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J Ayala

Publications and source records attributed to J Ayala.

33 records · Page 2Linked to original sources
PubMed1989

Endopeptidase 24.15 from rat testes. Isolation of the enzyme and its specificity toward synthetic and natural peptides, including enkephalin-containing peptides.

Endopeptidase 24.15, a metalloendopeptidase (EC 3.4.24.15) with an Mr of about 70,000, was purified to homogeneity from rat testes. The enzyme cleaves preferentially bonds on the carboxyl side of hydrophobic amino acids. Secondary enzyme-substrate interactions at sites removed from the scissile bond are indicated by the finding that a hydrophobic or bulky residue in the P3' position greatly contributes to substrate binding and catalytic efficiency. The isolated enzyme is inhibited by metal chelators and by thiols. Loss of enzymic activity after dialysis against EDTA can be restored by low concentrations of Zn2+ and Co2+ ions. The rate of reaction of the Co2+ enzyme with a synthetic substrate was higher than that of the Zn2+ enzyme. These results are consistent with the classification of the enzyme as a metalloendopeptidase. N-Carboxymethyl peptides that fulfil the binding requirements of the substrate recognition site of the enzyme act as potent competitive inhibitors. Biologically active peptides such as luteinizing hormone-releasing hormone, bradykinin and neurotensin are cleaved at sites consistent with the specificity of the enzyme deduced from studies with synthetic peptides. Dynorphin A (1-8)-peptide, beta-neoendorphin, metorphamide, and Metenkephalin-Arg6-Gly7-Leu8 are rapidly converted to the corresponding enkephalins. The testis enzyme is catalytically and immunologically closely related to the previously identified brain enzyme.

Animals↗
PubMed1989

Substrate specificity and inhibitors of a capillary injury-related protease from sheep lung lymph.

A serine protease (Mr 70,000 to 75,000) appearing in sheep lung lymph after capillary damage induced by Escherichia coli endotoxin, oleic acid, or air emboli, was studied for its specificity toward a series of synthetic peptide and thioester substrates containing an Arg residue in the P1 position. High specificity constants (kcat/Km) were generally obtained with substrates having two or more basic amino acid residues, and with those having a Gln residues in the P2 position. Secondary enzyme-substrate interactions at sites more removed from the scissile bond are of importance, since a few peptides with two basic residues were hydrolyzed slowly, and the site of cleavage of natural peptides was influenced by the amino acid sequence beyond the immediate vicinity of the hydrolyzed bond. The properties of the enzyme and its pattern of specificity distinguish it from enzymes of the clotting cascade, from components of the complement system, and from lung and skin tryptase. The enzyme was inactivated by p-amidinophenylmethanesulfonyl fluoride and by a series of mechanism-based isocoumarin derivatives, the most potent inhibitor being 4-chloro-7-guanidino-3-(2-phenylethoxy)isocoumarin. Enzyme solutions inactivated by reaction with isocoumarin inhibitors could be completely reactivated after 30 h by treatment with hydroxylamine at neutral pH. Formation of a stable sheep lymph acyl enzyme--in contrast to thrombin and other trypsin-like enzymes--is not followed by alkylation of an active site nucleophile that leads to irreversible enzyme inactivation. The high activity toward substrates with two basic residues suggests that the enzyme may potentially function in processing of precursors of bioactive peptides.

Animals↗
PubMed1989

Developmental and regional regulation of rab3: a new brain specific "ras-like" gene.

Recently, the expression of rab3, a new ras-like gene, has been shown to be restricted to brain tissues (Olofsson et al., 1988). This finding has prompted us to study the expression of rab3 in different brain regions of the developing mouse. The two transcripts corresponding to rab3 (1.8 and 1.3 kb) were first detected in the brains of E13 mouse embryos and were not randomly distributed. Highest levels were found in the mesencephalon, followed by the cortex, striatum, cerebellum, and brain stem in that order. In vitro, the expression of the 1.8-kb transcript was neuron specific, whereas the small transcript was present in neurons and astrocytes. This is the first report showing developmental and regional regulation of a nervous system-restricted ras-like gene. Based on the homologies found between the rab genes and YPT1 or sec-4, we suggest that the physiological role of rab3 might be related to the stabilization of the neuronal cytoskeleton or to post-Golgi vesicle transport and fusion.

Animals↗
PubMed1989

Developmental and regional expression of three new members of the ras-gene family in the mouse brain.

We have examined the expression in the mouse nervous system of three new members of the ras protooncogene family: rab1, rab2, and rab3. Each of these genes was transcribed into messenger RNAs with different molecular weights. These transcripts has specific developmental and regional patterns of expression. In particular, for the three genes, the ratio between the heavy and light mRNAs depended strongly on developmental stage and brain region. The use of pure neuronal and glial cultures revealed that the high molecular weight transcripts were enriched in neurons and that, in the case of rab2 and rab3, their expression increased with neuronal differentiation. These results are discussed considering the sequence identities between these genes and the yeast YPTI and sec-4 genes, which are known to be implicated in post-Golgi vesicular transport and cytoskeletal stabilization. We propose that the rab genes might be of importance in the regulation of these two processes within the developing and adult nervous system.

Aging↗
PubMed1984

Changes in protein synthesis and in RNA poly A+ population after treatment of Dictyostelium amoebae by 5-bromo-2'-deoxyuridine.

Incorporation of 5-bromodeoxyuridine (5-BUdR) into nuclear DNA severely interrupts the life cycle of Dictyostelium discoideum after the first generation of growth. Loose cellular aggregates are then formed, but no spore or stalk cells are detectable and no other morphological transformations are observed. The perturbation of gene expression in the life cycle has been studied at the protein level by two-dimensional gel electrophoresis after pulse labelling with 35S-methionine and also by changes in the patterns of polysomal messenger RNA population. The latter was monitored by hybridisation studies using specific cDNA probes for "vegetative" and "18 hr" messenger RNAs. In the presence of 5-BUdR major anomalies in polypeptide synthesis were observed after the loose aggregation stage. Some vegetative polypeptides, including actin, which are normally abundant only during growth to the aggregation stage, are oversynthesised during the period 12-24 hr after starvation. In this same interval the normal decline in the abundance of vegetative mRNA species was not observed. In marked contrast virtually half the normal "18 hr-specific polypeptides" were poorly synthesised. Likewise, the normal increase in abundance of the corresponding "18 hr-specific" poly A + RNA species in the polysomes did not occur. No major alteration in the timing of the appearance of new macromolecules during the cell cycle was observed in spite of extensive modification of gene expression by the incorporation of 5-BUdR into genomic DNA.

Bromodeoxyuridine↗
PubMed1978

[Gastric duplication with two cysts (author's transl)].

Gastric duplications are rare. The case of a two-year-old girl with double cystic tumors, and a wide connection with the stomach and duodenum, is described and commented upon. A partial resection with excision of the mucosa in the zone closely adhering to the duodenum, and "capitonage" of the raw surface was necessary. A year and a half later, the girl experiences no problems, and the radiological study is practically normal.

Child, Preschool↗
PubMed1977

Activation parameters and molecular changes induced by substrate hydrolysis of the adenosine triphosphatase of Micrococcus lysodeikticus. A comparison of three different soluble forms of the enzyme.

The Arrhenius plots for the active and low activity soluble forms of the ATPase purified from the membranes of Micrococcus lysodeikticus grown at 30 degrees C presented discontinuities at 30 and 33 degrees C, respectively. Their activation parameters differed, being highest for the low activity form of the enzyme. Both forms underwent changes in their molecular properties as a consequence of being enzymically active, i.e., upon incubation with substrates at an adequate temperature. These changes consisted of a decrease in the relative mobilities of some of their subunits in dodecyl sulphate polyacrylamide gel electrophoresis, and the temperature at which they occurred depended on the energy of activation of the particular form of the ATPase used. The low activity form required an incubation temperature of 50 degrees C, whereas for an active form 37 degrees C was sufficient.

Adenosine Triphosphatases↗
PubMed1976

Activation parameters of the adenosine triphosphatase of Micrococcus lysodeikticus. A comparison of the soluble and membrane-bound forms of the enzyme.

The Arrhenius plots for the membrane-bound ATPase and its soluble form purified from Micrococcus lysodeikticus, presented discontinuities near 30 degrees C at pH 7.5. Glycerol-containing lipids were not responsible for these discontinuities. The values of the enthalpies of activation were 12 (soluble) and 22 (membrane-bound) kcal/mol (50.2 and 92.0 kJ/mol) above 30 degrees C and 42 (soluble) and 29 (membrane-bound) kcal/mol (175.7 and 121.3 kJ/mol) below that temperature. The results suggested that both molecular forms of the ATPase were able to adopt at least two different structures, above and below the critical temperature. Of the two, only the high-temperature structure seemed to be enzymically active. In the case of lipid-dependent ATPases, such as the Escherichia coli enzyme, the transition between both enzyme structures probably occurred with simultaneous "melting" of their lipid microenvironment.

Adenosine Triphosphatases↗
PubMedDate not supplied

Brain macrophages synthesize interleukin-1 and interleukin-1 mRNAs in vitro.

Amoeboid microglial cells (brain macrophages) were purified from early post-natal mouse brain cultures. The percentage of cells stained with an anti-Mac-1 antibody was greater than 95%. Stimulation of these brain macrophages by lipopolysaccharides induced the synthesis of interleukin-1 (IL-1), which, in part, remained associated with the cell surface and, in part, was released into the culture medium. In contrast, pure primary astrocyte cultures and cell lines of transformed or immortalised astrocytes did not synthesise significant amounts of IL-1, demonstrating that amoeboid microglia and not astrocytes synthesise IL-1 in vitro. These physiological data were confirmed by RNA hybridisation studies showing that, on LPS treatment, brain macrophages synthesise significant amounts of IL-1 alpha and IL-1 beta mRNAs.

Animals↗
PubMedDate not supplied

[Genetic environment of CTX-M-2 in Klebsiella pneumoniae isolates from hospitalized patients in Uruguay].

We studied two CTX-M-2-producing Klebsiella pneumoniae clinical strains, K96005 and K13, isolated from hospitalized patients in Uruguay, during 1996 and 2003, respectively. The genomic surroundings of bla(CTX-M-2) were characterized by PCR-mapping and DNA sequencing. Our results show that blaCTX-M-2 is included in a complex class-1 integron (InK13), associated with an orf513 in both isolates. The genetic array of the integron, aac(6')-lb, bla(OxA,2), orfD (gene cassette region), associated with an orf513-bla(CTX-M-2), seems to be widely disseminated over the Rio de la Plata region.

Base Sequence↗
PubMedDate not supplied

Serum insulin in clomiphene responders and nonresponders with polycystic ovarian disease.

OBJECTIVE: To investigate insulin levels in women with polycystic ovarian disease (PCOD) who are responders or nonresponders to clomiphene citrate (CC). DESIGN: Open and prospective study. SETTING: Outpatient infertility clinic of a third level medical institution. PATIENTS: Ten healthy women (group 1) and 35 PCOD women classified as responders (group 2 n = 10) or nonresponders (group 3 n = 25) on the basis of serum progesterone > or = 19 nmol/L in response to CC repeatedly administered in doses up to 250 mg/day for five days; they were further subdivided if body mass index (BMI) was below (N) or above (H) mean + 3 SD of group 1. INTERVENTIONS: Blood samples were obtained in a 100-g, 2-hours oral glucose tolerance test (OGTT). MAIN OUTCOME MEASURES: Serum glucose, insulin, free testosterone (free-T), dehydroepiandrosterone sulphate (DHEA-S), and androstenedione (A) were determined in the samples OGTT and the areas under the curve (AUC) were calculated. RESULTS: Group 3 had higher BMI, basal LH, and insulin and AUC insulin than groups 1 and 2; free-T was higher in groups 2 and 3 than in group 1, and basal PRL was higher in group 2 compared to groups 1 and 3. When BMI < or = 25.4 kg/m2 (mean + 1 SD of group 1) 77% of the PCOD cases responded (10 out of 13) whereas none with BMI > 25.4 responded to CC (n = 22) irrespectively of basal insulin concentration or AUC insulin. CONCLUSION: Moderate to excessive overweight seems more frequently and closely associated to a negative CC response in women with PCOD than hyperinsulinemia.

Adult↗