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J Sanchez

Publications and source records attributed to J Sanchez.

At least 199 records · Page 11Linked to original sources
PubMed1988

Genetic fusion of a non-toxic heat-stable enterotoxin-related decapeptide antigen to cholera toxin B-subunit.

A decapeptide highly homologous to the STa Escherichia coli heat-stable enterotoxin and to several other heat-stable enterotoxins was fused genetically to the amino-end of the B-subunit of cholera toxin (CTB) and the hybrid protein gene expressed from a tacP overexpression system. The STa-related decapeptide used, which was encoded by a synthetic oligodeoxynucleotide, contained a single mutation which substituted a disulfide-linked cysteine by alanine. After its fusion to CTB the decapeptide was able to both react with and to give rise to anti-STa antibodies. Expression of the decapeptide-CTB hybrid by non-toxigenic Vibrio cholerae resulted in its full secretion into the extracellular milieu from where it could then be readily purified by single-step affinity chromatography using immobilized GM1 ganglioside. Bacteria producing this non-toxic, immunogenic decapeptide-CTB toxoid might be useful for the development of oral vaccines against diarrhea caused by E. coli and other bacteria producing immunologically related heat-stable enterotoxins, and as a source of immunoreagents for methods used to diagnose disease caused by these bacteria.

Amino Acid Sequence↗
PubMed1988

Hybrid enterotoxin LTA::STa proteins and their protection from degradation by in vivo association with B-subunits of Escherichia coli heat-labile enterotoxin.

Chimeric proteins exhibiting antigenic determinants of the heat-labile enterotoxin (LT) and heat-stable (STa) enterotoxins on the same molecule may provide a means to obtain immunoprophylactic and diagnostic reagents for Escherichia coli-caused diarrhea. We recently showed that fusion of two different lengths of the STa gene to the C end of the A-subunit of LT (LTA) results in LTA::STa fusion proteins as monitored by GM1-ELISA [Sanchez et al.: FEBS Lett. 208 (1986) 194-198]. Here we determine the approximate molecular size of the LTA::STa fusion proteins and provide further evidence of their hybrid nature by immunoblot analysis. Using this technique we also demonstrate that to obtain detectable amounts of these recombinant proteins it is essential to coexpress them with the respective B-subunit of LT (LTB). We propose that this dependence on coexpression reflects the association between the LTA::STa hybrids and LTB subunits. The resulting LTA::STa/LTB complexes were found in the E. coli periplasm. This indicated that the exported hybrids, once associated with LTB, were stabilized and formed molecules that behaved essentially as native LT. The protective effect exerted by the B-subunit might conceivably be extended to other LTA-derived hybrid proteins, thus allowing the fusion of other foreign peptides to LTA and their subsequent recovery in the same fashion.

Amino Acid Sequence↗
PubMed1988

In vitro neurite extension by granule neurons is dependent upon astroglial-derived fibroblast growth factor.

When grown in the absence of astroglial cells, purified mouse cerebellar granule neurons survive less than 36 hr and do not extend neurites. Here we report that low concentrations of basic fibroblast growth factor (bFGF, 1-25 ng/ml) maintained the viability and promoted the differentiation of purified granule neurons. The effect of bFGF on granule cell neurite outgrowth was dose dependent. Neurite outgrowth was stimulated markedly in the presence of 1-25 ng/ml bFGF, but effects were not seen below 1 ng/ml or above 50 ng/ml. When affinity-purified antibodies against bFGF (1-5 micrograms/ml) were added either to purified granule cells or to co-cultures of neurons and astroglial cells, process extension by granule neurons was severely impaired. The inhibition of neurite outgrowth in the presence of anti-bFGF antibodies was reversed by the addition of 25 ng/ml of exogenous bFGF. In addition to neuronotrophic effects, bFGF influenced the rate of growth of the astroglial cells. This result depended on whether the astroglia were grown in isolation from neurons, where low doses of bFGF (10-25 ng) stimulated glial growth, or in coculture with neurons, where much higher doses of bFGF (100-250 ng/ml) were needed for glial mitogenesis. Immunoprecipitation of lysates from 35S-labeled cerebellar astroglial cells with anti-bFGF antibodies revealed a single band after SDS-PAGE at 18,000 Da, the molecular weight of bFGF. These results indicate that glial cells synthesize bFGF and are possibly an endogenous source of bFGF in cerebellar cultures. Thus, astroglial cells synthesize soluble factors needed for neuronal differentiation.

Animals↗
PubMed1988

Coordinated assembly of multisubunit proteins: oligomerization of bacterial enterotoxins in vivo and in vitro.

In this paper we study the assembly, in vivo and in vitro, of a family of hexameric, heat-labile enterotoxins produced by diarrheagenic bacteria. The toxins, which consist of an A subunit and five B subunits, are assembled by a highly coordinated process that ensures secretion of the holotoxin complex. We show that (i) oxidation of cysteine residues in the B subunits is a prerequisite step for in vivo formation of B-subunit pentamers, (ii) reduction of dissociated B subunits in vitro abolishes their ability to reassemble, (iii) the kinetics of B-pentamer assembly in vivo can be mimicked under defined conditions in vitro, (iv) A subunits cannot associate with fully assembled B pentamers in vitro, and (v) A subunits cause an approximately 3-fold acceleration in the rate of B-subunit pentamerization in vivo, implying that A subunits play a coordinating role in the pathway of holotoxin assembly. The last finding is likely to be of general significance, since it provides a mechanism for preferentially excluding or favoring certain intermediates in the assembly of multisubunit proteins.

Cysteine↗
PubMed1988

An exocytoplasmic endonuclease with restriction function in Streptomyces antibioticus.

Streptomyces antibioticus produces a strong endo-DNase which is located between the cytoplasmic membrane and the cell wall. All DNA substrates assayed, including the chromosomal DNA of this species and several bacteriophage DNAs, were completely degraded in vitro by the enzyme. The rate of synthesis of the nuclease depended on the growth medium. In NBG medium, in which the enzyme is not produced, the size of lytic plaques of several actinophages was larger than that in GYM or GAE medium, in which synthesis of the nuclease takes place late in growth. In addition, one of the phages assayed, phi A6, showed a diminution of its efficiency of plating in GYM medium with respect to that in NBG medium; another phage, phi A9, grew in NBG medium but not in the other two media. It is postulated that the presence of the host nuclease, together with the capability of the particular phage to absorb on S. antibioticus of different growth phases, determines the efficiency of growth and the plaque size of the phages on productive media. This hypothesis was confirmed when the growth of phi A6 and phi A9 in a mutant of S. antibioticus lacking the endonuclease activity was analyzed. It is concluded that the enzyme can assume, under some circumstances, a role in in vivo restriction.

Bacteriophages↗
PubMed1988

Open cross-over comparison of tulobuterol and fenoterol in asthmatic adult patients.

The objective of this open randomized cross-over study was to compare the clinical efficacy and safety of a recently introduced beta-2 mimetic, tulobuterol, with fenoterol in asthma patients. The study length was four weeks with each drug, with a seven-day washout period between treatment courses. Spirometric tests were carried out every 14 days; laboratory tests and an electrocardiograph were performed at the beginning and end of each treatment course, and a daily diary of salbutamol aerosol use and adverse reactions was kept. Pulmonary function tests and registration of pulse rate and arterial pressure were performed on days 1, 14 and 28 of both treatment courses, before the morning dose and 3 h after administration of the drug. No statistically significant changes were detected in laboratory tests, pulse rate or arterial pressure. The only adverse reaction noted was transient tremor which appeared in three cases with tulobuterol and in two cases with fenoterol. Spirometric tests revealed increases in all parameters with both drugs, although in the comparison between groups no overall statistically significant differences were found. All patients required inhaled salbutamol with both of the drug treatments, and there was a significant increase (p less than 0.05) in its use during the fenoterol treatment course. With both tulobuterol and fenoterol, inhaled salbutamol was mainly used within 2 h before and 1 h after each dose. It is concluded that tulobuterrol (2 mg, twice daily) was at least as effective as fenoterol (2.5 mg, thrice daily), while its clinical effect was longer-lasting. It is doubtful, however, that it provides coverage for 12 h in the type of patients selected.

Adult↗
PubMed1988

Metabolic enzymatic activities in the intercostal and serratus muscles and in the latissimus dorsi of middle-aged normal men and patients with moderate obstructive pulmonary disease.

The glycolytic and oxidative enzyme activities (lactate dehydrogenase (LDH), hexokinase (HK), citrate synthase (CS) and 3-hydroxyacyl-CoA-dehydrogenase (HAD] were measured in the fifth internal and external intercostal muscles, in the vertical and horizontal parts of the serratus, an accessory inspiratory muscle, and in a non-respiratory muscle, the latissimus dorsi (LD) of twenty middle-aged men: nine subjects with normal lung function and eleven patients with moderate chronic obstructive pulmonary disease (COPD). In the normal subjects the enzyme activities of the respiratory muscles were similar to those of the LD, and there were no differences between the internal and the external intercostal muscles. In the COPD patients the metabolic activities of HK, CS and HAD were higher in both intercostals than in LD. Furthermore, there was a significant increase in these enzymatic activities as compared to the intercostals of the normal subjects. These data support the hypothesis that the internal and external intercostal muscles play a more important role in COPD patients than in normal subjects. They are consistent with the hypothesis that COPD has an endurance training effect on both intercostal muscles which could compensate for diaphragmatic disuse.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1987

Mithramycin selectively inhibits transcription of G-C containing DNA.

Mithramycin induces a reversible inhibition of cellular RNA synthesis without affecting DNA synthesis. The authors have shown this drug induces myeloid differentiation of HL-60 promyelocytic leukemia cells and is an effective agent in certain patients with chronic granulocytic leukemia. In order to investigate the mechanism by which this drug inhibits RNA synthesis we have compared the effect of mithramycin on RNA synthesis by whole cells, isolated nuclei, and RNA synthesis by isolated E. coli RNA polymerase and eukaryotic RNA polymerase II. Exposure of HL-60 cells to mithramycin at concentrations of 4.6 X 10(-7) m or higher for 48 hours causes an almost immediate inhibition of RNA synthesis (up to 85% at 4 hours) with only modest cytotoxicity at these concentrations. Endogenous RNA synthesis by isolated nuclei can be inhibited by mithramycin only at high concentrations (greater than 10(-5) m), suggesting that mithramycin primarily may inhibit initiation, rather than elongation. Mithramycin inhibits in vitro transcription of salmon sperm DNA by E. coli RNA polymerase at DNA:drug ratios similar to those required for RNA synthesis inhibition in whole cells. Similar DNA binding studies with synthetic oligonucleotides demonstrate that mithramycin is a potent inhibitor of transcription of Poly dG.dC by E. coli RNA polymerase but has no effect on transcription of Poly dA.dT. The rapid inhibition of whole cell and isolated RNA polymerase transcription, and the relative insensitivity of isolated nuclei, suggest mithramycin may interact with specific DNA sequences in order to inhibit the initiation of RNA synthesis in intact cells.

Base Composition↗
PubMed1986

Immunoactive chimeric ST-LT enterotoxins of Escherichia coli generated by in vitro gene fusion.

Two different lengths of the gene encoding Escherichia coli heat-stable toxin (STa) were fused to the carboxy end of the gene coding for the E. coli heat-labile toxin A-subunit (LTA). The hybrid genes directed expression of chimeric LTA-STa proteins. Association of these chimeras with native heat-labile toxin B-subunit (LTB) resulted in protein complexes that bound to GM1 ganglioside and thereby could be assayed in a GM1 ELISA. The complexes reacted with monoclonal antibodies against either LTA, LTB or STa indicating that the STa and LT epitopes remained immunologically intact after fusion. Genetically constructed chimeric proteins exhibiting LT and STa antigens on the same molecule may represent a promising approach to development of broadly protective immunoprophylactic agents and/or useful immunodiagnostic reagents for diarrhoeal diseases caused by enterotoxinogenic E. coli.

Antibodies, Monoclonal↗
PubMed1986

Ventral herniorrhaphy aided by pneumoperitoneum.

Twenty-four patients with large abdominal incisional hernias were alternately treated with preoperative pneumoperitoneum. The insufflation was performed on an outpatient basis each day for approximately one week prior to operation. The pneumoperitoneum-treated group was spared the necessity of developing abdominal wall flaps and presented a much easier peritoneal dissection. The operative time was 50 minutes in the 12 pneumoperitoneum-treated patients compared with an average of 150 minutes in the standard repair-treated group. There were no infections in the pneumoperitoneum-treated group compared with five (42%) in the other group. The postoperative stay of the pneumoperitoneum-treated group averaged 3.5 days compared with 12.5 days for the standard repair-treated group. Pneumoperitoneum is a valuable adjunct in the repair of large ventral hernias.

Adult↗
PubMed1986

Influence of laying on iron metabolism in quail.

Haematological and iron content values for liver, spleen, intestines, blood, ovary and eggs were determined in female quails, from the prelaying state to full laying. A drop in hematocrit, haemoglobin and in the liver stores occurred at the onset of laying (48 to 51 d of age), but these values recovered after 5 to 10 d. The iron lost in eggs (0.3 mg Fe/egg) was responsible for this. An active homeostatic control must be necessary to supply adequate iron for both eggs and blood formation. Quails fed on an iron-deficient diet distributed iron preferentially to the haematopoietic processes, at the expense of the iron content of the eggs and a subsequent reduction of viability.

Animal Nutritional Physiological Phenomena↗
PubMed1986

Basic mechanisms of metastasis.

Metastatic disease is responsible for the majority of deaths caused by cancer. The process of metastasis is an orderly, stepwise process that results in the selection of cells that possess the capability to establish viable metastases. These cells must be locally invasive and be able to survive the physical traumas of dissemination and normal host defenses. Once metastatic cells have been arrested in a capillary bed, they must be able to invade the host organ parenchyma and survive in that milieu. Studies in a number of model systems have documented the phenotypic alterations in cells that have "metastatic potential." These differences may stem from normal tumor cell heterogeneity and surprisingly reflect only minor differences in gene expression. The role of activated oncogenes in metastasis is unclear, but a number of laboratories have documented that transfection with activated Ha-Ras results in increased metastatic potential. An increased understanding of the genetic basis of metastatic potential may suggest new directions for intervening in this deadly process.

Humans↗
PubMed1986

Retractile mesenteritis involving the colon: barium enema, sonographic, and CT findings.

Retractile mesenteritis is a rare disorder characterized by either focal or diffuse thickening of the mesentery due to chronic inflammation and fibrosis. Six cases are reported in which stenosis of the colon, an uncommon complication of retractile mesenteritis, was the predominant radiologic finding. Diagnosis was suggested by the tapered, serrated appearance of the stenotic segment on barium enema complemented by the findings on sonography and CT.

Adult↗