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Biomedical subjects

J Blanco

Publications and source records attributed to J Blanco.

At least 127 records · Page 7Linked to original sources
PubMed1999

Angiotensin-converting enzyme is upregulated in the proximal tubules of rats with intense proteinuria.

Persistent proteinuria is considered a deleterious prognostic factor in most progressive renal diseases. However, the mechanisms by which proteinuria induces renal damage remain undetermined. Since proximal tubular cells possess all the machinery to generate angiotensin II (Ang II), we approached the hypothesis that proteinuria could elicit the renal activation of the renin-angiotensin system in a model of intense proteinuria and interstitial nephritis induced by protein overload. After uninephrectomy (UNX), Wistar-Kyoto rats received daily injections of 1 g BSA or saline for 8 days. The mean peak of proteinuria was observed at the fourth day (538+/-89 versus 3+/-1 mg/24 h in UNX controls; n=12; P<0.05) and was increased during the whole study period (at the eighth day: 438+/-49 mg/24 h; n=12; P=NS). Morphological examination of the kidneys at the end of the study showed marked tubular lesions (atrophy, vacuolization, dilation, and casts), interstitial infiltration of mononuclear cells, and mesangial expansion. In relation to UNX control rats, renal cortex of BSA-overloaded rats showed an increment in the gene expression of angiotensinogen (2.4-fold) and angiotensin-converting enzyme (ACE) (2.1-fold), as well as a diminution in renin gene expression. No changes were observed in angiotensin type 1 (AT1) receptor mRNA expression in both groups of rats. By in situ reverse transcription-polymerase chain reaction and immunohistochemistry, ACE expression (gene and protein) was mainly localized in proximal and distal tubules and in the glomeruli. By immunohistochemistry, angiotensinogen was localized only in proximal tubules, and AT1 receptor was localized mainly in proximal and distal tubules. In the tubular brush border, an increase in ACE activity was also seen (5. 5+/-0.5 versus 3.1+/-0.7 U/mg protein x10(-4) in UNX control; n=7; P<0.05). Our results show that in the kidney of rats with intense proteinuria, ACE and angiotensinogen were upregulated, while gene expression of renin was inhibited and AT1 was unmodified. On the whole, these data suggest an increase in Ang II intrarenal generation. Since Ang II can elicit renal cell growth and matrix production through the activation of AT1 receptor, this peptide may be responsible for the tubulointerstitial lesions occurring in this model. These results suggest a novel mechanism by which proteinuria may participate in the progression of renal diseases.

Animals↗
PubMed1998

Biochemical characterization of the SecA protein of Streptomyces lividans--interaction with nucleotides, binding to membrane vesicles and in vitro translocation of proAmy protein.

The SecA protein of Streptomyces lividans was purified to near electrophoretic homogeneity by means of FPLC from an overproducing strain harbouring plasmid pULA400, in which the secA gene (Blanco, J., Coque, J. J. R. & Martín, J. F. (1996) Gene (Amst.) 176, 61-65) was expressed from the strong promoter of the Streptomyces griseus saf gene. The native form of SecA was shown to be a dimer (Mr 209 kDa) by gel filtration. It crossreacted with antibodies raised against Escherichia coli or Bacillus subtilis SecA proteins. Purified S. lividans SecA showed a low endogenous ATPase activity that was stimulated by addition of a S. lividans lipid fraction. SecA contains a high-affinity and a low-affinity nucleotide-binding site (NBS). [Alpha-32P]ATP could be crosslinked by ultraviolet radiation at the high-affinity site. The intrinsic tryptophan fluorescence of SecA decreased on addition of increasing concentrations of ADP and reached a saturation level at about 1 microM (the range of saturation at the NBS I). The calculated Kd of the high-affinity binding site for ADP was 150 nM. Millimolar concentrations of ATP or ADP did not render the S. lividans SecA protein resistant to V8 protease degradation, in contrast to what occurs with the E. coli and B. subtilis SecA proteins. SecA was found to bind to urea-washed S. lividans membrane vesicles with high-affinity, i.e. 10 nM. SecA-dependent binding of E. coli SecB to membrane vesicles was observed when E. coli SecA was used, but not with the S. lividans SecA, suggesting that this interaction may be specific for the Gram-negative bacteria. An in vitro translocation system has been developed using inverted membrane vesicles of S. lividans. SecA supported in vitro translocation of proAmy into S. lividans membrane vesicles in an ATP-dependent manner.

Adenosine Diphosphate↗
PubMed1998

Dipeptidyl-peptidase IV-beta--further characterization and comparison to dipeptidyl-peptidase IV activity of CD26.

Dipeptidyl peptidase IV-beta (DPP IV-beta) is a novel protein which shows a peptidase activity similar to the T-cell-activation antigen CD26. To further characterize this DPP IV-beta and confirm its cell surface expression, we have developed a purification strategy using the CD26- cell line C8166. The purification process includes biotinylation of cell surface proteins before preparation of cell extracts and processing by gel-filtration, ion-exchange and lectin chromatographies. Consistent with the molecular mass of DPP IV-beta estimated by gel-filtration chromatography, the final purified fraction, manifesting a typical DPP IV activity, showed a major biotinylated 75-80-kDa band in SDS/PAGE, thus suggesting the monomeric nature of this enzyme. Kinetic parameters of DPP IV-beta and the sensitivity to a new family of irreversible DPP IV inhibitors, were studied in comparison to CD26. Both enzymes followed a Michaelis kinetics with different Km values for Gly-Pro-NH-Np (NH-Np, para-nitroanilide) hydrolysis (0.28+/-0.05 mM and 0.12+/-0.02 mM). More significant differences were observed in the sensitivity to inhibitors, which exerted a much higher activity on CD26 than on DPP IV-beta. These differences permitted us to study DPP IV-beta expression in CD26-expressing cells, showing the expression of this new enzyme in all lymphoid cells tested, and a rapid enhancement in phytohemagglutinin-stimulated or protein-A-stimulated peripheral blood mononuclear cells. Our results indicate that, although DPP IV-beta and CD26 are coexpressed and manifest a typical DPP IV activity, there are distinct features in their catalytic activities that may confer to each enzyme a complementary role in peptide processing.

Biotinylation↗
PubMed1998

Identification of V3 loop-binding proteins as potential receptors implicated in the binding of HIV particles to CD4(+) cells.

The binding of human immunodeficiency virus (HIV) type 1 particles to CD4(+) cells could be blocked either by antibodies against the V3 loop domain of the viral external envelope glycoprotein gp120, or by the V3 loop mimicking pseudopeptide 5[Kpsi(CH2N)PR]-TASP, which forms a stable complex with a cell-surface-expressed 95-kDa protein. Here, by using an affinity matrix containing 5[Kpsi(CH2N)PR]-TASP and cytoplasmic extracts from human CEM cells, we purified three V3 loop-binding proteins of 95, 40, and 30 kDa, which after microsequencing were revealed to be as nucleolin, putative HLA class II-associated protein (PHAP) II, and PHAP I, respectively. The 95-kDa cell-surface protein was also isolated and found to be nucleolin. We show that recombinant preparations of gp120 bind the purified preparations containing the V3 loop-binding proteins with a high affinity, comparable to the binding of gp120 to soluble CD4. Such binding is inhibited either by 5[Kpsi(CH2N)PR]-TASP or antibodies against the V3 loop. Moreover, these purified preparations inhibit HIV entry into CD4(+) cells as efficiently as soluble CD4. Taken together, our results suggest that nucleolin, PHAP II, and PHAP I appear to be functional as potential receptors in the HIV binding process by virtue of their capacity to interact with the V3 loop of gp120.

Amino Acid Sequence↗
PubMed1998

Increased rate of HIV-1 entry and its cytopathic effect in CD4+/CXCR4+ T cells expressing relatively high levels of CD26.

The role of the T-cell activation antigen CD26 was evaluated in viral entry and infection of CD4(+)/CXCR4(+) cells by the lymphotropic HIV-1 Lai isolate. For this purpose, CEM T cells, which are permissive to HIV infection and express low levels of CD26, were used to establish by transfection four groups of cell clones expressing either low, high, and very high levels of CD26, or expressing the anti-sense RNA of CD26. Entry was monitored by the detection of proviral DNA synthesis and the kinetics of virus production, whereas the cytopathic effect was demonstrated by the occurrence of apoptosis. HIV entry and infection were consistently accelerated by at least 24 to 48 h in clones expressing high levels of CD26 compared to the parental cells or to the clones expressing low levels of CD26. Interestingly, infection of clones expressing very high levels of CD26 was not accelerated and showed a kinetics of infection similar to that of low CD26 expressing clones. Moreover, HIV infection was significantly reduced in the clones expressing CD26 anti-sense RNA. In the different clones, apoptosis was dependent on the severity of virus infection and occurred after the accumulation of HIV envelope glycoproteins. Our results demonstrate that with equivalently expressed levels of CD4 and CXCR4 in cell lines established from CEM cells, relatively high levels of CD26 contribute to an increased rate of HIV entry, infection, and apoptosis. Furthermore, they point out that overexpression of CD26 in a given cell line may lead to a negative effect on HIV infection. Consequently, CD26 appears to regulate HIV entry and apoptosis, processes which are critical for viral pathogenesis.

CD4 Antigens↗
PubMed1998

Specific and irreversible cyclopeptide inhibitors of dipeptidyl peptidase IV activity of the T-cell activation antigen CD26.

The dipeptidyl peptidase IV (DPP IV) activity of CD26 is characterized by its post-proline-cleaving capacity that plays an important but not yet understood role in biological processes. Here we describe a new family of specific and irreversible inhibitors of this enzyme. Taking into account the substrate specificity of DPP IV for P2-P1><-P1' cleavage, we have designed and synthesized cyclopeptides c[(alphaH2N+)-Lys-Pro-Aba-(6-CH2-S+R2)-Glyn] 2TFA- (Aba = 3-aminobenzoic acid, R = alkyl) possessing a proline at the P1 position and a lysine in the P2 position, which allows the closing of the cycle on its side chain. These molecules show a free N-terminus, necessary for binding to the CD26 catalytic site, and a latent quinoniminium methide electrophile, responsible for inactivation. Treatment of c[alphaZ-Lys-Pro-Aba-(6-CH2-OC6H5)-Glyn], obtained by peptide synthesis in solution, with R2S/TFA simutaneously cleaved the Z protecting group and the phenyl ether function and led to a series of cyclopeptide sulfonium salts. These cyclopeptides inhibited rapidly and irreversibly the DPP IV activity of CD26, with IC50 values in the nanomolar range. Further studies were carried out to investigate the effect of the modification of the ring size (n = 2 or 4) and the nature of the sulfur substituents (R = Me, Bu, Oct). Cycle enlargement improved the inhibitory activity of the methylsulfonio cyclopeptide, whereas the increase of the alkyl chain length on the sulfur atom had no apparent effect. Other aminopeptidases were not inhibited, and a much weaker activity was observed on a novel isoform of DPP IV referred to as DPP IV-beta. Thus, this new family of irreversible inhibitors of DPP IV is highly specific to the peptidase activity of CD26.

Animals↗
PubMed1998

Enhanced expression of alpha(1,3)-fucosyltransferase genes correlates with E-selectin-mediated adhesion and metastatic potential of human lung adenocarcinoma cells.

Alpha(1,3)- and alpha(1,4)-fucosylated oligosaccharides such as sialyl-Lewis(x) (sialyl-Le(x)) and sialyl-Lewis(a) (sialyl-Le(a)) have been reported to participate in tumor cell adhesion to activated endothelium. We examined by cytofluorometry the expression of Le(x), sialyl-Le(x), sialyl-Le(x) dimeric, Le(a), and sialyl-Le(a) on the surface of two human lung adenocarcinoma cell lines with different lung colonization potential. High expression levels of all of these antigens were detected in the metastatic HAL-8Luc cells, whereas the closely related nonmetastatic HAL-24Luc cells only expressed the sialyl-Le(a) and sialyl-Le(x) dimeric antigens, both at lower level than in HAL-8Luc cells. Five alpha(1,3)-fucosyltransferases (alpha(1,3)-Fuc-T) controlling the synthesis of these molecules have been identified to date (Fuc-TIII-Fuc-IVII). The expression of these five genes was also higher in the metastatic cells than in the nonmetastatic counterparts as was shown by Northern blot analysis. In vitro adhesion assays showed that only the metastatic cell line adheres significantly to E-selectin-expressing human endothelial cells. Moreover, monoclonal antibody (mAb) blockade of E-selectin completely abolished tumor cell binding. Adhesion inhibition experiments using mAbs against sialylated fucosylated oligosaccharides expressed on tumor cells indicated that these antigens are involved in the binding. Anti-sialyl-Lex(x) mAb (CSLEX-1) inhibited adhesion by 85%; it had the most pronounced inhibitory effect. These findings suggest that the overexpression of alpha(1,3)-Fuc-T genes in the metastatic HAL-8Luc cells, compared with HAL-24Luc cells, results in an enhanced surface display of fucosylated oligosaccharides, which contributes to the adhesive capacity of these cells to the activated endothelium and correlates with their lung colonization potential.

Adenocarcinoma↗
PubMed1998

Serotypes of Escherichia coli isolated from septicaemic chickens in Galicia (northwest Spain).

The purpose of this study was to establish the serogroups of Escherichia coli that cause avian colibacillosis in Spain. The serogroups of 625 avian E. coli isolated between 1992 and 1993 were determined. The 458 E. coli from chickens with septicaemia belonged to 62 different O serogroups; however, 59% were of one of 18 serogroups (O1, O2, O5, O8, O12, O14, O15, O18, O20, O53, O78, O81, O83, O102, O103, O115, O116 and O132). These 18 serogroups were also determined as an important percentage (29%) of control isolates from faeces of healthy birds. Nevertheless, a significant difference (59% versus 29%; P < 0.001) was observed. Furthermore, the serogroups O12, O14, O18, O53, O78, O81, O102, O115, O116 and O132 were almost exclusively identified among septicaemic E. coli (31% versus 3%; P < 0.001). The high prevalence of O18, O81, O115, O116 and O132 isolates was not expected and may indicate the emergence of five new serogroups associated with avian colibacillosis not yet reported.

Animals↗
PubMed1998

Distribution and characterization of faecal necrotoxigenic Escherichia coli CNF1+ and CNF2+ isolated from healthy cows and calves.

Faecal swabs obtained from a random sample of 268 cows and 90 calves on 19 Lugo (northwestern Spain) farms were examined for necrotoxigenic Escherichia coli (NTEC) producing the cytotoxic necrotizing factors type 1 (CNF1) and type 2 (CNF2). We found NTEC CNF1+ and CNF2+ on 11% and 95% of the farms, respectively, NTEC producing CNF2 were significantly more frequently isolated from calves (58%) than from cows (17%) (P < 0.001). The proportion of animals colonized with CNF2+ strains on each farm ranged from 0% to 60%. NTEC strains producing CNF2 isolated from healthy cattle belonged to 27 O serogroups; however, 64% were of one of 12 serogroups (O2, O8, O8-O75, O14, O15, O55, O86, O88, O115, O121, O147, and O168). Furthermore, the serogroups determined in CNF2+ strains isolated from cows (O2, O8, and O14) were different from those found in NTEC producing CNF2 isolated from calves (O8-O75, O15, O55, O86, O88, O115 and O147).

Animals↗
PubMed1998

Prevalence and characteristics of necrotoxigenic Escherichia coli CNF1+ and CNF2+ in healthy cattle.

From February to July of 1994, 328 faecal samples from 32 herds were collected and examined for necrotoxigenic Escherichia coli (NTEC). Strains producing the cytotoxic necrotizing factors type 1 (CNF1) and type 2 (CNF2) were found on 4 and 63% of the farms, respectively. The proportion of animals infected within each herd varied from 0 to 38%. NTEC producing CNF2 were significantly more frequently isolated from calves (24%; 17 of 71) than from cows (4%; 11 of 257) (chi 2corr. 25.088; P < 0.001). Although the bovine CNF2+ strains belonged to 16 different serogroups, 5 (O15, O77, O88, O142 and O153) accounted for 44% of strains. This study confirmed that healthy cattle are a reservoir of NTEC producing CNF2, and revealed that CNF2+ strains are more frequently carried by calves than by adult cows.

Agriculture↗
PubMed1998

Chromosome 21 disomy in the spermatozoa of the fathers of children with trisomy 21, in a population with a high prevalence of Down syndrome: increased incidence in cases of paternal origin.

Between April 1991 and December 1994, epidemiological studies detected a population with a high prevalence of Down syndrome in El Vallès, Spain. Parallel double studies were carried out to determine the parental and the meiotic origins of the trisomy 21, by use of DNA polymorphisms, and to establish the incidence of disomy 21 in the spermatozoa of the fathers of affected children, by use of multicolor FISH. Results show that the overall incidence of chromosome 21 disomy in the fathers of affected children was not significantly different from that in the control population (0.31% vs. 0.37%). However, analysis of individual data demonstrates that two cases (DP-4 and DP-5) with significant increases of disomy 21 (0. 75% and 0.78% vs. 0.37%) correspond to the fathers of the two individuals with Down syndrome of paternal origin. DP-5 also had a significant increase of sex-chromosome disomies (0.69% vs. 0.37%) and of diploid spermatozoa (1.13% vs. 0.24%).

Chromosome Aberrations↗
PubMed1998

Efficiency of MicroSort flow cytometry for producing sperm populations enriched in X- or Y-chromosome haplotypes: a blind trial assessed by double and triple colour fluorescent in-situ hybridization.

Using fluorescent in-situ hybridization (FISH) we have evaluated, on a blind basis, the efficiency of flow cytometry to separate human X- and Y-chromosome bearing spermatozoa. Our data demonstrate that human spermatozoa can be sorted to a purity of 80-90% for X spermatozoa and of 60-70% for Y spermatozoa. Our results using triple FISH fully agree with the sorting treatment used in each case and corroborate the efficiency of the flow sorting technique for sperm sex selection. In these limited samples (200-500 sperm/donor), the frequencies of disomic or diploid spermatozoa were not increased when comparing the sorted samples with unselected samples or with our control series.

Cell Separation↗