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To investigate the pathogenesis of hyperlipidemia-induced atherosclerosis, we examined age-dependent changes in platelet activity, blood coagulation and fibrinolysis in susceptibility to a high cholesterol diet (HCD) feeding in male ICR mice. Pretreatment of platelet-rich-plasma from HCD feeding mice for 3 days with epinephrine (300 microM) resulted in a marked enhancement of adenosine 5'-diphosphate (ADP: 0.1 microM) or collagen (0.7 microgram/ml)-stimulated aggregation compared with the same in control mice. Yohimbine as alpha 2-adrenergic blocker antagonized these aggregations in a dose-dependent manner. A significant increase in plasma total cholesterol and VLDL (very low-density lipoprotein)-LDL (low-density lipoprotein)-cholesterol and the liver/body weight ratio was observed in mice fed on HCD for 3 months (3-month HCD mice). In the early phase of this experiment, a significant increase in fibrinogen was observed. In the middle phase, increases in the activity of antithrombin III (ATIII) and alpha 2-plasmin inhibitor (alpha 2-Pl) followed. Plasminogen content gradually decreased in both normal diet and HCD mice throughout the experiment. The activity of plasminogen activator inhibitor (PAI) decreased in 3-month HCD mice. Morphological observation of the aortic arch from 3-month HCD mice revealed apparent atheromatous plaques not seen in control mice. These results suggest that 3-month HCD mice can be a convenient hyperlipidemia-induced atherosclerotic model and the changes in platelet activity, coagulation and fibrinolysis in the early phase may be a cause of pathologic changes in this model.

No abstract available.

The importance of the receptor level in breast cancer as an indicator of hormone response has been extensively studied for more than 20 years. Besides cytosol-based ligand-binding assays (dextran-coated charcoal assay, DCC), new methods using monoclonal antibodies raised against estrogen and progesterone receptors allow for the detection of receptors both in cytosol extracts (enzyme immunoassay, EIA) and in tissue sections (immunocytochemical assay, ICA). The biochemical assays (DCC and EIA) as well as the immunochemical detection (ICA) have specific qualities and produce original information which is useful for the therapeutic decision. While DCC gives a measure of the receptor level, whatever the real source of synthesis (normal and/or neoplastic tissue), ICA locates the positive cells and their relative proportion in the tumor. Both methods present their own advantages and disadvantages which are summarized in this study.

The cellular and subcellular distribution of HAP1 was examined in rat brain by light and electron microscopic immunocytochemistry and subcellular fractionation. HAP1 localization was also determined in human postmortem tissue from control and Huntington's disease (HD) cases by light microscopic immunocytochemistry. At the cellular level, the heterogeneity of HAP1 expression was similar to that of huntingtin; however, HAP1 immunoreactivity was more widespread. The subcellular distribution of HAP1 was examined using immunogold electron microscopy. Like huntingtin, HAP1 is a cytoplasmic protein that associates with microtubules and many types of membranous organelles, including mitochondria, endoplasmic reticulum, tubulovesicles, endosomal and lysosomal organelles, and synaptic vesicles. A quantitative comparison of the organelle associations of HAP1 and huntingtin showed them to be almost identical. Within HAP1-immunoreactive neurons in rat and human brain, populations of large and small immunoreactive puncta were visible by light microscopy. The large puncta, which were especially evident in the ventral forebrain, were intensely HAP1 immunoreactive. Electron microscopic analysis revealed them to be a type of nucleolus-like body, which has been named a stigmoid body, that may play a role in protein synthesis. The small puncta, less intensely labeled, were primarily mitochondria. These results indicate that the localization of HAP1 and huntingtin is more similar than previously appreciated and provide further evidence that HAP1 and huntingtin have localizations consistent with roles in intracellular transport. Our data also suggest, however, that HAP1 is not present in the abnormal intranuclear and neuritic aggregates containing the N-terminal fragment of mutant huntingtin that are found in HD brains.

Heterodimeric cell surface receptor integrin is widely expressed in the nervous system, but its specific role during axon development has not been directly tested in vivo. We show that the Drosophila nervous system expresses low levels of positron-specific (PS) integrin subunits alphaPS1, alphaPS2, and betaPS during embryonic axogenesis. Furthermore, certain subsets of neurons express higher levels of integrin mRNAs than do the rest. Null mutations in either the alphaPS1 or alphaPS2 subunit gene cause widespread axon pathfinding errors that can be rescued by supplying the wild-type integrin subunit to the mutant nervous system. In contrast, misexpressing either the alphaPS1 or alphaPS2 integrin subunit in all neurons leads to no obvious axon pathfinding errors. We propose that integrin does not itself serve as either a "clutch" constituting molecule or a specific growth cone "receptor," as proposed previously, but rather as part of a molecular network that cooperatively guarantees accurate axon guidance.

The primary all-trans --> 13-cis photoisomerization of retinal in bacteriorhodopsin has been investigated by means of quantum chemical and combined classical/quantum mechanical simulations employing the density matrix evolution method. Ab initio calculations on an analog of a protonated Schiff base of retinal in vacuo reveal two excited states S1 and S2, the potential surfaces of which intersect along the reaction coordinate through an avoided crossing, and then exhibit a second, weakly avoided, crossing or a conical intersection with the ground state surface. The dynamics governed by the three potential surfaces, scaled to match the in situ level spacings and represented through analytical functions, are described by a combined classical/quantum mechanical simulation. For a choice of nonadiabatic coupling constants close to the quantum chemistry calculation results, the simulations reproduce the observed photoisomerization quantum yield and predict the time needed to pass the avoided crossing region between S1 and S2 states at tau1 = 330 fs and the S1 --> ground state crossing at tau2 = 460 fs after light absorption. The first crossing follows after a 30 degrees torsion on a flat S1 surface, and the second crossing follows after a rapid torsion by a further 60 degrees. tau1 matches the observed fluorescence lifetime of S1. Adjusting the three energy levels to the spectral shift of D85N and D212N mutants of bacteriorhodospin changes the crossing region of S1 and S2 and leads to an increase in tau1 by factors 17 and 10, respectively, in qualitative agreement with the observed increase in fluorescent lifetimes.

Some nonspecific, DNA-binding proteins, like the linker histones, precipitate DNA upon binding. This is a poorly understood process that limits analysis of such nucleoprotein complexes using standard gel electrophoresis. To circumvent this problem, low concentrations of glutaraldehyde were used to crosslink the linker histones to DNA; then the partially crosslinked complexes were solubilized in SDS2 and separated by SDS-PAGE. Differential detection was accomplished using two different silver staining protocols that preferentially stained either proteins or nucleic acids. A technique was developed which allows the relative proportion of linker histones and DNAs in each detected band to be determined, and is referred to as differential staining SDS-PAGE (DS-SDS-PAGE). DS-SDS-PAGE provides a novel, non-isotopic means for characterizing multiple nucleoprotein bands separated by polyacrylamide gel electrophoresis. In applying this method to a model linker histone-DNA study, we were able to detect both protein-DNA and protein-protein contacts that are important in linker histone assembly onto DNA.

<h4>Background & aims</h4>The diagnosis of hemochromatosis is now possible for C282Y homozygous patients using noninvasive molecular genetic tests. The aim of this study was to define noninvasive factors predictive of severe fibrosis (bridging fibrosis or cirrhosis) to avoid unnecessary liver biopsies in such patients.<h4>Methods</h4>Clinical and biological data were recorded at the time of diagnosis in 197 French C282Y homozygous patients, 52 (26%) of whom had severe fibrosis. Variables significantly linked to severe fibrosis using univariate analysis were entered into a multivariate stepwise analysis. These variables were combined to obtain a simple index allowing for prediction of severe fibrosis.<h4>Results</h4>Serum ferritin, hepatomegaly, and serum aspartate aminotransferase were selected using multivariate analysis. Their combination applied to the 96 patients with ferritin level of </=1000 microgram/L, normal aspartate aminotransferase values, and absence of hepatomegaly showed that no severe fibrosis was encountered in this subgroup of patients. The results were validated in 113 C282Y homozygous patients in Canada with a good reproducibility of negative prediction but a poor reproducibility of the positive prediction of severe fibrosis.<h4>Conclusions</h4>In C282Y homozygous patients, the diagnosis of severe fibrosis relies on liver biopsy, but absence of severe fibrosis can be accurately predicted in most patients on the basis of simple clinical and biochemical variables.

Transcripts for a new form of Sox5, called L-Sox5, and Sox6 are coexpressed with Sox9 in all chondrogenic sites of mouse embryos. A coiled-coil domain located in the N-terminal part of L-Sox5, and absent in Sox5, showed >90% identity with a similar domain in Sox6 and mediated homodimerization and heterodimerization with Sox6. Dimerization of L-Sox5/Sox6 greatly increased efficiency of binding of the two Sox proteins to DNA containing adjacent HMG sites. L-Sox5, Sox6 and Sox9 cooperatively activated expression of the chondrocyte differentiation marker Col2a1 in 10T1/2 and MC615 cells. A 48 bp chondrocyte-specific enhancer in this gene, which contains several HMG-like sites that are necessary for enhancer activity, bound the three Sox proteins and was cooperatively activated by the three Sox proteins in non-chondrogenic cells. Our data suggest that L-Sox5/Sox6 and Sox9, which belong to two different classes of Sox transcription factors, cooperate with each other in expression of Col2a1 and possibly other genes of the chondrocytic program.

Mild to moderate hepatic iron overload is frequent in patients with chronic viral hepatitis (CH). We evaluated the role of hemochromatosis (HFE) gene mutations and other acquired factors in the development of iron overload in these patients. We studied 110 patients with chronic B or C viral hepatitis (31 women, 79 men), including 20 with cirrhosis, and 139 controls. Hepatic iron was evaluated by semiquantitative analysis in all the patients, and hepatic iron concentration (HIC) was determined in 97 of them (26 women, 71 men). C282Y and H63D mutations were sought in all the subjects by a polymerase chain reaction-restriction assay. The frequency of HFE genotypes and alleles did not differ in patients and controls. No relation was detected between hepatic iron stores and HFE gene mutations in women. In men, all C282Y heterozygotes had iron overload, and the H63D mutation was significantly more frequent in patients with more marked hepatic siderosis than in those with mild or no siderosis (P = .0039) and in controls (P = .0008). Heavy alcohol intake and hepatic cirrhosis were also associated with increased hepatic iron stores in the men. In the 71 men in whom HIC was measured, multiple regression analysis showed that this variable was related independently only to alcohol intake and HFE gene mutations. We suggest that in patients with CH, iron accumulates in the liver as the result of an interplay between genetic and acquired factors, and that increased liver iron stores may influence progression toward liver fibrosis.

<h4>Background</h4>Major surgery, such as esophagectomy, activates inflammatory responses and the coagulation system, and this activation is characterized by release of inflammatory cytokines and a decrease in antithrombin-III (AT-III), respectively. Preoperative glucocorticoid administration has been reported to suppress circulatory cytokine levels after major surgery.<h4>Patients and methods</h4>A total of 28 patients underwent esophagectomy for esophageal carcinoma; 14 of them were given 10 mg/kg of methylprednisolone intravenously upon induction of anesthesia and 14 served as controls. Circulating levels of tumor necrosis factor-alpha (TNF-alpha), interleukin 6 (IL-6), polymorphonuclear (PMN) elastase, thrombin-antithrombin III complex (TAT), AT-III, and albumin were measured before and immediately after the operation and on postoperative days (PODs) 1, 3, 5, and 7.<h4>Results</h4>TNF-alpha, IL-6, and TAT levels significantly increased after esophagectomy in both groups. AT-III and albumin decreased to their minimum levels on POD 1 and POD 3, respectively. Methylprednisolone treatment effectively inhibited the increases in TNF-alpha and IL-6 and the decreases in AT-III and albumin, but did not inhibit the increases in PMN-elastase and TAT levels. There were significant correlations between AT-III, IL-6, and albumin levels.<h4>Conclusions</h4>These results suggest that methylprednisolone pretreatment attenuates the decrease in AT-III by reducing IL-6 production postoperatively.

Deletions of tumour-suppressor genes can be detected by loss of heterozygosity (LOH) studies, which were performed on 23 cases of adenocarcinoma of the oesophagus, using 120 microsatellite primers covering all non-acrocentric autosomal chromosome arms. The chromosomal arms most frequently demonstrating LOH were 3p (64% of tumours), 5q (45%), 9p (52%), 11p (61%), 13q (50%), 17p (96%), 17q (55%) and 18q (70%). LOH on 3p, 9p, 13q, 17p and 18q occurred mainly within the loci of the VHL, CDKN2, Rb, TP53 and DCC tumour-suppressor genes respectively. LOH on 5q occurred at the sites of the MSH3 mismatch repair gene and the APC tumour-suppressor gene. 11p15.5 and 17q25-qter represented areas of greatest LOH on chromosomes 11p and 17q, and are putative sites of novel tumour-suppressor genes. LOH on 9p was significantly associated with LOH on 5q, and tumours demonstrating LOH at both the CDKN2 (9p21) and MSH3 (5q11-q12) genes had a significantly higher fractional allele loss than those retaining heterozygosity at these sites. Six of nine carcinomas displaying microsatellite alterations also demonstrated LOH at CDKN2, which may be associated with widespread genomic instability. Overall, there are nine sites of LOH associated with oesophageal adenocarcinoma.

<h4>Background</h4>The clinical association of hemochromatosis and infertility is rare. Hemochromatosis may affect fertility through a variety of mechanisms.<h4>Case</h4>A 44-year-old man and his 36-year-old wife presented with primary infertility of 7 years' duration. The husband was diagnosed as having idiopathic hemochromatosis, abnormal glucose tolerance, and hypogonadism accompanied by impotence, retrograde ejaculation, and azoospermia. Treatment consisted of phlebotomies followed by gonadotropins, which corrected retrograde ejaculation and improved semen characteristics. Concomitant pelvic factors in the woman were corrected endoscopically. After failure of pregnancy with ovulation stimulation and intrauterine inseminations, a singleton pregnancy was achieved by in vitro fertilization, augmented with intracytoplasmic sperm injection.<h4>Conclusion</h4>This case underscores the need to consider advanced reproductive technologies after the failure of specific, first-line therapeutic options in infertile couples.

We obtained mice deficient for major histocompatibility complex (MHC) molecules encoded by the H-2K and H-2D genes. H-2 KbDb -/- mice express no detectable classical MHC class I-region associated (Ia) heavy chains, although beta2-microglobulin and the nonclassical class Ib proteins examined are expressed normally. KbDb -/- mice have greatly reduced numbers of mature CD8+ T cells, indicating that selection of the vast majority (>90%) of CD8+ T cells cannot be compensated for by beta2-microglobulin-associated molecules other than classical H-2K and D locus products. In accord with the greatly reduced number of CD8+ T cells, spleen cells from KbDb -/- mice do not generate cytotoxic responses in primary mixed-lymphocyte cultures against MHC-disparate (allogeneic) cells. However, in vivo priming of KbDb -/- mice with allogeneic cells resulted in strong CD8+ MHC class Ia-specific allogeneic responses. Thus, a minor population of functionally competent peripheral CD8+ T cells capable of strong cytotoxic activity arises in the complete absence of classical MHC class Ia molecules. KbDb -/- animals also have natural killer cells that retain their cytotoxic potential.

Multiplex polymerase chain reaction amplification and genotyping by fluorescent probe melting temperature (Tm) was used to simultaneously detect multiple variants in the hereditary hemochromatosis gene. Homogenous real-time analysis by fluorescent melting curves has previously been used to genotype single base mismatches; however, the current method introduces a new probe design for fluorescence resonance energy transfer and demonstrates allele multiplexing by Tm for the first time. The new probe design uses a 3'-fluorescein-labeled probe and a 5'-Cy5-labeled probe that are in fluorescence energy transfer when hybridized to the same strand internal to an unlabeled primer set. Two hundred and fifty samples were genotyped for the C282Y and H63D hemochromatosis causing mutations by fluorescent melting curves. Multiplexing was performed by including two primer sets and two probe sets in a single tube. In clinically defined groups of 117 patients and 56 controls, the C282Y mutation was found in 87% (204/234) of patient chromosomes, and the relative penetrance of the H63D mutation was 2.4% of the homozygous C282Y mutation. Results were confirmed by restriction enzyme digestion and agarose gel electrophoresis. In addition, the probe covering the H63D mutation unexpectedly identified the A193T polymorphism in some samples. This method is amenable to multiplexing and has promise for scanning unknown mutations.

A subset of hereditary and sporadic colorectal carcinomas is defined by microsatellite instability (MSI), but the spectra of gene mutations have not been characterized extensively. Thirty-nine hereditary nonpolyposis colorectal cancer syndrome carcinomas (HNPCCa) and 57 sporadic right-sided colonic carcinomas (SRSCCa) were evaluated. Of HNPCCa, 95% (37/39) were MSI-positive as contrasted with 31% (18/57) of SRSCCa (P < 0.000001), but instability tended to be more widespread in SRSCCa (P = 0.08). Absence of nuclear hMSH2 mismatch repair gene product by immunohistochemistry was associated with germline hMSH2 mutation (P = 0.0007). The prevalence of K-ras proto-oncogene mutations was similar in HNPCCa and SRSCCa (30% (11/37) and 30% (16/54)), but no HNPCCa from patients with germline hMSH2 mutation had codon 13 mutation (P = 0.02), and two other HNPCCa had multiple K-ras mutations attributable to subclones. 18q allelic deletion and p53 gene product overexpression were inversely related to MSI (P = 0.0004 and P = 0.0001, respectively). Frameshift mutation of the transforming growth factor beta type II receptor gene was frequent in all MSI-positive cancers (85%, 46/54), but mutation of the E2F-4 transcription factor gene was more common in HNPCCa of patients with germline hMSH2 mutation than in those with germline bMLH1 mutation (100% (8/8) versus 40% (2/5), P = 0.04), and mutation of the Bax proapoptotic gene was more frequent in HNPCCa than in MSI-positive SRSCCa (55% (17/31) versus 13% (2/15), P = 0.01). The most common combination of mutations occurred in only 23% (8/35) of evaluable MSI-positive cancers. Our findings suggest that the accumulation of specific genetic alterations in MSI-positive colorectal cancers is markedly heterogeneous, because the occurrence of some mutations (eg, ras, E2F-4, and Bax genes), but not others (eg, transforming growth factor beta type II receptor gene), depends on the underlying basis of the mismatch repair deficiency. This genetic heterogeneity may contribute to the heterogeneous clinical and pathological features of MSI-positive cancers.

The mechanisms by which mutant huntingtin induces neurodegeneration were investigated using a cellular model that recapitulates features of neurodegeneration seen in Huntington's disease. When transfected into cultured striatal neurons, mutant huntingtin induces neurodegeneration by an apoptotic mechanism. Antiapoptotic compounds or neurotrophic factors protected neurons against mutant huntingtin. Blocking nuclear localization of mutant huntingtin suppressed its ability to form intranuclear inclusions and to induce neurodegeneration. However, the presence of inclusions did not correlate with huntingtin-induced death. The exposure of mutant huntingtin-transfected striatal neurons to conditions that suppress the formation of inclusions resulted in an increase in mutant huntingtin-induced death. These findings suggest that mutant huntingtin acts within the nucleus to induce neurodegeneration. However, intranuclear inclusions may reflect a cellular mechanism to protect against huntingtin-induced cell death.

Defined nucleosomal arrays reconstituted from core histone octamers and twelve 208 bp tandem repeats of Lytechinus 5S rDNA (208-12 nucleosomal arrays) possess the ability to form an unstable folded species in MgCl2 whose extent of compaction equals that of canonical higher-order 30 nm diameter chromatin structures [Schwarz, P. M., and Hansen, J. C. (1994) J. Biol. Chem. 269, 16284-16289]. To address the mechanistic functions of linker histones in chromatin condensation, purified histone H5 has been assembled with 208-12 nucleosomal arrays in 50 mM NaCl. Novel purification procedures subsequently were developed that yielded preparations of 208-12 chromatin model systems in which a majority of the sample contained both one histone octamer per 5S rDNA repeat and one molecule of histone H5 per histone octamer. The integrity of the purified 208-12 chromatin has been extensively characterized under low-salt conditions using analytical ultracentrifugation, quantitative agarose gel electrophoresis, electron cryomicroscopy, and nuclease digestion. Results indicate that histone H5 binding to 208-12 nucleosomal arrays constrains the entering and exiting linker DNA in a way that produces structures that are indistinguishable from native chicken erythrocyte chromatin. Folding experiments performed in NaC1 and MgC12 have shown that H5 binding markedly stabilizes both the intermediate and extensively folded states of nucleosomal arrays without fundamentally altering the intrinsic nucleosomal array folding pathway. These results provide new insight into the mechanism of chromatin folding by demonstrating for the first time that distinctly different macromolecular determinants are required for formation and stabilization of higher-order chromatin structures.

A growing number of non-MHC-encoded class I-related molecules have been shown to perform diverse, yet essential, functions. These include T cell presentation of bacterially derived glycolipidic Ags by CD1, transcytosis of maternal IgG by the neonatal Fc receptor, enriched presence and plausible function within exocrine fluids of the Zn-alpha2-glycoprotein, subversion of NK cytolytic activity by the CMV UL18 gene product, and, finally, crucial involvement in iron homeostasis of the HFE gene. A recently described member of this family is the MHC class-I related (MR1) gene. The most notable feature of MR1 is undoubtedly its relatively high degree of sequence similarity to the MHC-encoded classical class I genes. The human chromosome 1q25.3 MR1 locus gives rise not only to the originally reported 1,263-bp cDNA clone encoding a putative 341-amino acid polypeptide chain, but to many additional transcripts in various tissues as well. Here we define the molecular identity of all human and murine MR1 isoforms generated through a complex scenario of alternative splicing, some encoding secretory variants lacking the Ig-like alpha3 domain. Moreover, we show ubiquitous transcription of these MR1 variants in several major cell lineages. We additionally report the complete 18,769-bp genomic structure of the MR1 locus, localize the murine orthologue to a syntenic segment of chromosome 1, and provide evidence for conservation of a single-copy MR1 gene throughout mammalian evolution. The 90% sequence identity between the human and mouse MR1 putative ligand binding domains together with the ubiquitous expression of this gene favor broad immunobiologic relevance.

<h4>Background</h4>Proteins belonging to the phosphatidylethanolamine-binding protein (PEBP) family are highly conserved throughout nature and have no significant sequence homology with other proteins of known structure or function. A variety of biological roles have previously been described for members of this family, including lipid binding, roles as odorant effector molecules or opioids, interaction with the cell-signalling machinery, regulation of flowering plant stem architecture, and a function as a precursor protein of a bioactive brain neuropeptide. To date, no experimentally derived structural information has been available for this protein family. In this study we have used X-ray crystallography to determine the three-dimensional structure of human PEBP (hPEBP), in an attempt to clarify the biological role of this unique protein family.<h4>Results</h4>The crystal structures of two forms of hPEBP have been determined: one in the native state (at 2.05 A resolution) and one in complex with cacodylate (at 1.75 A resolution). The crystal structures reveal that hPEBP adopts a novel protein topology, dominated by the presence of a large central beta sheet, and is expected to represent the archaetypal fold for this family of proteins. Two potential functional sites have been identified from the structure: a putative ligand-binding site and a coupled cleavage site. hPEBP forms a dimer in the crystal with a distinctive dipole moment that may orient the oligomer for membrane binding.<h4>Conclusions</h4>The crystal structure of hPEBP suggests that the ligand-binding site could accommodate the phosphate head groups of membrane lipids, therefore allowing the protein to adhere to the inner leaf of bilipid membranes where it would be ideally positioned to relay signals from the membrane to the cytoplasm. The structure also suggests that ligand binding may lead to coordinated release of the N-terminal region of the protein to form the hippocampal neurostimulatory peptide, which is known to be active in the development of the hippocampus. These studies are consistent with a primary biological role for hPEBP as a transducer of signals from the interior membrane surface.

Huntington's disease is one of a growing number of hereditary neurodegenerative disorders caused by expansion of a polyglutamine stretch at the NH2 terminus of huntingtin. To explore whether polyglutamine-expanded huntingtin induces neuronal toxicity, I examined the expression of the full-length of huntingtin with 16, 48, or 89 polyglutamine repeats in a rat hippocampal neuronal cell (HN33). Expression of mutated huntingtin with 48 or 89 polyglutamine repeats stimulated c-Jun amino-terminal kinases (JNKs) activity and induced apoptotic cell death in HN33 cells while expression of normal huntingtin with 16 polyglutamine repeats had no toxic effect. The JNK activation precedes apoptotic cell death and co-expression of a dominant negative mutant form of stress-signaling kinase (SEK1) nearly completely blocked activation of JNKs and neuronal apoptosis mediated by mutated huntingtin. Taken together, my studies demonstrate that expression of polyglutamine-expanded huntingtin induces neuronal apoptosis via activation of the SEK1-JNK pathway.

The HIV-1 Tat protein is a potent chemoattractant for monocytes. We observed that Tat shows conserved amino acids corresponding to critical sequences of the chemokines, a family of molecules known for their potent ability to attract monocytes. Synthetic Tat and a peptide (CysL24-51) encompassing the "chemokine-like" region of Tat induced a rapid and transient Ca2+ influx in monocytes and macrophages, analogous to beta-chemokines. Both monocyte migration and Ca2+ mobilization were pertussis toxin sensitive and cholera toxin insensitive. Cross-desensitization studies indicated that Tat shares receptors with MCP-1, MCP-3, and eotaxin. Tat was able to displace binding of beta-chemokines from the beta-chemokine receptors CCR2 and CCR3, but not CCR1, CCR4, and CCR5. Direct receptor binding experiments with the CysL24-51 peptide confirmed binding to cells transfected with CCR2 and CCR3. HIV-1 Tat appears to mimic beta-chemokine features, which may serve to locally recruit chemokine receptor-expressing monocytes/macrophages toward HIV producing cells and facilitate activation and infection.

Whereas a single gene encodes acetylcholinesterase (AChE) in vertebrates and most insect species, four distinct genes have been cloned and characterized in the nematode Caenorhabditis elegans. We found that ace-1 (mapped to chromosome X) is prominently expressed in muscle cells whereas ace-2 (located on chromosome I) is mainly expressed in neurons. Ace-x and ace-y genes are located in close proximity on chromosome II where they are separated by only a few hundred base pairs. The role of these two genes is still unknown.

Four putative quantitative trait loci (QTLs) that influence susceptibility to the induction of lung adenomas by urethane in an F2 cross between A/J and BALB/cOlaHsd have been mapped. Following microsatellite typing of mice with resistant and susceptible phenotypes at 97 microsatellite marker loci, a major locus was identified on chromosome 18 with a lod score of 15. This was responsible for an 8- to 10-fold increase in tumor multiplicity in males and females, respectively, having the AA and CC genotypes at the D18Mit188 marker locus. It mapped close to Dcc (deleted in colorectal cancer). A locus on chromosome 4 (lod score 6.5) had the resistant allele in strain A/J and the susceptible allele in BALB/c, with a 14-fold difference in tumor multiplicity between mice of the AA and CC genotypes. This mapped close to the Cdkn2a (cyclin-dependent kinase inhibitor 2A) locus, which is commonly deleted in mouse lung tumors. Two loci with smaller effects (lod scores 3.03 and 3.25) were identified on chromosomes 1 and 11. There was also significant sexual dimorphism in tumor multiplicity both among 151 F2 hybrids and among 52 mice resulting from a backcross to strain A/J, with males having higher tumor counts than females.

OX40 (CD134) is a member of the tumor necrosis factor (TNF) receptor superfamily first identified as a rat T cell activation marker. In the present study, we identified the rat ligand for OX40 (OX40L) by molecular cloning. Rat OX40L cDNA was cloned from a HTLV-1-transformed rat T cell line by cross-hybridization with mouse OX40L cDNA. The predicted rat OX40L polypeptide is composed of 199 amino acids, showing 80.9 and 43.3% homology to mouse and human OX40L, respectively. Expression of rat OX40L mRNA was found in HTLV-1-transformed rat T cell lines. Expression of OX40L on the cell surface of these HTLV-1-transformed rat T cell lines was also demonstrated by flow cytometric analysis with a soluble fusion protein composed of the extracellular region of the Fc portion of human IgG (OX40-Ig). To explore the function of rat OX40L, we generated cDNA transfectants stably expressing rat OX40L. The rat OX40L transfectants exhibited a potent costimulatory activity for proliferation and IL-2 production of anti-CD3-stimulated rat T cells. These results indicated that rat OX40L can provide an efficient costimulation for rat T cells and that it may be involved in HTLV-1-associated pathologies in the rat system as has been suggested in the human system.

The development of colonic carcinoma is associated with the mutation of a specific set of genes. One of these, DCC (deleted in colorectal cancer), is a candidate tumour-suppressor gene, and encodes a receptor for netrin-1, a molecule involved in axon guidance. Loss of DCC expression in tumours is not restricted to colon carcinoma, and, although there is no increase in the frequency of tumour formation in DCC hemizygous mice, reestablishment of DCC expression suppresses tumorigenicity. However, the mechanism of action of DCC is unknown. Here we show that DCC induces apoptosis in the absence of ligand binding, but blocks apoptosis when engaged by netrin-1. Furthermore, DCC is a caspase substrate, and mutation of the site at which caspase-3 cleaves DCC suppresses the pro-apoptotic effect of DCC completely. These results indicate that DCC may function as a tumour-suppressor protein by inducing apoptosis in settings in which ligand is unavailable (for example, during metastasis or tumour growth beyond local blood supply) through functional caspase cascades by a mechanism that requires cleavage of DCC at Asp 1,290.

The expression of Ca2+ channel alpha1E isoforms has been analyzed in different cell lines, embryoid bodies and tissues. The comparison of the different cloned alpha1E cDNA sequences led to the prediction of alpha1E splice variants. Transcripts of two cloned alpha1E isoforms, which are discriminated by a carboxy terminal 129-bp sequence, have been detected in different cell lines and tissues. Transcripts of the shorter alpha1E isoform have been assigned to the rat cerebrum and to neuron-like cells from in vitro, differentiated embryonic stem cells. The shorter isoform is the major transcript amplified from total RNA by reverse transcription (RT)-PCR and visualized on the protein level by Western blotting with common and isoform-specific antibodies. Transcripts of the longer alpha1E isoform have been identified in mouse, rat and human cerebellum, in in vitro, differentiated embryoid bodies, in the insulinoma cell lines INS-1 (rat) and betaTC-3 (mouse), in the pituitary cell line AtT-20 (mouse) when grown in 5 mM glucose, and in islets of Langerhans (rat) and kidney (rat and human). The detection of different isoforms of alpha1E in cell lines and tissues shows that the wide expression of alpha1E has to be specified by identifying the corresponding isoforms in each tissue. In islets of Langerhans and in kidney, a distinct isoform called alpha1Ee has been determined by RT-PCR, while in cerebellum a set of different alpha1E structures has been detected, which might reflect the functional heterogeneity of cerebellar neurons. The tissue-specific expression of different isoforms might be related to specific functions, which are not yet known, but the expression of the new isoform alpha1Ee in islets of Langerhans and kidney leads to the suggestion that alpha1E might be involved in the modulation of the Ca2+-mediated hormone secretion.

An increasing number of neurodegenerative diseases, including Huntington's disease (HD), have been found to be caused by a CAG/polyglutamine expansion. We have generated a mouse model of HD by the introduction of exon 1 of the human HD gene carrying highly expanded CAG repeats into the mouse germ line. These mice develop a progressive neurological phenotype. Neuronal intranuclear inclusions (NII) that are immunoreactive for huntingtin and ubiquitin have been found in the brains of symptomatic mice. In vitro analysis indicates that the inclusions are formed through self aggregation via the polyglutamine repeat into amyloid-like fibrils composed of a cross beta-sheet structure that has been termed a polar zipper. Analysis of patient material and other transgenic lines has now shown NII to be a common feature of all of these diseases. In the transgenic models, inclusions are present prior to the onset of symptoms suggesting a causal relationship. In contrast, neurodegeneration occurs after the onset of the phenotype indicating that the symptoms are caused by a neuronal dysfunction rather than a primary cell death.

The mechanism by which aggregated polygins cause the selective neurodegeneration in Huntington's disease (HD) is unknown. Here, we show that the SH3GL3 protein, which is preferentially expressed in brain and testis, selectively interacts with the HD exon 1 protein (HDex1p) containing a glutamine repeat in the pathological range and promotes the formation of insoluble polyglutamine-containing aggregates in vivo. The C-terminal SH3 domain in SH3GL3 and the proline-rich region in HDex1p are essential for the interaction. Coimmunoprecipitations and immunofluorescence studies revealed that SH3GL3 and HDex1p colocalize in transfected COS cells. Additionally, an anti-SH3GL3 antibody was also able to coimmunoprecipitate the full-length huntingtin from an HD human brain extract. The characteristics of the interaction between SH3GL3 and huntingtin and the colocalization of the two proteins suggest that SH3GL3 could be involved in the selective neuronal cell death in HD.

Recent advances highlight the versatility and complexity of this highly conserved axon and cell migration guidance system. Characterization of netrin mutant phenotypes in worm, fly and mouse all suggest that netrins play local as well as long-range roles in guidance. Evidence from multiple sources also indicates that the netrin receptor DCC can mediate both attractive and repulsive responses to netrins.

The synthesis of four ester derivatives of 7-theophylline-acetic acid and glycols by DCC/DMAP-mediated esterification under mild conditions was studied. The structures of the synthesized compounds were proved by microanalyses, UV-, IR-, and 1H-NMR data. Acute toxicity assessment of the compounds in mice showed that compounds 2a-d are less toxic than aminophylline. It was shown by in vivo experiments that 2a-d had depressive action on CNS (increasing of hexobarbital sleeping time and decreasing of spontaneous locomotor activity), and they did not influence to a statistically significant extent the normal 24 hour diuresis of rats (except 2c). The results of cardiovascular screening in rats show that compounds 2a and 2c decreased the heart rate of rats. A pharmacological study of the in vitro broncholytic effect (IC50 and pD2 values) of the derivatives and aminophylline showed that 2c exhibited good broncholytic effect in vitro especially in acetylcholine and serotonin induced guinea pig tracheal contraction. It was demonstrated that compounds 2b,c exerted a stronger inhibitory effect on the enzymic activity of phosphodiesterase in concentration 2 x 10(-3) M than aminophylline.

<h4>Background</h4>Allelic association case-control analysis of a deletion/insertion polymorphism in the serotonin transporter-linked polymorphic region (5-HTTLPR) has suggested associations with unipolar disorder, bipolar disorder, depression, and Alzheimer's disease. Moreover, the heterozygous long form of the 5-HTTLPR has been associated with increased levels of mRNA for the serotonin transporter (5-HTT) and increased serotonin uptake in lymphoblastic cell lines. This study attempts to determine whether there is an association between 5-HTTLPR genotype and schizophrenia or the binding of [3H]paroxetine to the human hippocampal 5-HTT.<h4>Materials and methods</h4>DNA from the cerebellum from 58 schizophrenic and 62 control subjects was used to genotype the 5-HTTLPR. In addition, the relationship between 5-HTTLPR genotype and the affinity and density of [3H]paroxetine binding to the hippocampal 5-HTT was assessed.<h4>Results</h4>There were no associations between 5-HTTLPR allotype or genotype and/or the parameters of [3H]paroxetine binding to the hippocampal 5-HTT.<h4>Conclusions</h4>Our data suggest that 5-HTTLPR genotype neither confers an increased susceptibility for schizophrenia nor dictates the expression of the 5-HTT in the human hippocampus.

Serrated adenoma has been recently proposed as a distinct histological lesion of the colorectum. This study examined p53 immunoreactivity, mutations of exons 5-8 of the p53 gene, codon 12 of the Ki-ras gene by PCR-SSCP analyses, and microsatellite instability in 19 serrated adenomas, ten adenocarcinomas in/with serrated adenomas, 23 hyperplastic nodules, four hyperplastic polyps and 29 tubular adenomas of the colorectum. Eleven of 11 (100 per cent) serrated adenomas had p53 immunoreactivity and all six (100 per cent) adenocacinomas in/with serrated adenomas exhibited moderate to severe p53 immunoreactivity. It was confirmed that 9 of 19 (47 per cent) serrated adenomas and 5 of 10 (50 per cent) adenocarcinomas in/with serrated adenomas harboured p53 gene mutations. On the other hand, no p53 gene mutation was detected in the other colorectal lesions. Meanwhile, 11 (58 per cent) serrated adenomas and six (60 per cent) adenocarcinomas in/with serrated adenomas had Ki-ras gene mutations, as also did 9 of 23 (39 per cent) hyperplastic nodules, 3 of 4 (75 per cent) hyperplastic polyps, and 12 of 29 (41 per cent) tubular adenomas. Microsatellite instability was detected in one (5 per cent) serrated adenoma and one (10 per cent) adenocarcinoma in a serrated adenoma. The other lesions did not show microsatellite instability. Serrated adenomas had significantly frequent p53 gene mutations compared with hyperplastic lesions or tubular adenomas (p < 0.005). On the other hand, they did not exhibit significant differences in mutations of the Ki-ras gene or in microsatellite instability. Genetic changes were then examined in small parts of serrated adenomas, such as the upper or lower parts of crypts, to determine the extent of gene mutations by using a microdissection technique. Exon 15 of the APC gene and the DCC gene, in addition to the p53 and Ki-ras genes and microsatellite instability, were analysed. Identical mutations of the p53 gene were found in both invasive adenocarcinomas and adjacent serrated adenomas by direct sequencing, suggesting single clonal origins for those lesions. Mutations of the APC gene and microsatellite instability were heterogeneous in some lesions. No loss of heterozygosity (LOH) of the DCC gene was found. These findings suggest that mutations of the p53 gene are the most characteristic genetic alterations in serrated adenomas, as a relatively early event in a multistep carcinogenic pathway of this type of colorectal lesion, that might be distinct from the ordinary adenoma-carcinoma sequence or from carcinogenesis via mutations of mismatch repair genes.