The effect of mild and moderate renal impairment on the pharmacokinetics of pridopidine, a new drug for Huntington's disease.

AIM: Pridopidine, a new oral drug for treatment of patients with motor symptoms associated with Huntington's Disease (HD) is currently under development. In steady-state conditions, pridopidine elimination is mediated primarily through renal excretion. This study evaluated single dose and steady-state pharmacokinetics (PK) of a daily dose of pridopidine in subjects with mild and moderate renal impairment and matched healthy subjects. METHODS: Subjects with mild renal impairment (n = 12), moderate impairment (n = 12), or their matched healthy controls (n = 25) participated in this study. Subjects received a single dose of pridopidine (45 mg) on day 1 and a multiple dose cycle of 45 mg once daily on days 5-18. Blood and urine samples were collected on days 1 and 18 for PK analysis. RESULTS: Mild renal impairment did not affect the PK of pridopidine whilst an increase in exposure was seen in subjects with moderate renal impairment. Subjects with moderate impairment showed reduced plasma clearance (by 44%) and had 68% higher AUC (90% CI 1.22, 2.30) and 26% higher Cmax (90% CI 1.02, 1.56) values than those with normal renal function at steady-state. Pridopidine was safe and well tolerated in healthy subjects and in subjects with mild and moderate renal impairment. CONCLUSIONS: Mild renal impairment has no impact on exposure to pridopidine while moderately impaired renal function resulted in higher pridopidine concentrations.

Implementation of structured physical activity in the pediatric stem cell transplantation.

The peripheral blood stem cell transplantation (PBSCT) represents a specific, but stressful therapy for hemato-oncological diseases. While for adults, data suggest positive eff ects for a supportive sport therapy, this question is not evaluated sufficiently for children. The objective of this study was to examine the integration of sports activity into pediatric PBSCT and to indicate attainable results. This 2-step case-control-study included 23 children and adolescents from the PBSCT: During the isolation phase 13 patients trained 3 times per week on a cycle ergometer and passed a course with different sports equipment. Apart from recording physiologic adaptations, quality of live was inquired in a pre-post design using questionnaires. Guided interviews according to necessity and requirements for sports activity at the PBSCT unit completed the evaluation and were used for the intervention as well as for the control group (n = 10) without sports therapy. On the ergometer, patients trained average 25 min with 0.6 watt / kg. In the majority, a loss of muscular power could be avoided. Quality of life and fatigue symptoms improved by trend. Interview analysis showed general acceptance of physical activity during PBSCT. After initial skepticism due to the additional burden, our implementation study showed the feasibility of supportive sports therapy in PBSCT. Quality and flexibility of the equipment should be higher than normal and different physical and psychological conditions of the patients should be anticipated and integrated into the training program.

Macrophage migration inhibitory factor (MIF) gene polymorphisms are associated with increased prostate cancer incidence.

Recurrent or persistent inflammation has emerged as an important factor in cancer development. Overexpression of macrophage migration inhibitory factor (MIF), an upstream regulator of innate immunity with pleiotropic effects on cell proliferation, has been implicated in prostate cancer (CaP). Two polymorphisms in the promoter of the MIF gene (-173G to C transition and seven copies of the -794 CATT repeat) are associated with increased MIF expression in vivo and poor prognosis in autoimmune diseases. We conducted a retrospective analysis of 131 CaP patients and 128 controls from a group of Veterans' Administration patients undergoing routine prostate-specific antigen screening. Patients with CaP were enrolled regardless of treatment. Inclusion criteria for the control group were absence of documented diagnosis of cancer and/or chronic inflammation within patient computerized records. Logistic regression demonstrated a significant association between CaP and the -173G/C, the -173C/C and the -794 7-CATT MIF polymorphisms (P<0.001). Patients with the -794 7-CATT allele had an increased risk of CaP recurrence at 5 years. Individuals with -173G/C, -173C/C and -794 7-CATT MIF genotypes have an increased incidence of CaP and these genotypes may serve as an independent marker for cancer recurrence.

Antibody levels against Chlamydia pneumoniae and outcome of roxithromycin therapy in patients with acute myocardial infarction. Results from a sub-study of the randomised Antibiotic Therapy in Acute Myocardial Infarction (ANTIBIO) trial.

BACKGROUND: Results of studies concerning prevention of cardiovascular disease by treatment with macrolide antibiotics targeting C. pneumoniae infection are still controversial. This study describes the results of different tests for infection with C. pneumoniae as well as the effect of treatment with roxithromycin in patients with acute myocardial infarction (AMI) in relation to their serostatus against C. pneumoniae. METHODS: We analysed blood of 160 patients who came from the ANTIBIOtic therapy after an AMI ( ANTIBIO-) study, a prospective, randomised, placebo-controlled, double-blind study to investigate the effect of roxithromycin 300 mg/OD for 6 weeks in patients with an AMI. Anti- Chlamydia IgG-, IgA-, and IgM-antibodies of these patients were analysed by means of different test systems. RESULTS: There was a good correlation between the two IgG and IgA methods (r = 0.900, p < 0.001 and r = 0.878, p < 0.001, respectively), but marked differences in the prevalence of positive tests. This resulted in only moderate concordance values, as expressed by the Kappa coefficients, for IgG kappa = 0.611 (95% CI = 0.498-0.724, p < 0.001) and for IgA kappa = 0.431 (95% CI: 0.322-0.540, p < 0.001). No significant association between positive C. pneumonia titers and the combined clinical endpoint during the 12 month follow-up could be found. In all test systems used, patients with positive anti- C. pneumoniae titers did not benefit from roxithromycin therapy (p = ns). CONCLUSION: Depending on the test system used, there are large differences in the prevalence of anti- C. pneumoniae seropositive patients. Clinical events during the 12 month follow-up after AMI did not depend on serostatus against C. pneumoniae and treatment with roxithromycin did not influence these events, independently of the serostatus against C. pneumoniae. However, the power of this subgroup analysis was low to detect small but significant differences.

The impact of different intensities of regular donor plasmapheresis on humoral and cellular immunity, red cell and iron metabolism, and cardiovascular risk markers.

BACKGROUND AND OBJECTIVES: Major studies are still lacking on the impact of differing intensities of long-term donor plasmapheresis, not only on total serum protein, albumin and immunoglobulin G (IgG), but also on humoral and cellular immunity, red cell and iron metabolism, and biochemical cardiovascular risk markers. MATERIALS AND METHODS: Three groups of donors, comprising 483 individuals undergoing differing intensities of long-term serial plasmapheresis, were entered into a cross-sectional study. A fourth control group consisted of 100 non-donors. In addition to measuring total protein, albumin and IgG levels, we determined parameters of humoral and cellular immunity, red cell and iron metabolism and recognized biochemical cardiovascular risk factors. RESULTS: The median annual net amount of plasma donated by the three donor groups was 37, 16 and 10 l, respectively (P < 0.0001). Donors had significantly lower total serum protein, albumin and IgG levels than non-donors (P < 0.0001), but the intensity of plasmapheresis had no influence on those parameters. Like non-donors, all plasma donors had normal humoral and cellular immunity. No increased rates of iron store depletion were observed in the three groups of plasma donors. Plasma donors were not at increased cardiovascular risk. CONCLUSIONS: Regular donor plasmapheresis of up to 45 l of plasma per year appears to be as safe as more moderate plasmapheresis programmes, with respect to the parameters analysed in this study. Individuals donating under these conditions did not develop impaired humoral and cellular immunity, iron store depletion, or increased cardiovascular risk with regard to established biochemical risk markers. Prospective studies are required to determine more exactly than in retrospective analyses the reasons why donors withdraw from plasmapheresis programmes.

Effectiveness of the glycoprotein IIb/IIIa antagonist abciximab during percutaneous coronary interventions (PCI) in clinical practice at a single high-volume center.

OBJECTIVE: Randomized controlled trials (RCTs) showed that the glycoprotein (GP) IIb/IIIa antagonist abciximab is able to reduce ischemic complications during percutaneous transluminal coronary interventions (PCIs). Its effectiveness in daily clinical practice in unselected patients remains to be determined. DESIGN, SETTING AND PATIENTS: From 7/1997 until 12/2000, 3310 PCIs were performed at the Heart Center Ludwigshafen. Out of them, 1076 (32.5%) patients were nonrandomly treated with a GP IIb/ IIa antagonist. Patients who were treated with abciximab were matched with patients not treated with abciximab. The matching procedure resulted in 590 pairs of patients. RESULTS: Patients treated with abciximab were more likely to have a history of former PCI (13.7% versus 8.8%, p=0.008) or coronary artery bypass surgery (19.2% versus 12.8%, p=0.003). There were no differences in concomitant diseases, left ventricular function, number of vessels diseased or target vessel. However, patients treated with abciximab had a higher rate of more complex stenosis (> or =B2; 94.4% versus 80.7%, p<0.001) and a longer x-ray exposition (median 486 s versus 422 s, p<0.001). Treatment with abciximab was associated with a significantly lower incidence of the combined endpoint of death, reinfarction or stroke during the hospital stay (2.4% versus 4.4%, p=0.039). This was confirmed after adjustment for confounding parameters (p=0.034). There was no increase in the rate of severe bleeding in the abciximab group (p=0.347). After one year the rates for the combined endpoint were 8.5% in the control group and 6.2% in the abciximab group (univariate analysis, p=0.134; multivariate analysis, p=0.143). CONCLUSION: Treatment with abciximab during PCI in daily clinical practice at a high volume center in patients with a high rate of acute coronary syndromes seems to be as effective as shown in RCTs.

Cytokine manipulation of explanted Dupuytren's affected human palmar fascia.

INTRODUCTION: Dupuytren's disease plagues human hands and digits producing fibrotic nodules and fascial cords with resultant debilitating flexion contracture deformities. Interest in this condition is great but because the disease is specific to humans and study has been hampered by the lack of an in vivo model. By utilizing an in vivo "nude" rat model it is possible to maintain and study explanted Dupuytren's contracted palmar fascia for prolonged periods of time. MATERIALS AND METHODS: Human specimens were divided into four, one for in vitro analysis, and three for model explantation. The explanted tissue was perfused with either transforming growth factor beta-2 (TGFbeta2), its antibody, or a control vehicle. Explant biopsies were obtained at 30 and 60 days and compared to tissue prior to explantation. Immunohistochemistry of collagen I and III, DNA synthesis, protein production, and fibroblast kinetics were serially determined. RESULTS: Perfusion of explanted Dupuytren's tissue by TGFbeta2 upregulated collagen I and III from biopsies obtained from the explants at 30 days when compared to vehicle control (P < 0.001). Perfusion with antibody prevented this upregulation when compared to vehicle control (P < 0.001). Cell cultures derived from fibroblasts obtained from biopsies of the explants perfused with TGFbeta2 increased DNA synthesis, protein production and fibroblast kinetics. CONCLUSION: These findings paralleled those from other fibroproliferative disorders suggesting a role for TGFbeta2 in the pathogenesis of Dupuytren's contracture as well as possible novel treatment approaches.

COX-2 specific inhibitor, NS-398, increases macrophage migration inhibitory factor expression and induces neuroendocrine differentiation in C4-2b prostate cancer cells.

BACKGROUND: Cyclooxygenase-2 (COX-2) is an inducible enzyme involved in the conversion of arachadonic acid to prostaglandins and other eicosaniods. Persistent COX-2 expression is associated with multiple forms of cancer.Therefore, there is much interest in COX-2 specific, non-steroidal anti-inflammatory drug use for cancer chemotherapy. The mechanism by which these drugs inhibit tumor growth and progression is unclear, and our knowledge about their potential to prevent or treat prostate cancer is inadequate. MATERIALS AND METHODS: The effects of NS-398, a selective COX-2 inhibitor, on human prostate carcinoma cell line LNCaP and the LNCaP subline C4-2b were investigated in this study. NS-398 effects on apoptosis were examined by caspase-3 activity increase, as well as internucleosomal cleavage. ELISA and PCR were used to determine inhibitor effects on macrophage migration inhibitory factor (MIF) and COX-2 production. RESULTS: At 10 microM, NS-398 treatment resulted in increased production of COX-2 and the pro-inflammatory cytokine, MIF by the C4-2b LNCaP subline. NS-398 (10 microM) induces apoptosis in LNCaP cells, but not in the more aggressive, androgen-unresponsive C4-2b cells. The C4-2b cells were observed to continue to proliferate when treated with NS-398 and continued to retain malignant phenotype characteristics. NS-398 treatment resulted in C4-2b cell differentiation into an unusual neuroendocrine-like cell. These neuroendocrine-like cells produced both epithelial (cytokeratin 18 and prostate specific antigen) and neuronal (neuron-specific enolase and chromogranin A) proteins. Furthermore, this C4-2b cellular response to NS-398 was mediated by NF-kappa beta transcription factor activation. CONCLUSIONS: These data suggest that COX-2 inhibition induces NF-kappa beta transcription factor activation, which subsequently induces pro-inflammatory protein expression (COX-2 and MIF) and neuroendocrine differentiation in the LNCaP C4-2b subline. These data provide further evidence that pro-inflammatory protein expression may play an important role in prostate cancer progression.

Increased stability of macrophage migration inhibitory factor (MIF) in DU-145 prostate cancer cells.

Macrophage migration inhibitory factor (MIF) has been localized to the glandular epithelium of the prostate and stimulates the in vitro growth of prostate epithelial cells. [35S]Methionine labeling of MIF protein was used to determine if prostate cells synthesize and secrete this cytokine. The results demonstrated that the DU-145 prostate cancer cells secrete about twice the amount of a more stable protein compared with normal prostate epithelial cells. To investigate if differences in MIF mRNA levels account for the differences in MIF protein secreted by these cells, mRNA stability was analyzed by [3H]uridine incorporation. Following a 12-h pulse, DU-145 cells were found to contain four times the amount of [3H]uridine-labeled MIF mRNA, and this message exhibited a longer half-life than the message found in normal cells (33 h and 19 h, respectively). Nuclear run-on experiments confirmed that the MIF gene is transcribed at a greater rate (1.8-fold) in the DU-145 prostate cancer cells. This study documents, for the first time, that human prostate epithelial cells synthesize and secrete this cytokine. These results indicate that the increased levels of MIF found in prostate cancer cells is likely due to the increased protein and mRNA stability as exhibited by DU-145 cells.

Macrophage migration inhibitory factor increases MMP-2 activity in DU-145 prostate cells.

Macrophage migration inhibitory factor (MIF) is a cytokine expressed by a number of different cell types and has been detected in prostatic glandular epithelial cells by immunohistochemistry. The goal of this study was to determine if in vitro cultured prostate cells produce this protein and some of the effects of MIF on these cells. Proliferation of normal prostate cells, the BPH-1 and DU-145 established cell lines in the presence of MIF were assessed. ELISA was used to screen conditioned medium for the production of MIF, matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2). Zymogram electrophoresis gels determined the activities of secreted MMP-2. The amount of MIF in the conditioned medium detected after 72 h of growth in normal, BPH-1 and DU-145 cells was 2.9, 5.2 and 10.2 ng/ml/10(6)cells respectively. Exogenous addition of MIF (25 ng/ml) to cells cultured in vitro stimulated proliferation of all the cell types tested. MIF addition to proliferating DU-145 cells resulted in a two-fold increase in the relative amount of active MMP-2 as determined by zymogram gel analysis of conditioned medium.

Sequential cytokine therapy for pressure ulcers: clinical and mechanistic response.

OBJECTIVE: To compare the healing response of sequential topically applied cytokines to that of each cytokine alone and to a placebo in pressure ulcers, and to evaluate the molecular and cellular responses. SUMMARY BACKGROUND DATA: Because of a deficiency of cytokine growth factors in chronic wounds and the reversal of impaired healing in animal models, pressure ulcer trials have been performed with several exogenously applied growth factors. Because single-factor therapy has not been uniformly successful, combination or sequential cytokine therapy has been proposed. Laboratory data have suggested that sequential treatment with granulocyte-macrophage/colony-stimulating factor (GM-CSF)/basic fibroblast growth factor (bFGF) might augment the previously reported effect of bFGF alone. METHODS: A masked, randomized pressure ulcer trial was performed comparing sequential GM-CSF/bFGF therapy with that of each cytokine alone and with placebo during a 35-day period. The primary measure was wound volume decrease over time. Cytokine wound levels and mRNA levels were serially determined. Fibroblast-populated collagen lattices (FPCLs) were constructed from serial fibroblast biopsies. Cellular ultrastructure was evaluated by electron microscopy. Changes in ease of surgical closure and its relative cost were determined. RESULTS: Ulcers treated with cytokines had greater closure than those in placebo-treated patients. Patients treated with bFGF alone did the best, followed by the GM-CSF/bFGF group. Patients treated with GM-CSF or bFGF had higher levels of their respective cytokine after treatment. Patients with the greatest amount of healing showed higher levels of platelet-derived growth factor (PDGF) on day 10 and transforming growth factor beta (TGFbeta1) on day 36. Message for the bFGF gene was upregulated after treatment with exogenous bFGF, suggesting autoinduction of the cytokine. FPCLs did not mimic the wound responses. Ultrastructure of wound biopsies showed response to bFGF. Treatment with any of the cytokines improved the wound by allowing easier wound closure. This was most marked for the bFGF-alone treatment, with a cost savings of $9,000 to $9,200. CONCLUSIONS: Treatment with bFGF resulted in significantly greater healing than the other treatments in this trial. The clinical response appeared to be related to upregulation of the bFGF message and to increased levels of PDGF-AB, bFGF, and TGFbeta1 in the wounds and changes in ultrastructure. The resultant improvements could be correlated with cost savings.

Expression of macrophage migration inhibitory factor in the human prostate.

The expression of macrophage migration inhibitory factor (MIF) in prostate tissue was investigated by immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), and Northern blot analysis using a prostate tissue bank. MIF expression was examined in each of the following established prostate tissue categories: prepubertal, pubertal, adult normal, benign hyperplastic (BPH), focal carcinoma within the prostate, and metastatic prostate cancer. IHC showed that all samples tested were positive for MIF protein, which localized to the glandular epithelial cells with no apparent staining of stroma. The most intense staining was observed in the metastatic prostatic adenocarcinoma and the human prostatic adenocarcinoma cell line LNCaP. Using quantitative ELISA, MIF expression was found to be at least three times higher in metastatic adenocarcinoma than in normal, BPH, or focal carcinoma in the adult prostate. This study is the first to report that prostate glandular epithelial cells express MIF. The exact role of MIF in prostate development and disease progression requires further study.

Preliminary studies on the isolation and characterization of predominant prostatic proteins.

BACKGROUND: The increasing incidence of prostate cancer demands that we give our full attention not only to the etiology and prevention of this common type of cancer, but also to the diagnosis and prognostic course of this disease. In an effort to develop new prostatic tumor markers that could be useful to the physician at the current state of our knowledge in the diagnosis and prognosis of this disease, our laboratories have undertaken an effort to isolate and characterize the nature of the major proteins in the normal prostate and in prostatic neoplasia. METHODS: In this preliminary study, tissue was obtained from open prostatic surgery in patients with a pre- and postoperative diagnosis of benign prostatic hyperplasia. The initial fractionation and separation of the proteins was achieved through the use of ultrafiltration of homogenates followed by SDS-PAGE. Initial analysis of four prominent protein bands was accomplished by amino acid sequencing, and identified by a search in GeneBank data base. RESULTS: Two proteins previously identified in prostatic tissue were prostate specific antigen (M(r) 26,496) with 240 amino acid residues and beta-inhibin (M(r) 10,704) with 94-amino acid residues. A third protein was identified as human cysteine rich protein (hCRP). This protein functions as a DNA binding protein and has previously been postulated to contain four putative zinc fingers and to play a fundamental role in cellular function. Ubiquitin, the fourth major protein identified was a 76-amino acid polypeptide whose function is to target other proteins for destruction. CONCLUSIONS: hCRP and ubiquitin are reported as being found in high levels in prostatic tissue for the first time.

Enhanced expression of macrophage migration inhibitory factor in prostatic adenocarcinoma metastases.

OBJECTIVES: Determining the genetic changes associated with the development of metastatic prostate cancer is of utmost importance in patient prognosis and therapy. Our goal is to identify genes whose enhanced expression is associated with metastatic prostate cancer. METHODS: Total ribonucleic acid was isolated from prostatic tissue exhibiting no histologic evidence of carcinoma and from a prostatic adenocarcinoma lymph node metastasis. The differential display polymerase chain reaction (DD-PCR) technique was used to isolate genes that exhibited increased expression in the metastatic tissue sample. Isolated PCR products were cloned, sequenced, and identified by screening complementary deoxyribonucleic acid (cDNA) databases. RESULTS: Using DD-PCR, we identified three cDNA clones that exhibit enhanced expression in metastatic prostatic tissue. Two of these cDNA clones have not been identified because they show no homology to known database sequences. The third cDNA is 166 base pairs in length and exhibits 93% homology to nucleotides 662 to 828 of human macrophage migration inhibitory factor (MIF). Slot blot analysis using RNA from various prostate-derived sources suggests that increased expression of MIF is associated with metastatic prostate cancer. CONCLUSIONS: These results show that the DD-PCR technique is applicable for the identification and cloning of human genes that exhibit enhanced expression in prostate cancer metastases. These results indicate the possibility that MIF production by prostate cancer cells plays a role in the development of metastases. The enhanced expression of MIF by prostate cancer cells may be a potential prognostic marker for metastatic prostate cancer.

Cell cycle regulation of the glyceraldehyde-3-phosphate dehydrogenase/uracil DNA glycosylase gene in normal human cells.

The cell cycle regulation of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)/uracil DNA glycosylase (UDG) gene was examined in normal human cells. Steady state RNA levels were monitored by Northern blot analysis using a plasmid (pChug 20.1) which contained the 1.3 kb GAPDH/UDG cDNA. The biosynthesis of the 37 kDa GAPDH/UDG protein was determined using an anti-human placental GAPDH/UDG monoclonal antibody to immunoprecipitate the radiolabeled protein. Increases in steady state GAPDH/UDG mRNA levels were cell cycle specific. A biphasic pattern was observed resulting in a 19-fold increase in the amount of GAPDH/UDG mRNA. The biosynthesis of the 37 kDa GAPDH/UDG protein displayed a similar biphasic regulation with a 7-fold increase. Pulse-chase experiments revealed a remarkably short half life of less than 1 hr. for the newly synthesized 37 kDa protein, comparable to that previously documented for a number of oncogenes. GAPDH/UDG mRNA levels were markedly reduced at 24 hr. when DNA synthesis was maximal. These results define the GAPDH/UDG gene as cell cycle regulated with a characteristic temporal sequence of expression in relation to DNA synthesis. The cell cycle synthesis of a labile 37 kDa monomer suggests a possible regulatory function for this multidimensional protein. Further, modulation of the GAPDH/UDG gene in the cell cycle may preclude its use as a reporter gene when the proliferative state of the cell is not kept constant.

Proliferative dependent regulation of the glyceraldehyde-3-phosphate dehydrogenase/uracil DNA glycosylase gene in human cells.

The relationship between the proliferative dependent expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)/uracil DNA glycosylase (UDG) gene and the induction of uracil DNA glycosylase activity was examined in human cells. Three different cell types were studied to determine whether the growth-dependent regulation of this multifunctional gene was a common characteristic of human cells. These included WI-38 normal embryonic lung fibroblasts, a Japanese Bloom's syndrome non-transformed skin fibroblast cell strain (GM-05289) and a lymphoblastoid cell line transformed by the Epstein-Barr virus. The Japanese Bloom's syndrome cells displayed the altered immunoreactivity with marker monoclonal antibody 40.10.09 which characterizes cells from this human genetic disorder. In noncycling human cells Northern blot analysis using a plasmid (pChug 20.1) which contained the human GAPDH/UDG cDNA revealed a single 1.6 kb transcript. In each case, the expression of this gene was increased during cell proliferation. This increase in GAPDH/UDG gene expression was identical to that observed for UDG enzyme activity. Further, using anti-human UDG monoclonal antibodies, there was a growth-dependent rise in immunoreactivity suggesting an increase in the level of antigenic protein. These results demonstrate that: (i) the expression of the GAPDH/UDG gene was dependent on the proliferative state of the cell; and (ii) a correlation existed between the transcription of this gene and the level of uracil DNA glycosylase enzyme activity.

A human nuclear uracil DNA glycosylase is the 37-kDa subunit of glyceraldehyde-3-phosphate dehydrogenase.

We have isolated and characterized a plasmid (pChug 20.1) that contains the cDNA of a nuclear uracil DNA glycosylase (UDG) gene isolated from normal human placenta. This cDNA directed the synthesis of a fusion protein (Mr 66,000) that exhibited UDG activity. The enzymatic activity was specific for a uracil-containing polynucleotide substrate and was inhibited by a glycosylase antibody or a beta-galactosidase antibody. Sequence analysis demonstrated an open reading frame that encoded a protein of 335 amino acids of calculated Mr 36,050 and pI 8.7, corresponding to the Mr 37,000 and pI 8.1 of purified human placental UDG. No homology was seen between this cDNA and the UDG of herpes simplex virus, Escherichia coli, and yeast; nor was there homology with the putative human mitochondrial UDG cDNA or with a second human nuclear UDG cDNA. Surprisingly, a search of the GenBank data base revealed that the cDNA of UDG was completely homologous with the 37-kDa subunit of human glyceraldehyde-3-phosphate dehydrogenase. Human erythrocyte glyceraldehyde-3-phosphate dehydrogenase was obtained commercially in its tetrameric form. A 37-kDa subunit was isolated from it and shown to possess UDG activity equivalent to that seen for the purified human placental UDG. The multiple functions of this 37-kDa protein as here and previously reported indicate that it possesses a series of activities, depending on its oligomeric state. Accordingly, mutation(s) in the gene of this multifunctional protein may conceivably result in the diverse cellular phenotypes of Bloom syndrome.

Isolation and characterization of the human uracil DNA glycosylase gene.

A series of anti-human placental uracil DNA glycosylase monoclonal antibodies was used to screen a human placental cDNA library in phage lambda gt11. Twenty-seven immunopositive plaques were detected and purified. One clone containing a 1.2-kilobase (kb) human cDNA insert was chosen for further study by insertion into pUC8. The resultant recombinant plasmid selected by hybridization a human placental mRNA that encoded a 37-kDa polypeptide. This protein was immunoprecipitated specifically by an anti-human placental uracil DNA glycosylase monoclonal antibody. RNA blot-hybridization (Northern) analysis using placental poly(A)+ RNA or total RNA from four different human fibroblast cell strains revealed a single 1.6-kb transcript. Genomic blots using DNA from each cell strain digested with either EcoRI or Pst I revealed a complex pattern of cDNA-hybridizing restriction fragments. The genomic analysis for each enzyme was highly similar in all four human cell strains. In contrast, a single band was observed when genomic analysis was performed with the identical DNA digests with an actin gene probe. During cell proliferation there was an increase in the level of glycosylase mRNA that paralleled the increase in uracil DNA glycosylase enzyme activity. The isolation of the human uracil DNA glycosylase gene permits an examination of the structure, organization, and expression of a human DNA repair gene.

Thrombospondin promotes platelet aggregation.

Thrombospondin (TSP), isolated from human platelets, promotes aggregation of both nonstimulated platelets and platelets stimulated with thrombin or ADP. The TSP-promoted aggregation is specific since a monoclonal antibody against TSP inhibits the effect of exogenously added TSP and inhibits thrombin-induced platelet aggregation in the absence of added TSP. Several lines of evidence suggest that TSP mediates its effect on aggregation of nonstimulated and stimulated platelets through different platelet-surface receptor systems. The TSP-promoted aggregation of nonstimulated platelets was inhibited by a monoclonal antibody to platelet glycoprotein IV (GPIV), but not by a monoclonal antibody to the fibrinogen receptor, GPIIb-IIIa. In contrast, the antibody to GPIIb-IIIa totally inhibited the TSP-potentiated aggregation of thrombin-stimulated platelets, whereas the antibody to GPIV has no effect. Thus, these studies suggest that TSP promotes platelet aggregation by at least two mechanisms--one dependent on and one independent of the platelet fibrinogen receptor system.