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The phenotype of mouse thymic B cells and their capacity to induce T cell negative selection in vitro were analyzed. Thymic B cells expressed B cell markers such as IgM, Fc gamma receptor, CD44, heat-stable antigen, LFA-1 and CD40. In addition, they were positive for the activation molecule CD69 and displayed high levels of B7-2. Although thymic B cells expressed CD5 on their surface, no CD5-specific mRNA was detected. Moreover, thymic B cells induced a stronger deletion of TCR-transgenic (TG) thymocytes than splenic B cells, which had low CD69 and B7-2 levels. Interestingly, CD40-activated splenic B cells up-regulated CD69 and B7-2 and acquired a capacity to induce T cell deletion comparable to that of thymic B cells. Moreover, thymic B cells from CD40-deficient mice displayed lower CD69 and B7-2 levels than control thymic B cells, and lower capacity to induce the deletion of TCR TG thymocytes. These results support the hypothesis that CD40-mediated activation of thymic B cells determines a high efficiency of antigen presentation, suggesting that within the thymus B cells may play an important role in the elimination of autoreactive thymocytes.

An in vitro gut model was used to investigate the bioavailability of calcium, magnesium, cadmium, chromium, iron, manganese, and lead in the Mexican (maize) tortilla. The samples (4 g) were digested in quartz tubes using concentrated nitric acid (12 mL) at 65 degrees C (2 h) and then at 120 degrees C (4 h). Total element concentration was determined by atomic absorption spectrometry. Enzymatic hydrolysis was carried out in two steps: first, with pepsin in a hydrochloric acid medium (pH 1.8) and then after neutralization with sodium bicarbonate (pH 6.0), with the mixture of pancreatine, amylase, and bile salts extract. Elements under study were determined in the supernatant by atomic absorption spectrometry. The bioavailable fraction of each element in tortilla was evaluated as the percentage of total element content found in the solution after enzymolysis. The obtained results showed relatively low bioavailability of the selected elements (from 2% to 32%), which possibly may be ascribed to the presence of dietary fiber in tortilla.

MCF-7 cells migrate through vitronectin-coated filters in response to insulin-like growth factor I (IGF-I); migration is inhibited by the matrix metalloproteinase (MMP) inhibitor BB-94, but not by the serine proteinase inhibitor aprotinin. MMP-9 was identified in the conditioned medium of MCF-7 cells; in addition, fluorescence-activated cell sorting analysis revealed its presence on the cell surface, where MMP-9 activity was also found using a specific fluorogenic peptide. Furthermore, the messenger RNA encoding MMP-9 was detected in MCF-7 cells by PCR. The IGF-I concentration leading to maximal MCF-7 invasion produces an increase in cell surface proteolytic activity after short incubation periods. At 18 h, however, preincubation of MCF-7 cells with IGF-I produces at 18 h a dose-dependent decrease in cell-associated MMP-9 activity and an increase in soluble MMP-9. MCF-7 invasion is dependent on the alpha(v)beta5 integrin, a vitronectin receptor. The levels of alpha(v)- and beta5-subunits expressed in MCF-7 cells depend on the IGF-I concentration, which triggers an increase in both of these subunits. Based on these results, we suggest that IGF-I-induced MCF-7 cell migration is mediated by the MMP-9 activity on the cell surface and by alpha(v)beta5 integrin.

We have cloned the murine CCR6 receptor and its ligand, the beta-chemokine mMIP-3alpha. Calcium mobilization assays performed with mCCR6 transfectants showed significant responses upon addition of mMIP-3alpha. Murine MIP-3alpha RNA is expressed in thymus, small intestine and colon, whereas mCCR6 RNA is expressed in spleen and lymph nodes. RT-PCR analysis of FACS-sorted lymphoid and antigen presenting cell subsets showed mCCR6 expression mainly in B cells, CD8- splenic dendritic cells and CD4+ T cells. The cloning and functional characterization of the mCCR6 and mMIP-3alpha will allow the study of the role of these proteins in mouse models of inflammation and immunity.

A structural model is presented for family 32 of the glycosyl-hydrolase enzymes based on the beta-propeller fold. The model is derived from the common prediction of two different threading methods, TOPITS and THREADER. In addition, we used a correlated mutation analysis and prediction of active-site residues to corroborate the proposed model. Physical techniques (circular dichroism and differential scanning calorimetry) confirmed two aspects of the prediction, the proposed all-beta fold and the multi-domain structure. The most reliable three-dimensional model was obtained using the structure of neuraminidase (1nscA) as template. The analysis of the position of the active site residues in this model is compatible with the catalytic mechanism proposed by Reddy and Maley (J. Biol. Chem. 271:13953-13958, 1996), which includes three conserved residues, Asp, Glu, and Cys. Based on this analysis, we propose the participation of one more conserved residue (Asp 162) in the catalytic mechanism. The model will facilitate further studies of the physical and biochemical characteristics of family 32 of the glycosyl-hydrolases.

Stromelysin 1 (ST1) is a member of the matrix metalloproteinase (MMP) family probably involved in extracellular matrix degradation. Stromelysin 3 (ST3), considered by sequence homology to be a member of the MMP family of proteases, is specifically expressed in the stroma adjacent to the invasive tumoral cells, but its role in cancer progression remains to be elucidated. Genes encoding ST1 and ST3 were expressed in lepidopteran insect cells using the baculovirus expression vector system. Recombinant baculoviruses were obtained after cloning the full-length cDNA of ST1 and ST3 in plasmids pBacPAK1 and pBacPAK9, respectively. Sf9 insect cells infected with the recombinant baculovirus overexpressed the zymogen proST1 (60 kDa) in an insoluble form, a peak of expression being reached from 24 h postinfection. After solubilization in 8 M urea, and further refolding, activation, and purification, 0.3 mg of mature ST1 (30 kDa), purified to 90% homogeneity, was obtained per 5 x 10(8) infected cells. Recombinant ST1 exhibited proteolytic activity on alpha2-macroglobulin, casein, fibronectin, alpha1-antitrypsin, and laminin. The recombinant zymogen proST3 (55 kDa) was expressed as a soluble form in insect cells, maximal expression occurring at 72 h postinfection. After purification to 95% homogeneity, 2.5 mg of proST3 was obtained per 5 x 10(8) infected cells. A number of proteases including plasmin, urokinase, and ST1 were shown to be able to cleave proST3 giving rise to defined bands of 50-30 kDa. The ST3 mature form of 45 kDa (mST3) was also expressed in the baculovirus system and the obtained protein, 2. 5 mg per 5 x 10(8) infected cells purified to 80% homogeneity, was shown to be active on both casein degradation and alpha2-macroglobulin entrapment assays. Our results suggest that the baculovirus system offers a convenient and efficient means to produce ST1 and ST3 in order to carry out further biochemical studies., (Copyright 1998 Academic Press.)

To determine the nature of the mechanism by which certain derived ruthenium (Ru) complexes induce regression in tumour growth, we have investigated the possibility that this mechanism was associated with an increase of superoxide anion (O2-. production by phagocytic cells, which are usually found in tumour nodes. Here we present evidence that a newly synthesized complex, Ru3+-propylene-1, 2-diaminotetra-acetic acid (Ru-PDTA), derived from Ru and the sequestering ligand (PDTA), specifically stimulates O2-. production. This increase was associated with the translocation of cytosolic factors p47(phox) and p67(phox) of NADPH oxidase to the plasma membrane. The Ru-PDTA-complex-dependent O2-. production was abrogated by staurosporine, partially inhibited by diphenylene iodonium, and it was insensitive to pertussis toxin or dibutyryl cyclic AMP pretreatment. An increase of cytosolic Ca2+ levels were also detected in neutrophils treated with the Ru-PDTA complex. Also, Ru-PDTA complex induced the phosphorylation of tyrosine residues of several proteins as assessed by Western blotting. Present data are consistent with the possibility that Ru-PDTA-dependent antitumour effects are due in part to the complex's ability to stimulate the release of toxic oxygen metabolites from phagocytic cells infiltrating tumour masses.

To measure matrix metalloproteinase (MMP) activity in a large number of samples it is advisable to use easily automated methods. We have evaluated and compared the activity of stromelysin-1 (MMP-3), matrilysin (MMP-7), 72 kDa gelatinase A (MMP-2) and 92 kDa gelatinase B (MMP-9) by zymogram analysis and fluorescent substrate degradation assays. FITC-casein and the fluorogenic peptide Dnp-Pro-beta-cyclo-hexyl-Ala-Gly-Cys(Me)-His-Ala-Lys-(N-Me-Abz)-NH 2 were used as fluorescent substrates. FITC-casein was more efficiently degraded than the fluorogenic peptide by all MMPs tested except MMP-9. MMP-2 was not significantly able to degrade the fluorogenic peptide. Gelatin zymography was the most sensitive method to detect the activity of both gelatinases but quantitation problems compromise its use. The degradation of fluorogenic substrates by MMPs could be inhibited by the chelating agent EDTA and by the tissue inhibitor of metalloproteinases 2 (TIMP-2), an MMP-specific inhibitor. Fluorometric methods represent a good alternative for MMP activity measurement, especially when a large number of samples must be processed.

HLA-B27 and associate antigens incidence were studied in 620 cases of seronegative spondiloarthropathies (SNS) and 262 controls of a Venezuelan mestizo population from Zulla state between 1985 and 1995. The incidence of HLA-B27 was 20.96% of all cases of SNS. It was increased in patients with ankilosing spondylitis (AS) 33.33% and Relter's syndrome (RS) 30%, but not in uveitis (Uv) 20% an psoriatic arthropathy (PsA) 0%. The incidence in the control group was 4.2%. This results are lower than the previous description in Venezuelan mestizo and caucasic population but are close to the incidence described in population of West Africa with important contribution to admixture of the occidental part of Venezuela.

Two new human genes encoding putative G protein-coupled receptors were cloned from genomic DNA following a degenerate PCR strategy. The predicted amino acid sequences of the proteins encoded by these genes have the characteristic motifs of chemokine receptors. This prompted us to name these genes CKR-L1 and CKR-L3. Nevertheless, the identification of their ligands has not been possible using 16 human chemokines in three different assays. The RNA expression pattern of CKR-L1 and CKR-L3 is also similar to that generally found for chemokine receptors. To a different extent, these genes are expressed in spleen, lymph nodes, and CD4+, CD8+ and CD19+ lymphocytes. CKR-L1 expression was also detected in monocyte/macrophages. The homology to chemokine receptors suggests that CKR-L1 belongs to the family of beta chemokine receptors, while CKR-L3 is more similar to alpha chemokine receptors.

The export efficiency of a fusion of the Escherichia coli preOmpA signal peptide to human interleukin-6 can be significantly raised by coexpressing three different prlA alleles of sec Y along with wild type secE. The effect seems prlA-specific, as prlG1 (a prl allele of secE) does not affect the export of preOmpA-hIL-6. Coexpression of secD and secF also stimulates the export of the fusion protein.

A cDNA encoding the complete reading frame of a novel homeodomain-containing protein, named zhx-1, has been cloned from a mouse bone marrow stromal cell line. The ORF encodes a protein of 873 amino acids, in which two C2-H2 zinc-fingers and five homeodomains have been identified. These features classify zhx-1 as a member of the ZF class of homeodomain transcription factors, zhx-1 mRNA is widely expressed in adult mouse, although it is preferentially expressed in brain. At least, two homologous mRNAs are present in humans suggesting that zhx-1 is the first member of a novel subclass of homeodomain factors.

The baculovirus expression system was used to produce recombinant human matrilysin. Expression of promatrilysin reached a peak at 72 h post-infection. Most of the recombinant protein remained in the intracellular fraction in an insoluble form, which after renaturation was purified by S-Sepharose and Green A Dyematrex chromatography in order to remove host proteases. Active recombinant matrilysin degraded casein, type I and type IV collagens and fibronectin. Expression of recombinant human matrilysin using the baculovirus system represents a useful tool for obtaining large amounts of this metalloproteinase in order to carry out further biochemical studies and to screen for inhibitors.

Purpose: Some genetic factors associated to the HLA system phenotypes may allegedly predispose to the development of infection in patients exposed to the human immunodeficiency virus (HIV). So the aim of this study was to assess if certain HLA antigens are positive or negative risk factors in the development of AIDS in Zulia State., Patients and Methods: A total of 62 samples were studied, 31 from HIV seropositive subjects and 31 form healthy individuals. The patients were subclassified into four groups in accordance with Atlanta's CDC guidelines. Tests for histocompatibility including HLA-A-B-C, DR and DQ typing were performed with Terasaki's technique. VIH positivity was determined by ELISA and confirmed by Western Blot. The statistical evalub1p4n was performed with the chi 2 test for antigen frequency comparison, the relative risk (RR) was estimated with the Ryder and Svelgaard test, and the inferential analysis was made by means of non-parametric statistics., Results: Most patients were included in CDC's groups II and IV, 48.4% and 29.0%, respectively. Increased B35 and DQw2 and decreased B39 and DR2 antigens were found when comparing the HLA distribution in the sample and the antigenic frequency of the population. RR > 1 was observed in the infected patients A for A1, A3, A10, A11, B5, B7, B12, B14, B35, B61, CW4, DR4, DRW52 and DQW2 HLA antigens. A positive association between symptomatic infected patients and antigen B35 was present (X = 7.045)., Conclusion: The findings reported here suggest that antigen B35 is a major risk factor for the development of AIDS.

A new methodology for the identification of genes modulated by transcription factors in vivo is described. Mouse genomic DNA fragments bound by the thyroid hormone receptor (T3R) were selected and amplified in vitro. Subsequent hybridisation with biotinylated cDNA allowed the selection of those DNA fragments containing binding sites for T3R that corresponded to transcribed DNA. Expression analysis of the corresponding genes showed that more than 80% are indeed modulated by thyroid hormones in vivo in the liver. Together with the presence of consensus binding sites for T3R this result suggests that the selected DNA fragments may contain T3R transcriptional regulatory elements. This method, extensive to other ligand-modulated transcription factors, might be useful to all transcription factors with slight modifications.

Export of recombinant proteins to the periplasm of Escherichia coli is in many cases preferable to cytoplasmic production. However, when the protein is overexpressed, export efficiency decreases significantly and some advantages of the system are lost. This is what happens when attempting to produce recombinant human interleukin-6 (hIL-6) as a pre(OmpA) fusion in E. coli. Assuming that the host protein export machinery becomes overloaded, we have tested the effect of providing the host with additional copies of two key components of that machinery. Supplementation with a plasmid bearing prlA4 (secY allele) and secE genes increased the ratio of mature to precursor hIL-6 from 1.2 to 10.8. The increase in processing ratio was associated with the accumulation of a larger amount of total (mature plus precursor forms) hIL-6. Providing a plasmid-borne wild-type prlA was ineffective compared to prlA4 allele. This suggests that the PrlA protein, a component of the translocator, recognizes features at the mature portion of secretory substrates independently of those at the signal peptide portion.

Clinical studies have shown that continuous or intermittent scheduled administration of medroxyprogesterone acetate (MPA) reduces the bone marrow toxicity induced by antitumor drugs. This MPA myeloprotection has been attributed to an arrest of hematopoietic progenitors in a quiescent phase, although no in vitro studies have demonstrated such an effect of MPA. Human bone marrow cells were preincubated for 3 days with MPA (100 ng/mL) and then exposed to sublethal doses of adriamycin; LD50 was significantly increased in MPA-preincubated cells (896 +/- 172 ng/mL) vs. control cells (162 +/- 37 ng/mL); this protective effect of MPA was shown to be more efficient against S-phase-specific drugs such as 5-fluorouracil (5-FU) than against non-phase-specific drugs such as cisplatin. MPA did not protect several human leukemic cell lines from the cytotoxic action of adriamycin. "Suicide" assays showed that the percentage of myeloid progenitor cells (granulocyte-macrophage colony-forming units [CFU-GM]) in S-phase was significantly reduced from 67 +/- 2.5% (control cells) to 38 +/- 5.5% (24-hour MPA-preincubated cells). These results demonstrate in vitro that MPA exerts a cell cycle arrest of hematopoietic precursors, protecting them from the toxicity of chemotherapy.

Active penicillin acylase from Kluyvera citrophila strain ATCC 21,285 consists of two different alpha- and beta-subunits derived from a single precursor by post-translational processing. Using the chemical mutagen hydroxylamine we have treated plasmid pYKD59 containing the active penicillin acylase gene (pga) from K. citrophila and have generated different point mutant penicillin acylase genes, one producing a maturation deficient precursor. This point mutation has changed the glycine 310 residue of the precursor for a glutamic acid (residue number 21 of the mature beta-subunit). The introduction of a charged residue in this position did not prevent translocation of the precursor to the periplasm but the resultant molecule was not able to undergo subsequent post-translational modification to yield the active protein.

Between January 1984 and December 1987 a total of 178 AIDS cases were reported to the Colombian Ministry of Health. The location of these cases suggests that the human immunodeficiency virus (HIV) is widely distributed in Colombia. Most of those afflicted (97%) have been adult males. HIV seroprevalence studies of selected population groups revealed the highest antibody prevalence (5.65% in females, 22.5% in males) among individuals involved in high-risk behaviors who participated in a free AIDS testing program. High prevalences (from 0.6% to 3.9% in females, and 14.6% to 15.9% in males) were also found in patients (primarily female prostitutes and male homosexuals) attending clinics for sexually transmitted diseases in several urban areas. The number of AIDS cases in Colombia has doubled or tripled annually since reporting began in 1984, a pattern similar to that observed worldwide.