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Substituted cinnamoyl-tyramine derivatives were synthesized by DCC-coupling of substituted cinnamic acid with tyramine or tyramine methyl-1-ether to evaluate PAF-receptor binding antagonistic activities and inhibitory activities on PAF-induced platelet aggregation with interest on structure-activity relations. The results show that 3,4-dimethoxy-cinnamoyl tyramine-amide or its methyl ether have significant PAF-receptor binding antagonistic activity and platelet anti-aggregatory activities.

Genomic DNAs of various strains of Oxalobacter formigenes were subjected to restriction endonuclease fragment length polymorphism- and PCR-based amplification analyses with DNA probes and primers complementary to sequences within either the oxc gene, encoding oxalyl coenzyme A (oxalyl-CoA) decarboxylase, or the frc gene, encoding formyl-CoA transferase. Oligonucleotide probes based on nonconserved sequences of oxc or frc were able to divide O. formigenes strains into at least two groups, consistent with the current separation of O. formigenes strains into groups I and II on the basis of 16S rRNA sequence similarities and lipid content. In contrast, an oligonucleotide probe based on the conserved 5' end of oxc appeared to bind all group I and the majority of group II strains. PCR amplification of the oxc gene showed even greater sensitivity in detecting O. formigenes and provided support for further division of the strains into subgroups. In addition, these oligonucleotides failed to hybridize to or amplify PCR products from whole fecal DNA isolated from fresh stool samples from an individual not colonized with O. formigenes, indicating unique specificity. Thus, these DNA analyses permit both detection as well as classification of O. formigenes strains.

The deleted in colorectal cancer (DCC) gene, a candidate tumor suppressor gene on chromosome 18q21, encodes a neural cell adhesion molecule family protein that is most highly expressed in the nervous system. To address the hypothesis that DCC may play a role in glioma development and/or progression, we examined DCC expression by immunohistochemistry in 57 resected human astrocytic tumors. Overall, low-grade astrocytomas were predominantly DCC positive (15 of 16, or 94%), whereas high-grade tumors significantly less often expressed the DCC protein (27 of 41, or 66%; P = 0.03). We were able to directly assess the relationship between DCC expression and tumor progression in 15 patients who initially presented with a low-grade astrocytoma and subsequently recurred with a glioblastoma. Within this panel of paired lesions from the same patient, 14 of 15 (93%) low-grade tumors expressed the DCC protein, whereas only 7 of 15 (47%) corresponding glioblastomas were DCC positive. We also observed that secondary glioblastomas resulting from malignant progression of low-grade astrocytomas were more often DCC negative (8 of 15, or 53%) compared with primary or de novo glioblastomas (6 of 26, or 23%; P = 0.05). These findings implicate DCC inactivation in glioma progression and also demonstrate that DCC expression is preferentially, but not exclusively, lost in the genetic pathway to secondary glioblastoma multiforme.

Therapeutic hematopoietic stem cell transplantation has made great strides in recent years, providing curative therapy for many previously untreatable diseases. Nevertheless, the applicability and effectiveness of this procedure continues to be restricted by adverse immunoregulatory states, including graft rejection, graft vs. host disease (GvHD), and/or persistent immunodeficiency. Here, we provide evidence that long-term hematopoietic stem cell transplantation across major histocompatibility complex (MHC) barriers is possible in the human with limited adverse sequelae. We observed the rapid, complete, and stable replacement of recipient hematopoiesis and B lymphopoiesis with donor-derived cells approximately 6 weeks following orthotopic liver transplantation for hemochromatosis. Long-term T lineage reconstitution also occurred, but most intriguingly, derived almost exclusively from expansion of mature, memory/effector T cells from the transplanted liver. Although demonstrating both functional and molecular evidence of a simplified T cell receptor (TCR) repertoire and unable to become sensitized to "new" antigens (Ag), this patient demonstrated long-term clinical immunocompetence. Moreover, the transplanted T cells were effectively tolerant to host tissues as the patient did not manifest clinically significant GvHD off immunosuppressive therapy. These observations suggest that isolated memory/effector T cell populations have the potential of promoting stem cell engraftment in an allogeneic host without persistent GvHD, and to provide sufficient immune reconstitution to provide the recipient with long-term immune homeostasis.

The effects of the stoichiometric inhibitors tissue factor pathway inhibitor (TFPI), antithrombin-III (AT-III) and heparin cofactor-II (HC-II) on thrombin generation were evaluated in a reaction system composed of coagulation factors VIIa, X, IX, VIII, and V and prothrombin initiated by tissue factor (TF) and phospholipids. Initiation of the reaction in the absence of inhibitors resulted in explosive thrombin generation for factor VIIa.TF concentrations varying from 100 to 0.25 pM with the lag time or initiation phase of thrombin generation increasing from 0 to 180 s with decreasing factor VIIa.TF concentrations. During the propagation phase, prothrombin is quantitatively activated to 1.4 micro;M alpha-thrombin. At normal plasma concentration (2.5 nM) full-length recombinant TFPI prolonged the initiation phase of thrombin generation 2-fold, and the rate of thrombin generation in the propagation phase of the reaction was 25-50% that of the uninhibited reaction when the reaction was initiated with 1.25-20 pM factor VIIa.TF. Inhibition of the reaction by TFPI is associated with a delay in factor V activation. In the presence of TFPI no explosive thrombin generation was observed when factor VIII was omitted from reactions initiated by factor VIIa.TF concentrations </=20 pM. This indicates that in the presence of TFPI the factor IXa.factor VIIIa pathway becomes essential at low factor VIIa.TF concentrations. In the reconstituted system, AT-III (3.4 micro;M) did not prolong the initiation phase of thrombin generation when the reaction was initiated with 1.25 pM factor VIIa.TF, nor did AT-III delay factor V activation. The rate of thrombin formation in the presence of AT-III was reduced to 30% that of the uninhibited reaction, and the alpha-thrombin formed was rapidly inhibited subsequent to its generation. The addition of HC-II alone at its physiological concentration (1.38 micro;M) to the procoagulant mixture did not alter the rate or extent of thrombin generation. Subsequently, the thrombin formed was slowly inhibited by HC-II. The slow inactivation of thrombin by HC-II does not contribute to thrombin inhibition in the presence of AT-III. In contrast, the combination of physiological levels of AT-III and TFPI inhibited explosive thrombin generation initiated by 1.25 pM factor VIIa.TF completely. The absence of prothrombin consumption indicated that the combination of TFPI and AT-III is able to prevent the formation of prothrombinase activity at low factor VIIa.TF concentrations. The data indicate that TFPI potentiates the action of AT-III by decreasing the rate of formation and thus the amount of catalyst formed in the reaction, enabling AT-III to effectively scavenge the limited traces of factor IXa and factor Xa formed in the presence of TFPI. The initiation of thrombin generation by increasing factor VIIa.TF concentrations in the presence of physiological concentrations of TFPI and AT-III showed dramatic changes in the maximal rates of thrombin generation over small changes in initiator concentration. These data demonstrate that significant thrombin generation becomes a "threshold-limited" event with regard to the initiating factor VIIa.TF concentration in the presence of TFPI and AT-III.

Six inherited neurodegenerative diseases are caused by a CAG/polyglutamine expansion, including spinal and bulbar muscular atrophy (SBMA), Huntington's disease (HD), spinocerebellar ataxia type 1 (SCA1), dentatorubral pallidoluysian atrophy (DRPLA) Machado-Joseph disease (MJD or SCA3) and SCA2. Normal and expanded HD allele sizes of 6-39 and 35-121 repeats have been reported, and the allele distributions for the other diseases are comparable. Intergenerational instability has been described in all cases, and repeats tend to be more unstable on paternal transmission. This may present as larger increases on paternal inheritance as in HD, or as a tendency to increase on male and decrease on female transmission as in SCA1 (ref. 15). Somatic repeat instability is also apparent and appears most pronounced in the CNS. The major exception is the cerebellum, which in HD, DRPLA, SCA1 and MJD has a smaller repeat relative to the other brain regions tested. Of non-CNS tissues, instability was observed in blood, liver, kidney and colon. A mouse model of CAG repeat instability would be helpful in unravelling its molecular basis although an absence of CAG repeat instability in transgenic mice has so far been reported. These studies include (CAG) in the androgen receptor cDNA, (CAG) in the HD cDNA, (CAG) in the SCA1 cDNA, (CAG) in the SCA3 cDNA and as an isolated (CAG) tract.

Hemochromatosis is a hereditary iron-overload disease linked to HLA. The clinical expression of hemochromatosis is influenced by sex and age. However, other factors must account for the notorious heterogeneity of expression of the disease independent of sex, age, and HLA phenotype. The present study attempts to clarify some of these additional factors based on exhaustive statistical analysis of data collected from 43 selected patients with hemochromatosis. The statistical analysis focused on three groups of variables: the first group included variables reflecting the clinical expression of the disease; the second group represented the biochemical and hematological values at the time of diagnosis; and the third group consisted of the independent variables sex, age, HLA phenotype, and T-cell subset profile, i.e., the percentages and total numbers of CD4+ and CD8+ cells and the CD4-CD8 ratios. The results show that the relative expansion of the two main T-cell subsets, in the context of the HLA phenotype, correlates significantly with the clinical expression of hemochromatosis and the severity of iron overload. The present findings substantiate further the postulate that T cells have a role in the regulation of iron metabolism.

<h4>Background/aims</h4>Hereditary hemochromatosis (HH), which affects some 1 in 400 and has an estimated carrier frequency of 1 in 10 individuals of Northern European descent, results in multi-organ dysfunction caused by increased iron deposition, and is treatable if detected early. Using linkage-disequilibrium and full haplotype analysis, we have identified a 250kb region more than 3 megabases telomeric of the major histocompatibility complex (MHC) that is identical-by-descent in 85% of patient chromosomes. Within this region, we have identified a gene related to the MHC class I family, termed HLA-H, containing two missense alterations. One of these is predicted to inactivate this class of proteins and was found homozygous in 83% of 178 patients. A role of this gene in hemochromatosis is supported by the frequency and nature of the major mutation and prior studies implicating MHC class I-like proteins in iron metabolism.

<h4>Background</h4>Sporadic porphyria cutanea tarda is a skin disease associated with hepatic siderosis. Depletion of iron stores by phlebotomy is curative. The role of haemochromatosis genes in determining susceptibility to this disorder is controversial. We have examined the frequency in sporadic porphyria cutanea tarda of mutations (Cys282Tyr, His63Asp) in a novel MHC class-I-like gene, one of which (Cys282Tyr) is believed to cause haemochromatosis.<h4>Methods</h4>41 patients with sporadic porphyria cutanea tarda, in whom the frequency of microsatellite alleles that define the ancestral haemochromatosis haplotype had previously been determined, and 101 healthy blood donors were studied for the presence of the Cys282Tyr and His63Asp mutations. We used restriction-enzyme digestion of PCR-amplified genomic DNA.<h4>Findings</h4>The Cys282Tyr mutation occurred in 18 (44%) of patients compared with 11 (11%) of controls (relative risk 6.2, 95% CI 2.6-14.5, p = 0.00003). Seven (17%) patients, aged 48-79 years, were homozygotes. In 12 patients, the Cys282Tyr mutation was associated with markers of the HLA-A3-containing ancestral haemochromatosis haplotype. Ages at presentation were the same for those with or without the Cys282Tyr mutation. There was no difference in the frequency of the His63Asp mutation.<h4>Interpretation</h4>Inheritance of one or more haemochromatosis genes is an important susceptibility factor for sporadic porphyria cutanea tarda. Some homozygotes for the Cys282Tyr mutation present late in life with porphyria cutanea tarda, indicating that not all homozygotes present clinically with haemochromatosis. The relation between this genotype and disease needs further investigation.

Deleted in colorectal cancer (DCC), a candidate tumor-suppressor gene, has recently been found to encode a netrin receptor required for axon guidance in vitro. Mutations in Caenorhabditis elegans and Drosophila genes encoding DCC-related proteins affect axon guidance, and these phenotypes resemble those of mutations in netrin genes. Netrins and their DCC-related receptors thus play an evolutionarily conserved role in midline guidance, and DCC may be required more generally for cellular morphogenesis.

<h4>Background</h4>Genetic factors are probably important in the development of gastric carcinoma in young patients (younger than 40 years). The authors investigated early onset primary gastric adenocarcinomas for the presence of microsatellite instability, which is a phenotypic marker for the hereditary nonpolyposis colon carcinoma syndrome.<h4>Methods</h4>DNA was extracted from archival microdissected carcinoma and corresponding normal tissue from 10 British gastric carcinoma patients age 19 to 39 years at the time of diagnosis. A panel of 12 microsatellite loci were amplified by fluorescent polymerase chain reaction and analyzed using an automated DNA sequencer.<h4>Results</h4>There was no evidence of microsatellite instability. In contrast, allelic imbalance was recorded at D3S966, D3S1076, D10S197, D11S904, P53, NM23, and DCC microsatellite loci.<h4>Conclusions</h4>The authors reported ten cases of early onset gastric carcinoma that demonstrated allelic imbalance but no evidence of instability at microsatellite loci. It is unlikely that defective DNA mismatch repair is important in this group of young patients.

OX40 ligand (OX40L) and OX40 are members of the tumor necrosis factor and tumor necrosis factor receptor superfamilies, respectively. OX40L is expressed on activated B and T cells and endothelial cell lines, whereas OX40 is expressed on activated T cells. A construct for mouse OX40L was expressed as a soluble protein with domains 3 and 4 of rat CD4 as a tag (sCD4-OX40L). It formed a homotrimer as assessed by chemical cross-linking and gel filtration chromatography. Radiolabeled sCD4-OX40L bound to activated mouse T cells with a high affinity (KD = 0.2-0.4 nM) and dissociated slowly (koff = 4 x 10(-5) s-1). The affinity and kinetics of the OX40L/OX40 interactions were studied using the BIAcoreTM biosensor, which measures macromolecular interactions in real time. The extracellular part of the OX40 antigen was expressed as a soluble monomeric protein and immobilized on the BIAcore sensor chip. sCD4-OX40L bound the OX40 with a high affinity (KD = 3.8 nM), although this was lower than that determined on the surface of activated T cells (KD = 0.2-0.4 nM), where there is likely to be less restriction in mobility of the receptor. In the reverse orientation, sOX40 bound to immobilized sCD4-OX40L with a stoichiometry of 3.1 receptors to one ligand, with low affinity (KD = 190 nM) and had a relatively fast dissociation rate constant (koff = 2 x 10(-2) s-1). Thus if the OX40 receptor is cleaved by proteolysis, it will release any bound ligand and is unlikely to block re-binding of ligand to cell surface OX40 because of the low monomeric affinity.

Human XPG nuclease makes the 3' incision during nucleotide excision repair of DNA. The enzyme cleaves model DNA bubble structures specifically near the junction of unpaired DNA with a duplex region. It is not yet known, however, whether an unpaired structure is an intermediate during actual DNA repair. We find here that XPG requires opening of >5 bp for efficient cleavage. To seek direct evidence for formation of an open structure around a lesion in DNA during a nucleotide excision repair reaction in vitro, KMnO4 footprinting experiments were performed on a damaged DNA molecule bearing a uniquely placed cisplatin adduct. An unwound open complex spanning approximately 25 nucleotides was observed that extended to the positions of 5' and 3' incision sites and was dependent on XPA protein and on ATP. Opening during repair occurred prior to strand incision by XPG.

Many years ago it was shown that an infusion of tissue factor (TF) into rabbits causing only limited consumption of factor X and prothrombin resulted in extensive consumption of fibrinogen. More recently it was shown that an injection of a concentration of the factor X-activating fraction of Russell's viper venom (RVV-X) depleting rabbits of factor X resulted in only minimal consumption of both plasma prothrombin and fibrinogen. We report here experiments in which rabbits depleted of antithrombin III (ATIII) to different degrees were infused over 4 hours with a concentration of RVV-X, causing consumption of about 60% of plasma factor X. Similar minimal mean falls in plasma prothrombin and fibrinogen levels were observed in control rabbits given nonimmune goat IgG and in rabbits immunodepleted with goat anti-rabbit ATIII IgG to about 40% of normal plasma ATIII activity. However, if rabbits were immunodepleted to about 10% to 20% of normal plasma ATIII, then mean consumption of prothrombin was increased modestly and, more impressively, mean consumption of plasma fibrinogen was increased markedly. Whereas limited amounts of thrombin generated on the surface of phospholipid vesicles by factor VIIa/ TF can trigger extensive intravascular coagulation in rabbits with normal plasma ATIII levels, limited amounts of thrombin generated by reactions triggered by factor Xa formed in fluid phase did so only after plasma ATIII levels were markedly depleted. A possible reason for this difference is discussed.

The deduced amino acid sequence of Leishmania major sw3 cDNA reveals the presence of characteristic histone H1 amino acid motifs. However, the open reading frame is of an unusually small size for histone H1 (105 amino acids) because it lacks the coding potential for the central hydrophobic globular domain of linker histones present in other eukaryotes. Here, we provide biochemical evidence that the SW3 protein is indeed a L. major nuclear histone H1, and that it is differentially expressed during the life cycle of the parasite. Due to its high lysine content, the SW3 protein can be purified to a high degree from L. major nuclear lysates with 5% perchloric acid, a histone H1 preparative method. Using an anti-SW3 antibody, this protein is detected as a 17 kDa or as a 17/19 kDa doublet in the nuclear subfraction in different L. major strains. The nuclear localization of the SW3 protein is further supported by immunofluorescence studies. During in vitro promastigote growth, both the sw3 cytoplasmic mRNA and its protein progressively accumulate within parasites from early log phase to stationary phase. Within amastigotes, the high level of H1 expression is maintained but decreases when amastigotes differentiate into promastigotes. Together, these observations suggest that the different levels of this histone H1 protein could influence the varying degrees of chromatin condensation during the life-cycle of the parasite, and provide us with tools to study this mechanism.

No abstract available.

A previous study in this laboratory showed that depolymerized holothurian glycosaminoglycan (DHG) has two different antithrombin III (ATIII)-independent inhibitory effects on the in vitro blood coagulation system: heparin cofactor II (HCII)-dependent inhibition of thrombin, and ATIII- and HCII-independent inhibition of factor X activation by factor IXa-factor VIIIa complex (Nagase et al. Blood 85, 1527-1534, 1995). In the present study, we compared the antithrombotic effects of DHG in normal and in ATIII-deficient mice with those of unfractionated heparin (UFH) and low molecular weight heparin (LMWH). DHG, unlike UFH and LMWH, exerted an in vivo antithrombotic effect even in mice with decreased plasma ATIII activity (about 30% of normal). We then compared the anticoagulant and antithrombotic effects of DHG in mice with those of high molecular weight (HMW)-DHG, low molecular weight (LMW)-DHG, and dermatan sulfate (DS). In terms of in vitro anticoagulant activity assessed by use of purified human components, DHGs (DHG, HMW-DHG, and LMW-DHG) had different anti-thrombin activity in the presence of HCII and anti-factor Xase activities, which differences were dependent on the molecular weight. With respect to in vivo antithrombotic activity, DHG, HMW-DHG, and LMW-DHG showed almost the same inhibitory effect on acute thromboembolism in mice (minimum effective dose [MED]: > 0.3 mg/kg). Since the antithrombotic activities of DHGs were not correlated with the anticoagulant-specific activities, the contribution of the two anticoagulant activities to the in vivo antithrombotic effect of DHGs remains unknown. However, DHG was more effective against acute thromboembolism in mice than DS (MED > 1 or > 3 mg/kg), which showed no inhibitory activity toward factor Xase. Therefore, it seems that factor Xase inhibition contributes greatly to the antithrombotic effect of DHG and that DHG exerts this effect in mice mainly by inhibiting factor Xase.