Gene Literature Dashboard

The metabotropic glutamate receptors (mGluRs) mGluR4 and mGluR7 have been postulated to serve as presynaptic autoreceptors in the hippocampal formation. The cellular and synaptic localization of these proteins in the hippocampus, however, is not known. Polyclonal antibodies that specifically react with mGluR4a or mGluR7 were produced and used for immunocytochemical localization of these receptor proteins. Immunoblot analysis with mGluRs expressed in stably transfected baby hamster kidney cells suggests that each of the antibodies reacts specifically with the appropriate mGluR subtype but not with other mGluRs. Both antibodies recognized native proteins in rat brain with molecular weights similar to the molecular weights of the bands in mGluR-transfected cell lines. The distribution of mGluR4a immunoreactivity was fairly uniform in all brain regions. Immunoreactivity for mGluR7 was variable in different brain regions and closely paralleled the previously reported distribution of mGluR7 mRNA. Immunocytochemistry with light and electron microscopy (EM) revealed that immunoreactivities for mGluR4a and mGluR7 are widely but differentially distributed throughout the hippocampal formation. Staining with antibodies directed against mGluR4a was intense in cell bodies and dendrites of pyramidal cells, granule cells, and scattered interneurons. Analysis at the EM level revealed postsynaptic mGluR4a localization at asymmetrical (presumably glutamatergic) synapses and presynaptic localization at both asymmetrical and symmetrical synapses. Immunoreactivity for mGluR7 revealed largely presynaptic localization of this receptor at asymmetrical but not symmetrical synapses. These data are consistent with a largely presynaptic role of mGluR7 in the hippocampus and suggest that mGluR4 may have both presynaptic and postsynaptic functions.

Circular splicing has already been described on nuclear pre-mRNA for certain splice sites far apart in the multi exonic ETS-1 gene and in the single 1.2 kb exon of the Sry locus. To date, it is unclear how splice site juxtaposition occurs in normal and circular splicing. The splice site selection of an internal exon is likely to involve pairing between splice sites across that exon. Based on this, we predict that, albeit at low frequency, internal exons yield circular RNA by splicing as an error-prone mechanism of exon juxtaposition or, perhaps more interestingly, as a regulated mechanism on alternative exons. To address this question, the circular exon formation was analyzed at three ETS-1 internal exons (one alternative spliced exon and two constitutive), in human cell line and blood cell samples. Here, we show by RT-PCR and sequencing that exon circular splicing occurs at the three individual exons that we examined. RNase protection experiments suggest that there is no correlation between exon circle expression and exon skipping.

Ro RNPs are evolutionarily conserved ribonucleoprotein particles that consist of a small RNA, known as Y RNA, associated with several proteins, such as La, Ro60, and Ro52. The Y RNAs (Y1-Y5), which are transcribed by RNA polymerase III, have been shown to reside almost exclusively in the cytoplasm as Ro RNPs. To obtain more insight into the nuclear export pathway of Y RNAs, hY1 RNA export was studied in Xenopus laevis oocytes. Injection of various hY1 RNA mutants showed that an intact Ro60 binding site is a prerequisite for nuclear export, whereas the presence of an intact La binding site resulted in strong nuclear retention of hY1 RNA. Competition studies with various classes of RNAs indicated that, in addition to Ro60, another titratable factor was necessary for nuclear export of hY1 RNA. This factor appears also to be involved in nuclear export of tRNA. Because export of hY1 RNA could not be blocked by a synthetic peptide containing the recently identified nuclear export signal of the HIV-1 Rev protein, nuclear export of hY1 RNA does not seem to be dependent on a Rev-like nuclear export signal.

Seventy-four post menopausal patients with primary non-metastatic invasive ductal carcinomas of the breast were first treated with tamoxifen alone (30 mg p.o. daily) for 5 months. To study changes induced by tamoxifen, core biopsies before treatment and surgical specimens after hormonal therapy were assayed by immunohistochemistry for oestrogen (ER) and progesterone receptors (PR), pS2, GSTpi and c-erbB2. After tamoxifen, ER and PR significantly decreased in 60 and 44 cases respectively, whereas 11 and 19 cases showed no variation and 2 and 11 cases showed an increase (P<10(-4)). GSTpi and pS2 showed a significant increase in 43 and 41 cases, a decrease in 2 and 21 cases and no variation in 29 and 12 cases (P<10(-4) and P=0.04 respectively). c-erbB-2 showed no significant variation under tamoxifen, increased in only three cases and decreased in 13 cases. No relation was found between these variations and efficiency of hormone therapy. Our results allow a better knowledge of protein expression modifications occurring in breast cancer cells under tamoxifen therapy. They are also more consistent with clone selection rather than with phenotype modification.

Some oral cancers are preceded by premalignant lesions which include leucoplakia and erythroplakia. At present there are no reliable markers to identify lesions that may progress to malignancy. We have analysed 30 potentially malignant oral lesions for deletions at chromosomal regions that harbour tumour-suppressor genes for oral cancer. A total of 16 of 30 cases (53%) showed loss of heterozygosity (LOH) or allele imbalance at TP53, DCC, 3p21.3-22.1 or 3p12.1-13. These genetic alterations were detected in dysplastic lesions but not in histologically normal mucosa and may be early events in the carcinogenic process. A total of 64% of dysplastic lesions that recurred during the study showed LOH or allele imbalance in the initial biopsy and the number of genetic abnormalities increased in the tumours that developed. This type of molecular profiling may help to identify patients with lesions that may recur or acquire additional genetic events and progress to malignancy.

At least five adult-onset neurodegenerative diseases, including Huntingtin disease (HD), and dentatorubral-pallidoluysian atrophy (DRPLA) are produced by genes containing a variably increased CAG repeat within the coding region. The size range of the repeats is similar in all diseases; unaffected individuals have fewer than 30 CAG repeats, whereas affected patients usually have more than 40 repeats. The size of the inherited CAG repeat correlates with the severity and age of disease onset. The CAG triplet repeat produces a polyglutamine domain in the expressed proteins. All of these diseases are inherited in a dominant fashion, and a pathologic gain of function in gene carriers has been proposed. We sought to identify proteins in the brain that selectively interact with polyglutamine-domain proteins, hypothesizing that the polyglutamine domain may determine protein-protein interactions.

gp34, which we had identified as a target molecule of the trans-activation by Tax of human T-cell leukemia virus type I (HTLV-I), has been found to bind OX40, a member of the tumor necrosis factor receptor family, resulting in growth stimulation of activated T cells. We here demonstrate that not only gp34 (OX40L), but also OX40 can be transcriptionally activated by Tax. Three Tax-producing human T-cell lines carrying the HTLV-I genome expressed OX40 on their surfaces. Furthermore, Tax-induced transcriptional activation of OX40 was shown in Tax-inducible JPX-9 cells. These results demonstrate that both OX40 and its ligand (gp34) are constitutively expressed on the surfaces of Tax-expressing T lymphocytes, suggesting that the OX40L/OX40 system contributes to growth stimulation of the virus-infected T cells.

Colon carcinomas appear to arise from the cumulative effect of mutations to several genes (APC, DCC, p53, ras, hMLH1, and hMSH2). By using novel colonic epithelial cell lines derived from the Immorto mouse, named the YAMC (young adult mouse colon) cell line, and an Immorto-Min mouse hybrid, named the IMCE (Immorto-Min colonic epithelial) cell line, carrying the Apc min mutation, we investigated the effect of an activated v-Ha-ras gene on tumor progression. The YAMC and IMCE cell lines are normal colonic epithelial cell lines which are conditionally immortalized by virtue of expression of a temperature-sensitive simian virus 40 (SV40) large T antigen. Under conditions which permit expression of a functional SV40 large T antigen (33 degrees C plus gamma interferon), neither the YAMC nor the IMCE cell line grows in soft agar or is tumorigenic in nude mice. In vitro, when the SV40 large T antigen is inactivated (39 degrees C without gamma interferon), the cells stop proliferating and die. By infecting the YAMC and IMCE cell lines with a replication-defective psi2-v-Ha-ras virus, we derived cell lines which overexpress the v-Ha-ras gene (YAMC-Ras and IMCE-Ras). In contrast to the parental cell lines, under conditions in which the SV40 large T antigen is inactive, both the YAMC-Ras and IMCE-Ras cell lines continue to proliferate. Initally YAMC-Ras cells do not form tumors; however, tumors are visible after 90 days of incubation. IMCE-Ras cells form colonies in soft agar under both permissive and nonpermissive culture conditions. Furthermore, IMCE-Ras cells form tumors in nude mice within 3 weeks. The phenotype of the IMCE-Ras cell line thus clearly demonstrates that a defective Apc allele and an activated ras gene are sufficient to transform normal colonic epithelial cells and render them tumorigenic.

The inhibition of proteinase activity by members of the serine proteinase inhibitor (serpin) family is a critical regulatory mechanism for a variety of biological processes. Once formed, the serpin enzyme complexes (SECs) are removed from the circulation by a hepatic receptor. The present study suggests that this receptor is very likely the low density lipoprotein receptor-related protein (LRP), a prominent liver receptor. In vitro binding studies revealed that antithrombin III (ATIII)-thrombin, heparin cofactor II (HCII)-thrombin, and alpha1-antitrypsin (alpha1AT)-trypsin bound to purified LRP, and their binding was inhibited by the 39-kDa receptor-associated protein (RAP), an antagonist of LRP-ligand binding activity. In contrast, native or modified forms of the inhibitors were unable to bind to LRP. Mouse embryonic fibroblasts, which express LRP, mediate the cellular internalization leading to degradation of these SECs, while mouse fibroblasts genetically deficient in LRP showed no capacity to internalize and degrade these complexes. SECs were also degraded by HepG2 cells, and this process was inhibited by LRP antibodies, RAP, and chloroquine. The cellular-mediated uptake and degradation was specific for SECs; native or modified forms of the inhibitors were not internalized and degraded. Finally, in vivo clearance studies in rats demonstrated that RAP inhibited the clearance of ATIII-125I-thrombin complexes from the circulation. Together, these results indicate that LRP functions as a liver receptor responsible for the plasma clearance of SECs.

Activation of factor X by both the unactivated tissue factor:factor VII complex (TF:VII) and the activated tissue factor:factor VIIa complex (TF:VIIa) has been studied in the presence of tissue factor pathway inhibitor (TFPI), antithrombin III (ATIII), and heparin. At near-plasma concentrations of TFPI, ATIII, and factor X, factor X activation that occurs in response to TF:VII is essentially abolished in the presence of heparin (0.5 micromol/L). This effect requires both inhibitors, acting on different targets: (1) ATIII, which in the presence of heparin blocks the activation of TF:VII, and (2) TFPI, which inhibits the TF:VIIa that is generated. In the absence of ATIII, TFPI alone with heparin reduces but does not abolish factor X activation. Conversely, in the absence of TFPI, ATIII + heparin reduces but does not abolish TF:VIIa generation and allows continuing activation of factor X. These results indicated that when the unactivated TF:VII complex is the initiating stimulus, heparin-dependent reduction in the rate and extent of factor X activation requires both ATIII and TFPI. In contrast, if TF:VIIa is used to initiate activation, only TFPI is involved in its regulation.

The Escherichia coli mutant (ppk) lacking the enzyme polyphosphate kinase, which makes long chains of inorganic polyphosphate (poly P), is deficient in functions expressed in the stationary phase of growth. After 2 days of growth in a medium limited in carbon sources, only 7% of the mutants survived compared with nearly 100% of the wild type; the loss in viability of the mutant was even more pronounced in a rich medium. The mutant showed a greater sensitivity to heat, to an oxidant (H2O2), to a redox-cycling agent (menadione), and to an osmotic challenge with 2.5 M NaCl. After a week or so in the stationary phase, mutant survivors were far fewer in number and were replaced by an outgrowth of a small-colony-size variant with a stable genotype and with improved viability and resistance to heat and H2O2; neither polyphosphate kinase nor long-chain poly P was restored. Suppression of the ppk feature of heat sensitivity by extra copies of rpoS, the gene encoding the RNA polymerase sigma factor that regulates some 50 stationary-phase genes, further implicates poly P in promoting survival in the stationary phase.

The quantitation of estrogen and progesterone receptors (ER and PgR) has become the standard of care in the evaluation of patients with primary breast carcinoma. It has been demonstrated that ER and PgR detected by immunohistochemical methods in formalin-fixed paraffin-embedded tissue can be quantified by computerized image analysis. In this study, ER and PgR levels were determined by using an automated immunochemistry stainer (Ventana ES 320) and an image analyzer (CAS 200) in a series of 236 patients with stage I/II carcinoma of the breast. The degree of correlation of the ER and PgR levels determined by the dextran-coated charcoal method (DCC) with image analysis quantitation was high (r=0.75). The agreement between both methods was 77% for ER and 73% for PgR. Hormone receptor levels were correlated with prognosis as determined by overall survival. An ER level of 30 fmol/mg as determined by image analysis was established to stratify the patient population most effectively into favorable and unfavorable prognostic groups (P=0.003). An ER level of 20 fmol/mg for prognostic stratification reached statistical significance (P=0.03). The DCC method was not able to stratify the patients into prognostic groups at the traditionally accepted cutpoint of 10 fmol/mg (P=0.52). We conclude that when used in combination, automated immunohistochemistry and quantitative image analysis offer a favorable alternative to the DCC method in assessment of ER and PgR status in human mammary carcinoma. In addition, quantitative immunocytochemistry techniques may prove superior to the DCC method in specimens in which there is limited tumor volume (including fine-needle aspirates), stroma-rich tumors, and early-stage lesions including intraductal carcinoma.

Colorectal cancer (CRC) has a strong familial component. Candidate genes for colorectal cancer have been identified through mutations in four mismatch repair genes (hMSH2, hMLH1, hPMS1, and hPMS2) and genes that are deleted or mutated in tumors (DCC, APC, and p53). Linkage analysis of candidate loci/regions was performed in 10 kindreds ascertained for common colorectal cancer from the Utah Population Database. Evidence for linkage to candidate genes was assessed using two- or three-point logarithm of the odds ratio scores with markers spanning the region of localization. One kindred is linked to hMSH2 and also fits the criteria for hereditary nonpolyposis colorectal cancer, having early age of onset and high penetrance for CRC. The remaining nine kindreds are unlinked to the candidate genes tested. These kindreds have a later age of onset and a lower penetrance than hereditary nonpolyposis colorectal cancer kindreds. these results indicate that further unmapped susceptibility loci may be responsible for much of the familial aggregation of CRC.

No abstract available.

We have investigated the reactivity to two human histocompatibility leukocyte antigen (HLA) A11-restricted cytotoxic T lymphocyte (CTL) epitopes derived from amino acids 416-424 (IVTDFSVIK, designated IVT) and 399-408 (AVFDRKSVAK, designated AVF) of the Epstein-Barr virus (EBV) nuclear antigen (EBNA) 4. A strong predominance of CTL clones specific for the IVT epitope was demonstrated in polyclonal cultures generated by stimulation of lymphocytes from the EBV-seropositive donor BK with the autologous B95.8 virus-transformed lymphoblastoid cell line (LCL). This was not due to intrinsic differences of CTL efficiency since clones specific for the two epitopes lysed equally well A11-positive phytohemagglutinin blasts and LCLs pulsed with the relevant synthetic peptide. Irrespective of the endogenous levels of EBNA4 expression, untreated LCLs were lysed more efficiently by the IVT-specific effectors, suggesting that a higher density of A11-IVT complexes is presented at the cell surface. In accordance, 10-50-fold higher amounts of IVT peptides were found in high-performance liquid chromatography fractions of acid extracts corresponding to an abundance of about 350-12,800 IVT and 8-760 AVF molecules per cell. Peptide-mediated competition of CTL sensitization, transport assays in streptolysin-O permeabilized cells, and induction of A11 expression in the transporter associated with antigen presentation-deficient T2/A11 transfectant demonstrated that the IVT and AVF peptides bind with similar affinities to A11, are translocated with equal efficiency to the endoplasmic reticulum, and form complexes of comparable stability over a wide range of temperature and pH conditions. A rapid surface turnover of A11 molecules containing the AVF peptide was demonstrated in metabolically active T2/A11 cells corresponding to a half-life of approximately 3.5 as compared to approximately 2 h for molecules induced at 26 degrees C in the absence of exogenous peptides and >12 h for IVT-containing complexes. This difference in persistence is likely to determine the representation of individual class I-restricted CTL epitopes within the cell surface pool of molecules, and may be an important factor contributing to their immunogenicity.

Recent in vitro studies have established that activated B cells express OX40 ligand (L), a member of the tumor necrosis factor/nerve growth factor family of cytokines, and become stimulated to proliferate and secrete immunoglobulin (Ig) after cross-linking of OX40L by its counterreceptor OX40, which is expressed on activated T cells. In the present study we investigated the in vivo role of this receptor-ligand pair for the interaction of T and B cells in the course of the T-dependent B cell response against 2,4,6 trinitro-phenyl-keyhole limpet hemocyanin. First, we showed that OX40 is maximally expressed by T cells in the periarteriolar lymphoid sheath (PALS) 3 d after primary immunization. These OX40+ cells are located in close proximity to antigen-specific, activated B cells. Second, we demonstrated that blocking of OX40-OX40L interaction with polyclonal anti-OX40 antibody or with antibodies against certain peptide sequences within its extracellular domain resulted in a profound decrease of the anti-hapten IgG response, whereas the antihapten IgM response was grossly unchanged. Third, we showed that this antibody treatment leads to an inhibition of the development of PALS-associated B cell foci, whereas the formation of germinal centers remained intact. Finally, our data suggest that, whereas B cell memory development was not impaired by anti-OX40 administration, OX40-OX40L interaction seems to be crucial in the secondary immune response. We conclude from these data that the OX40-OX40L interaction in vivo is necessary for the differentiation of activated B cells into highly Ig-producing cells, but is not involved in other pathways of antigen-driven B cell differentiation such as memory cell development in the germinal centers.

Variant late infantile neuronal ceroid lipofuscinosis (vLINCL) is an autosomal recessive progressive encephalopathy of childhood enriched in the western part of Finland, with a local incidence of 1 in 1500. We recently assigned the locus for vLINCL, CLN5, to 13q21.1-q32. In the present study, the haplotype analysis of Finnish CLN5 chromosomes provides evidence that one single mutation causes vLINCL in the Finnish population. Eight microsatellite markers closely linked to the CLN5 gene on chromosome 13q were analyzed, to study identity by descent by shared haplotype analysis. One single haplotype formed by flanking markers D13S160 and D13S162 in strong linkage disequilibrium (P < .0001) was present in 81% of disease-bearing chromosomes. Allele 4 at the marker locus D13S162 was detected in 94% of disease-bearing chromosomes. To evaluate the age of the CLN5 mutation by virtue of its restricted geographical distribution, church records were used to identify the common ancestors for 18 vLINCL families diagnosed in Finland. The pedigrees of the vLINCL ancestors merged on many occasions, which also supports a single founder mutation that obviously happened 20 to 30 generations ago (i.e., approximately 500 years ago) in this isolated population. Linkage disequilibrium was detected with seven markers covering an extended genetic distance of 11 cM, which further supports the young age of the CLN5 mutation. When the results of genealogical and linkage disequilibrium studies were combined, the CLN5 gene was predicted to lie approximately 200 - 400 kb (total range 30 - 1360 kb) from the closest marker D13S162.

A review of the literature on pituitary function in haemochromatosis is presented. In morphological studies pituitary iron deposition, in particular in gonadotropic cells, has been demonstrated. In a number of case series, insufficiency of pituitary gonadotropic secretion with clinical hypogonadism was observed in 46% of the patients. Overt insufficiency of the other hormonal axes was infrequent. However, subclinical insufficiency of the growth hormone axis was present in 15%, of the lactotropic axis in 8%, of the thyroid axis in 4% and of the adrenocortical axis in 1.5% of the patients. Lactotropic, thyroid or adrenocortical insufficiency was usually associated with hypogonadism or growth hormone insufficiency. Investigation of pituitary function (in first line the gonadotropic and the somatotropic function) in patients with primary or secondary haemochromatosis is recommended on wide indications, in order to initiate relevant substitution therapy.

After menopause the haemostatic balance shifts towards a latent hypercoagulable state. To evaluate the effects of two regimens of transdermal estradiol (E2) combined with progestin on the balance between procoagulant factors and inhibitors, 255 women in physiological menopause for 1-5 years were randomly allocated to 1 year of treatment with cyclic transdermal E2 (50 micrograms/day for 21 days) plus medroxyprogesterone acetate (MPA) (10 mg/day from days 10 to 21), continuous transdermal E2 (50 micrograms/day for 28 days) plus MPA (10 mg/day from days 14 to 25), or placebo. Fibrinogen, factor VII (FVII), factor VIII:C (FVIII:C), antithrombin III (ATIII), protein C, protein S, heparin cofactor II (HCII) and plasminogen activator inhibitor (PAI-1) levels were measured at baseline and after 6 and 12 cycles. 167 women who took the treatment for at least 6 cycles were evaluable. The continuous treatment group had significantly lower final values of fibrinogen, FVII, ATIII, protein S and HCII than the placebo group; the cyclic treatment reduced fibrinogen in comparison with placebo but the difference was not significant. In conclusion, both regimens produce a clinically relevant decrease of fibrinogen levels; the continuous regimen affects also the levels of FVII and inhibitors suggesting that the haemostatic balance is shifted to a more physiological state.

Proteins of the immunoglobulin superfamily (IgSF) are involved in a variety of specific cell-cell interactions in the developing nervous system. We used a systematic approach, based on the PCR, to identify and characterize new members of this protein family in the developing chick nervous system. In this study, we report on CEPU-1, a new IgSF protein that is strongly expressed in developing Purkinje neurons in the cerebellum and is detectable on the dendrites, somata, and axons of these cells. The timing of CEPU-1 expression coincides with the growth of the dendritic tree--after the Purkinje cells have finished their migration from the ventricular zone. CEPU-1 is a 51 kDa glycoprotein comprised of three Ig-like domains of the C2 subcategory and is anchored to the plasma membrane via a post-translationally attached glycosyl-phosphatidylinositol moiety. The protein shows high sequence similarity to the neurotrimin glycoprotein (78% identity), to the opioid-binding cell adhesion molecule (or OBCAM; 68% identity), and to the limbic system-associated membrane protein (or LAMP; 54% identity). Our results suggest that CEPU-1 may serve as a cellular identification marker that is specific for developing Purkinje cells.

Seventy-four patients with PSS were evaluated with regard to plasma concentration of blood coagulation and fibrinolysis factors: fibrinogen (Fbg), prothrombin time (PT), active partial thromboplastin time (APTT), protein C, thrombin-antithrombin III complex (TAT), antithrombin-III (AT-III), factor XIII (XIII) fibrinopeptide A (FPA), alpha 1-antitrypsin (alpha 1-AT), plasminogen (Pmg), alpha 2-plasmin inhibitor plasmin complex (PIC), alpha 2-plasmin inhibitor (alpha 2-PI), alpha 2-macroglobulin (alpha 2-MG), fibrinopeptide B beta 15-42 (FPB beta-15-42) and soluble fibrin monomer complex (SFMC), FDP (fibrin degradation product) and D-dimer. They were also evaluated with regard to platelet-derived proteins: beta-thromboglobulin (beta-TG), platelet factor 4 (PF4), thromboxane B2 and 6-keto-prostaglandin F1 alpha (6KF). In the coagulation/fibrinolysis systems high plasma levels of TAT, AT-III, FPA, alpha 2-MG and FPB beta 15-42 could be demonstrated in more than 50% of total PSS patients. There was no statistical correlation between those of TAT and AT-III. Plasma levels of PIC, D-dimer, FDP and SFMC were not always high. There was no statistical correlation between those of TAT and PIC. These data lead us to consider that alpha 2-MG may play an important role for inhibiting PIC, which accelerates the conversion from fibrin into FDP. Subsequently, there were high plasma levels of FPB beta 15-42 converted from fibrin monomer. These data seem to be indicative of an involvement of coagulation and platelet disorder in PSS. These platelet-vessel system disorders might be closely related to the pathophysiology of PSS.

Positional cloning strategies for the hemochromatosis gene have previously concentrated on a target area restricted to a maximum genomic expanse of 400 kb around the HLA-A and HLA-F loci. Recently, the candidate region has been extended to 2-3 Mb on the distal side of the MHC. In this study, 10 coding sequences [hemochromatosis candidate genes (HCG) I to X] were isolated by cDNA selection using YACs covering the HLA-A/HLA-F subregion. Two of these (HCG II and HCG IV) belong to multigene families, as well as other sequences already described in this region, i.e., P5, pMC 6.7, and HLA class 1. Fingerprinting of the four YACs overlapping the region was performed and allowed partial localization of the different multigene family sequences on each YAC without defining their exact positions. Fingerprinting on cosmids isolated from the ICRF chromosome 6-specific cosmid library allowed more precise localization of the redundant sequences in all of the multigene families and revealed their apparent organization in clusters. Further examination of these intertwined sequences demonstrated that this structural organization resulted from a succession of complex phenomena, including duplications and contractions. This study presents a precise description of the structural organization of the HLA-A/HLA-F region and a determination of the sequences involved in the megabase size polymorphism observed among the A3, A24, and A31 haplotypes.

Inactivation of tumor suppressor genes is thought to be a critical step in tumorigenesis. The DCC (deleted in colorectal carcinoma) gene, located on the long arm of chromosome 18, has been shown to be frequently deleted in colorectal tumors. To investigate the involvement of allelic deletions on chromosome 18q in breast cancer tumorigenesis we analyzed 28 primary breast tumors and 28 colorectal tumors (24 carcinomas, 4 adenomas) with four different polymorphic DNA markers detecting RFLPs on chromosome 18q. In breast cancer we found loss of heterozygosity (LOH) in 4 of 27 (15%) informative cases whereas 15 of 25 (60%) colorectal tumors showed allelic deletions. In all cases of allelic loss the DCC locus or its proximal vicinity (locus SSAV1) were involved. LOH on chromosome 18q occurs both in breast and colorectal cancer, yet the frequency of these deletions in breast tumors is lower than in colorectal tumors. Moreover, in breast cancer these mutations were only detected in large and undifferentiated tumors.

The chemical immobilization of heparin onto polymeric materials through hydrophilic spacer groups was performed to improve the hemocompatibility of blood-contacting devices. Significant data have been gathered attesting to the biological activity of immobilized heparin in static in vitro studies (clotting times) and dynamic in vivo studies (thrombus formation). However, few studies have been performed to investigate the binding kinetics of spacer-immobilized heparin under flow (shear stress) with antithrombin III (ATIII) and thrombin. To help elucidate this binding mechanism, a mathematical model was developed which parallels experiments to measure protein binding and dissociation at the heparin immobilized surface under flow conditions. Heparinized tubing was prepared by chemically immobilizing a high-ATIII-affinity fraction of heparin onto the surface of poly(ethylene)-oxide grafted, poly(styrene-co-p-aminostyrene)-coated polyethylene tubing. ATIII was first bound onto the immobilized heparin, followed by the introduction of thrombin to interact with ATIII. The concentration of thrombin-ATIII complex (TAT) flowing from the tubing was determined, and the dissociation rate constants (kD) of TAT from immobilized heparin were calculated as a function of flow rate. The results indicate that the dissociation rate constant of TAT varied with flow rate, especially low flow rates, high flow rates, and turbulent flow. As the TAT complex dissociates from immobilized heparin, this "recovered" heparin is available for subsequent binding of more ATIII and thrombin. These in vitro mathematical results may help support mechanisms and hypotheses generated for the biological activity of spacer-immobilized heparin observed during long-term in vivo and ex vivo experiments.

Glutamine repeat expansion has been established as the mutation underlying five inherited neurodegenerative diseases. The mechanism by which this apparently universal mutation, in ubiquitously expressed proteins, causes highly selective neurodegeneration is unknown. The proteins containing the glutamine expansions are otherwise unrelated and likely to have different functions. Two recently published papers provide evidence of a conformational change occurring in polyglutamine expansions, which may allow novel interactions and is consistent with a toxic gain-of-function hypothesis. HAP1, a protein that interacts with huntingtin (Huntington's disease protein), has an expression profile that intriguingly mirrors the selective neurodegeneration seen in Huntington's disease.

Prothrombin activation fragment 1 + 2 (F1 + 2) and thrombin-antithrombin-III complexes (TAT) were measured in plasma of 34 patients with newly diagnosed essential hypertension. The patients with hypertension showed an increase in both F1 + 2 and TAT concentrations. The obtained results point to the intravascular thrombin generation in very early stages of essential hypertension.