overload occurs without cirrhosis and moderate iron overload can occur with cirrhosis. Previous estimates may have overestimated the problem because of the overdiagnosis of hemochromatosis in patients with alcoholic liver disease and chronic viral hepatitis. The objective of this study was to determine if a threshold for hepatic iron concentration leads to the development of cirrhosis in C282Y hemochromatosis.<h4>Methods</h4>This study included only hemochromatosis patients who were homozygotes for the C282Y mutation of the HFE gene and had undergone liver biopsy with hepatic iron concentration. Analysis of the thresholds for cirrhosis were determined using receiver operating characteristic (ROC) curve analysis.<h4>Results</h4>Data were available on 100 C282Y homozygotes (62 men, 38 women; mean age 51, range 18-74 yr). ROC curve analysis showed an area under the curve for hepatic iron concentration versus cirrhosis of 0.85 (95% CI = 0.75-0.96). The threshold for the prediction of cirrhosis was 283 micromol/g. At that threshold, the sensitivity was 85% and specificity 84%.<h4>Conclusions</h4>From this analysis, it appears that a hepatic iron concentration >283 micromol/g is associated with cirrhosis. However, the low sensitivity of this threshold suggests that other cofactors contribute to the development of cirrhosis in hemochromatosis. Early diagnosis is encouraged to initiate iron depletion before the development of cirrhosis.

The antidepressive actions of electroconvulsive shock (ECS) therapy are considered to involve altered neurotransmission of serotonin. In this study, we investigated the effects of acute and chronic ECS on 5-hydroxytryptamine (5-HT) transporter mRNA expression in rat raphe nucleus. We found that serotonin transporter (5-HTT) mRNA expression was decreased in 9 and 24 h after acute ECS and in 3, 9, 24 h and 2 weeks after chronic ECS in rat raphe nucleus. We presume that the adaptive change in 5-HTT mRNA expression is possibly related to the therapeutic efficacy of electroconvulsive therapy (ECT) on medication-resistant depression.

<h4>Objective</h4>To establish a sensitive method for measuring nontransferrin-bound iron (NTBI) in serum samples using graphite furnace atomic absorption spectrometry (GFAAS).<h4>Design and methods</h4>Nontransferrin-bound iron (NTBI) was chelated using nitrilotriacetic acid (NTA) and then ultrafiltered according to the method employed by Singh et al. [1]. Serum ultrafiltrates were diluted eightfold with distilled water. NTBI from the Fe-NTA complex present in the serum ultrafiltrate was measured using GFAAS.<h4>Results</h4>Nontransferrin-bound iron (NTBI) and other parameters were measured in seven patients diagnosed with hereditary hemochromatosis by liver biopsy. Total serum iron, NTBI and transferrin saturation values (ranging from 87% to 90%) were elevated for three of the seven hemochromatosis patients tested before therapeutic phlebotomy. Six of the seven hemochromatosis patients had undergone phlebotomy and revealed normal total serum iron, NTBI and transferrin saturation values. Nine test subjects (not diagnosed with hemochromatosis) with abnormally high total serum iron and/or ferritin concentrations exhibited normal NTBI values (< or =0.14 micromol/L to 0.29 micromol/L). The detection limit was 0.1 micromol/L for a 25 microL injection volume.<h4>Conclusions</h4>The GFAAS method presented here provides a sensitive assay to quantitate NTBI in serum samples. The method developed is 4 to 5 times more sensitive than the only other GFAAS method [2] and more than an order of magnitude more sensitive than other colorimetric methods [1,3]. Improvement in sensitivity over the other GFAAS method [2] may be accounted for by differences in sample preparation between this method and that of Nielsen et al. [2]. Serum ultrafiltrates in this study were diluted eightfold with distilled water and mixed with a magnesium nitrate matrix modifier before GFAAS analysis. NTBI results obtained from this study indicate that the plasma iron pool in hemochromatosis patients awaiting phlebotomy increases to a level at which transferrin's ability to bind iron becomes exhausted and elevated NTBI levels appear in the serum. NTBI can mediate the production of reactive oxygen species and may cause organ damage associated with iron overload.

The establishment of sister chromatid cohesion during S phase and its dissolution at the metaphase-anaphase transition are essential for the faithful segregation of chromosomes in mitosis [1-4]. Recent studies in yeast genetics and Xenopus biochemistry have identified a large protein complex, cohesin, that plays a key role in sister chromatid cohesion [5-10]. The cohesin complex consists of a heterodimeric pair of SMC (structural maintenance of chromosomes) subunits and at least two non-SMC subunits. This structural organization is reminiscent of that of condensin, another major SMC protein complex that drives chromosome condensation in eukaryotic cells [11]. Condensin has been shown to reconfigure and compact DNA in vitro by utilizing the energy of ATP hydrolysis [12]. Very little is known, however, about how cohesin works at a mechanistic level. Here we report the first set of biochemical activities associated with an intact cohesin complex purified from HeLa cell extracts. The cohesin complex binds directly to double-stranded DNA and induces the formation of large protein-DNA aggregates. In the presence of topoisomerase II, cohesin stimulates intermolecular catenation of circular DNA molecules. This activity is in striking contrast to intramolecular knotting directed by condensin [13]. Cohesin also increases the probability of intermolecular ligation of linear DNA molecules in the presence of DNA ligase. Our results are consistent with a model in which cohesin functions as an intermolecular DNA crosslinker and is part of the molecular "glue" that holds sister chromatids together [14].

Increased iron stores may play a role in the development of coronary heart disease (CHD) by increasing lipoprotein oxidation. Recently, mutations have been discovered in the gene (HFE) for hereditary hemochromatosis, an autosomal recessive condition of disordered iron metabolism, absorption, and storage. It is possible that people who carry HFE mutations have increased risk of CHD. We used a prospective case-cohort design (243 CHD cases and 535 non-cases) to determine whether the HFE C282Y mutation was associated with incident CHD in a population-based sample of middle-aged men and women. The frequencies of homozygosity and heterozygosity for the C282Y mutation in the ARIC study population were 0.2% (one homozygous person) and 6%, respectively. The C282Y mutation was associated with nonsignificantly increased risk of CHD (relative risk=1.60, 95% CI 0.9-2.9). After adjusting for other confounding risk factors (age, race, gender, ARIC community, smoking status, diabetes status, hypertension status, LDL cholesterol, HDL cholesterol, and triglycerides), the association became stronger (relative risk=2.70, 95% CI 1.2-6.1). However, a sensitivity analysis showed that this estimate of relative risk was somewhat unstable due to few subjects in some strata. Our prospective findings suggest that individuals carrying the HFE C282Y mutation may be at increased risk of CHD.

Site-specific acetylation of histone H4 by MOF is central to establishing the hyperactive male X chromosome in Drosophila. MOF belongs to the MYST family of histone acetyltransferases (HATs) characterized by an unusual C2HC-type zinc finger close to their HAT domains. The function of these rare zinc fingers is unknown. We found that this domain is essential for HAT activity, in addition to the established catalytic domain. MOF uses its zinc finger to contact the globular part of the nucleosome as well as the histone H4 N-terminal tail substrate. Point mutations that leave the zinc-finger structure intact nevertheless abolish its interaction with the nucleosome. Our data document a novel role of the C2HC-type finger in nucleosome binding and HAT activity.

Aurora/Ipl1-related kinases are a conserved family of enzymes that have multiple functions during mitotic progression. Although it has been possible to use conventional genetic analysis to dissect the function of aurora, the founding family member in Drosophila (Glover, D.M., M.H. Leibowitz, D.A. McLean, and H. Parry. 1995. Cell. 81:95-105), the lack of mutations in a second aurora-like kinase gene, aurora B, precluded this approach. We now show that depleting Aurora B kinase using double-stranded RNA interference in cultured Drosophila cells results in polyploidy. aurora B encodes a passenger protein that associates first with condensing chromatin, concentrates at centromeres, and then relocates onto the central spindle at anaphase. Cells depleted of the Aurora B kinase show only partial chromosome condensation at mitosis. This is associated with a reduction in levels of the serine 10 phosphorylated form of histone H3 and a failure to recruit the Barren condensin protein onto chromosomes. These defects are associated with abnormal segregation resulting from lagging chromatids and extensive chromatin bridging at anaphase, similar to the phenotype of barren mutants (Bhat, M.A., A.V. Philp, D.M. Glover, and H.J. Bellen. 1996. Cell. 87:1103-1114.). The majority of treated cells also fail to undertake cytokinesis and show a reduced density of microtubules in the central region of the spindle. This is accompanied by a failure to correctly localize the Pavarotti kinesin-like protein, essential for this process. We discuss these conserved functions of Aurora B kinase in chromosome transmission and cytokinesis.

Evidence suggests that antithrombin III (ATIII) exerts anti-inflammatory properties in addition to its anti-coagulative mechanisms. In animal models of sepsis, ATIII affected cytokine plasma concentrations with a decrease of pro-inflammatory cytokines. In addition to cytokines, excessive production of nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) might represent another important mediator of the cytotoxic events during sepsis. Regarding ATIII as a potential anti-inflammatory modulator, one may speculate that ATIII inhibits the synthesis of iNOS-derived NO. However, our data demonstrate that ATIII further stimulates iNOS gene expression when applied together with either interleukin-1 beta or the combination of lipopolysaccharide plus interferon-gamma. The most prominent synergistic effects on NO synthesis were found when ATIII was given at higher concentrations (1, 5, and 10 U/ml). Although the mechanisms of ATIII signal transduction remain to be established, intensification of interleukin-1 beta or interferon-gamma/lipopolysaccharide-induced NO synthesis by ATIII does not attribute to the anti-inflammatory properties of ATIII.

It is now more than 7 years since the genetic mutation causing Huntington's disease (HD) was first identified. Unstable CAG expansion in the IT15 gene, responsible for disease, is translated into an abnormally long polyglutamine (polyQ) tract near the N-terminus of the huntingtin protein. The presence of expanded polyQ in the mutant protein leads to its abnormal proteolytic cleavage with liberation of toxic N-terminal fragments that tend to aggregate, probably first in the cytoplasm. Subsequent nuclear translocation of the cleaved mutant huntingtin is associated with formation of intranuclear protein aggregates and neurotoxicity, probably involving apoptotic cascades. These processes, which can be experimentally modelled in cultured neuronal and non-neuronal cells, seem to underlie neurodegeneration in HD, and also other polyQ disorders, such as dentatorubro-pallidoluysian degeneration, spinal and bulbar muscular atrophy and the spinocerebellar ataxias. They do not, however, explain why within the corpus striatum and cerebral cortex certain nerve cells are susceptible to disease and others are not. In the human HD brain, vulnerable pyramidal neurones within the deeper layers of the cerebral cortex frequently contain large intranuclear inclusions composed of N-terminal fragments of huntingtin. Such inclusions are, however, rare within neurones of the striatum, even in the medium spiny neurones preferentially lost from this region. While inclusions per se do not seem to be neurotoxic, they may provide a surrogate marker of molecular pathology. Recent studies indicate that the nuclear accumulation of mutant huntingtin interferes with transcriptional events. Of particular importance may be the effect on the genes encoding neurotransmitter receptor proteins, especially those involved with glutamatergic neurotransmission. Such changes may trigger or facilitate a low-grade, chronic excitotoxicity of the glutamatergic cortical projection neurones on their target cells in the striatum, already partly compromised by the toxic effects of the HD mutation. This combination of insults, for anatomical reasons experienced predominantly by striatal projection neurones, would eventually cause their selective demise.

We have attempted to establish a systematic pathogenetic analysis of thrombophilia by including assays of antithrombin III(AT III), protein C(PC), protein S(PS), fibrinogen, plasminogen and heparin cofactor II by both functional and immunological methods as well as detecting lupus anticoagulants. Such a comprehensive scheme was instrumental in systematically identifying and confirming the pathogenesis of 164 cases which otherwise would have escaped detection since 1994 in our laboratory (Kyushu University Hospital). The analysis was conducted on 485 consecutive patients with venous thrombosis, arterial thrombosis and disorders in which small vessel thrombosis were implicated. Hundred and sixty four patients, (40% of the examined patients), were found to have low activities of PS, PC, ATIII etc. Among them, seventy five patients(46%) had low PS activity, and twenty nine(18%) had low PC activity. Genetic analyses performed on specimens with low PS/PC activities resulted in the confirmation of 24 genetic abnormalities. Such genetic abnormalities, however, does not solely lead to the pathogenesis of thromboses. We have found that some genetic polymorphisms, such as PS Tokushima, factor XII 46C allele, were also additional risk factors for thromboses.

Like most essential nutrients, Fe needs to be maintained in the body at a defined level for optimal health, with appropriate adaptation to varying Fe needs and supply. The primary mechanism for controlling Fe level is the regulation of Fe absorption. Several different proteins have been identified as contributors to the process. Despite a complex regulatory system, Fe disorders (both Fe deficiency and Fe overload) occur. Fe deficiency is a common problem worldwide, resulting from inadequate dietary Fe and blood loss. Complications include pre-term labour, developmental delay, and impaired work efficiency. No specific genetic syndromes causing isolated Fe deficiency have been described, but animal studies and clinical observations suggest that such a relationship may be a possibility. Conversely, the known causes of Fe overload are genetic. Fe overload is less common than Fe deficiency, but can result in serious medical complications, including cirrhosis, primary liver cancer, diabetes, cardiomyopathy and arthritis. The most common and best characterized syndrome of Fe overload is hereditary haemochromatosis (HHC), an autosomal recessive disorder. Mutations in the HFE protein cause HHC, but the clinical presentation is variable. Of particular interest is the factor that some FIFE genotypes appear to be associated with protection from Fe deficiency. Other genetic variants in the regulatory pathway may influence the likelihood of Fe deficiency and Fe overload. Studies of genetic variants in HFE and other regulatory proteins provide important tools for studying the biological processes in Fe regulation. This work is likely to lead to new insights into Fe disorders and potentially to new therapeutic approaches. It will not be complete, however, until coordinated study of both genetic and nutritional factors is undertaken.

The aim of the present study was to investigate the relationship between different measures of iron status, and the expression of CD2, and the activation markers CD25, CD71, CD45RO, HLADR CD38 within the Th-cell subset in patients with progressive transfusional iron overload. We estimated the expression of the activation surface markers on the Th cells of peripheral blood by flow cytometry from 22 multiply transfused patients. The number of CD2 binding sites (BS) on Th cells was significantly higher in the patients (82 917 +/- 30 801) than in age-matched normal controls (41 145 +/- 6989, P < 0.0001). When investigating whether this difference could be due to the iron overload we found the number of CD2 BS closely related to the iron saturation of serum transferrin (TfS) (R2 = 0.78, P < 0.001). The relationship to the serum ferritin concentration and to the number of blood units given was weaker, but also significant (R2 = 0.22, P < 0.027, respectively, R2 = 0.21, P < 0.032). Also the fraction of mature memory Th cells which express CD45RO at a high level was directly related to the TfS (R2 = 0.57, P < 0.0001), while the expression of CD38 within the Th cell fraction was inversely related to the TfS (R2 = - 0.43, P = 0.009). The expression of HLA-DR (but not of CD25 and CD71) was also directly related to the TfS (R2 = 0.29, P = 0.01). Our results show a clear, statistical relationship between the iron status and the expression of surface markers within Th cells in multiply transfused patients.

Plasma was subjected to methylene blue (MB) photochemical virus inactivation using the Maco Pharma Maco-Tronic system which allows three units to be illuminated together, thus reducing processing time. The plasma bag system used incorporates an integral membrane plasma filter and a dry MB pill which dissolves in the plasma to give a 1-microM concentration. There is computer-controlled processing and datalogging. In an assessment of 10 pools of Group A plasma, the losses of coagulation factors, following MB/light treatment, were 23% fibrinogen, 10% FV, 26% FVIII, 11% FIX and 13% FXI. Group O, Group B and Group AB plasmas were not tested. Von Willebrand factor (vWf) multimers showed no substantial change when treated with MB, and no losses were seen for antithrombin III (ATIII), protein C and vWf:Ag. Measurements of C3a, C5a, prothrombin fragment 1+2 and FXIIa indicated that there was no activation as a result of filtration.

No abstract available.

No abstract available.