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J Ayala

Publications and source records attributed to J Ayala.

At least 19 recordsLinked to original sources
PubMed2001

Individual somatic H1 subtypes are dispensable for mouse development even in mice lacking the H1(0) replacement subtype.

H1 linker histones are involved in facilitating the folding of chromatin into a 30-nm fiber. Mice contain eight H1 subtypes that differ in amino acid sequence and expression during development. Previous work showed that mice lacking H1(0), the most divergent subtype, develop normally. Examination of chromatin in H1(0-/-) mice showed that other H1s, especially H1c, H1d, and H1e, compensate for the loss of H1(0) to maintain a normal H1-to-nucleosome stoichiometry, even in tissues that normally contain abundant amounts of H1(0) (A. M. Sirotkin et al., Proc. Natl. Acad. Sci. USA 92:6434-6438, 1995). To further investigate the in vivo role of individual mammalian H1s in development, we generated mice lacking H1c, H1d, or H1e by homologous recombination in mouse embryonic stem cells. Mice lacking any one of these H1 subtypes grew and reproduced normally and did not exhibit any obvious phenotype. To determine whether one of these H1s, in particular, was responsible for the compensation present in H1(0-/-) mice, each of the three H1 knockout mouse lines was bred with H1(0) knockout mice to generate H1c/H1(0), H1d/H1(0), or H1e/H1(0) double-knockout mice. Each of these doubly H1-deficient mice also was fertile and exhibited no anatomic or histological abnormalities. Chromatin from the three double-knockout strains showed no significant change in the ratio of total H1 to nucleosomes. These results suggest that any individual H1 subtype is dispensable for mouse development and that loss of even two subtypes is tolerated if a normal H1-to-nucleosome stoichiometry is maintained. Multiple compound H1 knockouts will probably be needed to disrupt the compensation within this multigene family.

Animals↗
PubMed1999

Immunohistochemical localization of types 1 and 2 5alpha-reductase in human scalp.

The predominant form of 5alpha-reductase (5aR) in human scalp is 5aR1. None the less, clinical studies have shown that finasteride, a selective inhibitor of 5aR2, decreases scalp dihydrotestosterone and promotes hair growth in men with androgenetic alopecia. Immunolocalization studies were thus carried out to examine 5aR isozyme distribution within scalp and, in particular, to determine whether 5aR2 might be associated with hair follicles. 5aR2 was localized using both a rabbit polyclonal and a mouse monoclonal antibody. 5aR1 was detected with a mouse monoclonal antibody. The specificity of these reagents was demonstrated both by immunofluorescence and Western blot analyses of COS cells overexpressing human 5aR1 or 5aR2. When cryosections of scalp from men with androgenetic alopecia were stained with antibody against 5aR2, using immunoperoxidase avidin-biotin complex methodology, immunostaining was observed in the inner layer of the outer root sheath and, in more proximal regions of the follicle, in the inner root sheath. Staining was also prominent in the infundibular region of the follicle, with less intense staining extending throughout the granular layer of the epidermis. Some staining was also seen in sebaceous ducts. Similar results were obtained with both the polyclonal and monoclonal 5aR2 antibodies. In contrast, in scalp cryosections stained with antibody to 5aR1, no immunostaining was observed within hair follicles. Intense staining for the type 1 isozyme was, however, detected within sebaceous glands. Our immunolocalization data suggest that the results seen in clinical trials of men with male pattern hair loss treated with finasteride may be due, at least in part, to local inhibition of 5aR2 within the hair follicle.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1998

Endoscopic-assisted temporoparietal fascial flap dissection and harvesting: a feasibility preliminary cadaveric study.

Endoscopic procedures in plastic surgery have been applied in various aesthetic and reconstructive surgical techniques. The authors describe, in this preliminary report, a new surgical technique of endoscopic dissection of the temporoparietal fascial flap. A series of 6 fresh human cadavers (12 flaps) were dissected endoscopically. The surgical incisions, flap anatomy applicable to endoscopy, endoscopic surgical technique, and type of endoscopic setup is standardized for all flaps, allowing direct identification of the temporoparietal fascial layers and the major vascular pedicle. This endoscopic manipulation of the flap, without the traditional large scalp incision, permits local and free transfer of the temporoparietal fascial flap. Exposure of the flap by means of the conventional T or Y temporal incisions has several possible disadvantages, including an increased risk of blood loss, alopecia, and facial nerve injury. Endoscopic dissection and mobilization of the temporoparietal fascial flap can obviate the direct flap incision and yield a flap accessible to dissection and mobilization for additional clinical applications. This new, innovative, and minimally invasive endoscopic procedure may prove particularly applicable to future clinical applications of this type of fascial flap.

Cadaver↗
PubMed1998

Contribution of the Pmra promoter to expression of genes in the Escherichia coli mra cluster of cell envelope biosynthesis and cell division genes.

Recently, a promoter for the essential gene ftsI, which encodes penicillin-binding protein 3 of Escherichia coli, was precisely localized 1.9 kb upstream from this gene, at the beginning of the mra cluster of cell division and cell envelope biosynthesis genes (H. Hara, S. Yasuda, K. Horiuchi, and J. T. Park, J. Bacteriol. 179:5802-5811, 1997). Disruption of this promoter (Pmra) on the chromosome and its replacement by the lac promoter (Pmra::Plac) led to isopropyl-beta-D-thiogalactopyranoside (IPTG)-dependent cells that lysed in the absence of inducer, a defect which was complemented only when the whole region from Pmra to ftsW, the fifth gene downstream from ftsI, was provided in trans on a plasmid. In the present work, the levels of various proteins involved in peptidoglycan synthesis and cell division were precisely determined in cells in which Pmra::Plac promoter expression was repressed or fully induced. It was confirmed that the Pmra promoter is required for expression of the first nine genes of the mra cluster: mraZ (orfC), mraW (orfB), ftsL (mraR), ftsI, murE, murF, mraY, murD, and ftsW. Interestingly, three- to sixfold-decreased levels of MurG and MurC enzymes were observed in uninduced Pmra::Plac cells. This was correlated with an accumulation of the nucleotide precursors UDP-N-acetylglucosamine and UDP-N-acetylmuramic acid, substrates of these enzymes, and with a depletion of the pool of UDP-N-acetylmuramyl pentapeptide, resulting in decreased cell wall peptidoglycan synthesis. Moreover, the expression of ftsZ, the penultimate gene from this cluster, was significantly reduced when Pmra expression was repressed. It was concluded that the transcription of the genes located downstream from ftsW in the mra cluster, from murG to ftsZ, is also mainly (but not exclusively) dependent on the Pmra promoter.

Cell Division↗
PubMed1996

The non-penicillin-binding module of the tripartite penicillin-binding protein 3 of Escherichia coli is required for folding and/or stability of the penicillin-binding module and the membrane-anchoring module confers cell septation activity on the folded structure.

The ftsI-encoded multimodular class B penicillin-binding protein 3 (PBP3) is a key element of the cell septation machinery of Escherichia coli. Altered ftsI genes were overexpressed, and the gene products were analyzed with respect to the level of production, stability, penicillin affinity, and cell septation activity. In contrast to the serine beta-lactamases and low-molecular-mass PBPs which are autonomous folding entities, the S-259-to-V-577 penicillin-binding module of M-1-to-V-577 PBP3 lacks the amino acid sequence information for correct folding. The missing piece of information is provided by the associated G-57-to-E-258 non-penicillin-binding module which functions as a noncleaved, pseudointramolecular chaperone. Key elements of the folding information reside within the motif 1-containing R-60-to-W-110 polypeptide segment and within G-188-to-D-197 motif 3 of the n-PB module. The intermodule interaction is discussed in the light of the known three-dimensional structure (at 3.5-A [0.35-nm] resolution) of the analogous class B PBP2x of Streptococcus pneumoniae (S. Pares, N. Mouz, Y. Pétillot, R. Hakenbeck, and O. Dideberg, Nature Struct. Biol. 3:284-289, 1996). Correct folding and adoption of a stable penicillin-binding conformation are necessary but not sufficient to confer cell septation activity to PBP3 in exponentially growing cells. The in vivo activity of PBP3 also depends on the M-1-to-E-56 amino-terminal module which encompasses the cytosol, the membrane, and the periplasm and which functions as a noncleaved pseudo-signal peptide.

Amino Acid Sequence↗
PubMed1995

Cell-mediated catabolism of aggrecan. Evidence that cleavage at the "aggrecanase" site (Glu373-Ala374) is a primary event in proteolysis of the interglobular domain.

A rat chondrosarcoma cell line and primary bovine chondrocytes have been used to study cell-mediated aggrecan catabolism. Addition of 1 microM retinoic acid to chondrosarcoma cultures resulted in aggrecan proteolysis with the release of greater than 90% of the cell layer aggrecan into the medium within 4 days. NH2-terminal sequencing of chondroitin sulfate-substituted catabolic products gave a single major NH2-terminal sequence of ARGNVILTXK, initiating at Ala374. This showed that the proteinase, commonly referred to as "aggrecanase," which cleaves the Glu373-Ala374 bond of the interglobular domain of aggrecan (Sandy, J. D., Neame, P. J., Boynton, R. E., and Flannery, C. R. (1990) J. Biol. Chem. 266, 8683-8685), is active in this cell system. Aggrecan G1 domain, generated by cleavage of the interglobular domain, was also liberated during catabolism and this was characterized with three antipeptide antisera. Anti-CDAGWL was used as a general probe for G1 domain. Anti-FVDIPEN was used to specifically detect G1 domain with COOH terminus of Asn341, the form which is readily generated by cleavage of aggrecan by a wide range of matrix metalloproteinases. Anti-NITEGE antiserum was used to specifically detect G1 domain with COOH terminus of Gln373, the form which is the expected product of "aggrecanase"-mediated cleavage of aggrecan. Western blot analysis indicated that a single form of G1 domain of about 60 kDa was formed. G1 domain of this size reacted with both anti-CDAGWL and anti-NITEGE but not with anti-FVDIPEN. Similar experiments with primary bovine chondrocyte cultures, treated with either retinoic acid or interleukin 1, showed that two forms of catabolic G1 domain, of about 62 and 66 kDa, were formed. Both of these forms reacted on Western blots with anti-CDAGWL and also with anti-NITEGE. It is suggested that cell-mediated catabolism of the aggrecan interglobular domain in these culture systems, whether promoted by retinoic acid or interleukin 1, primarily involves cleavage of the Glu373-Ala374 bond by aggrecanase. The accumulation of G1 domain with a COOH-terminal of Glu373 shows that such aggrecanase-mediated cleavage can occur independent of the cleavage of the Asn341-Phe342 bond by matrix metalloproteinases.

Aggrecans↗
PubMed1995

Opposite effects of breakfast vs. oral glucose on circulating androgen levels in healthy women.

Since certain hormone abnormalities can be food-dependent as in some cases of nodular adrenal hyperplasia, as a first step to study other more frequent endocrine disorders, using a standard meal as means of a more physiologic stimulus, we investigated in healthy women whether or not the changes in serum glucose, insulin, cortisol, and androgens are different following a breakfast than after oral glucose alone. Ten women (group 1) ingested a 725 kilocalories (173 kiloJoules) breakfast and ten women (group 2) ingested a 100-g glucose load at 8:00 A.M. Serum glucose, insulin, cortisol, dehydroepiandrosterone sulfate (DHEAS), free testosterone (free-T), and androstenedione (A) were determined every 30 min for the next 2 h. Serum glucose rose higher in group 2 than in group 1 (p < or = 0.05), yet insulin increased similarly in both groups. In group 1 free-T and DHEAS increased at 30 min (p < or = 0.05) while cortisol decreased (p < or = 0.04); A did not change. On the contrary, in group 2 only a decrease in free-T (p < or = 0.01) was detected. In these healthy women, serum androgen changed inversely depending on the type and composition of the oral stimulus used. The simultaneous increase of serum insulin, free-T and DHEAS coinciding with a cortisol decrease may enhance insulin anabolic effects following breakfast, but not after oral glucose alone.

Administration, Oral↗
PubMed1994

Gene sequence and biochemical characterization of FOX-1 from Klebsiella pneumoniae, a new AmpC-type plasmid-mediated beta-lactamase with two molecular variants.

Klebsiella pneumoniae BA32, a clinical isolate from Buenos Aires, Argentina, was found to produce a plasmid-encoded beta-lactamase (FOX-1) which conferred resistance to broad-spectrum cephalosporins and cephamycins. Resistance could be transferred by conjugation or transformation into Escherichia coli K-12 via a 48.5-kb plasmid (pGLK1) that produced two FOX-1 molecular variants with isoelectric points of 6.8 and 7.2 and apparent molecular sizes of 37 and 35 kDa, respectively. The kinetic study revealed that the two variants had very similar substrate and inhibition profiles. These values resemble those of chromosomally mediated class C (group 1) cephalosporinases. The structural gene of FOX-1 (blaFOX-1) was cloned into a 2,270-bp PstI-PstI fragment and was expressed in E. coli TG1. The deduced 382-amino-acid sequence of FOX-1 exhibited a high degree of similarity with chromosomally encoded AmpC beta-lactamases of Pseudomonas aeruginosa, Serratia marcescens, Enterobacter cloacae, E. coli, and Citrobacter freundii. These findings suggest that FOX-1 is a plasmid-mediated AmpC-type beta-lactamase that is encoded by a single gene and that has two molecular variants.

Aged↗
PubMed1994

Rab3A and Rab3B carboxy-terminal peptides are both potent and specific inhibitors of prolactin release by rat cultured anterior pituitary cells.

Chimeric polypeptides composed of the homeodomain of Antennapedia and of the C-terminus of several low molecular weight GTP-binding proteins of the rab family have been found to translocate through the membrane of cells in culture and to accumulate in the cytoplasm and nucleus. We have used these chimeric peptides to study, in intact endocrine cells, a putative role for the C-terminal domain of rab proteins in the exocytotic process. We show that the internalization of 33- and 32-amino acid polypeptides corresponding to the C-terminal domains of rab3A and rab3B blocks calcium-triggered PRL release from adult rat anterior pituitary cells maintained in primary culture. This effect is specific to rab3 since it is not observed after internalization of either rab1 or rab2 C-terminal peptides. In addition, we demonstrate that this inhibition does not require the geranylgeranylation of the internalized C-termini. As rab3B normally shows a permissive effect on exocytosis in PRL-secreting cells, we suggest that the C-terminal domains of rab3A and rab3B contain structural elements that compete with endogenous rab3 necessary for calcium-induced exocytosis.

Amino Acid Sequence↗
PubMed1994

Dengue virus. Detection and pathogeny.

Sequences front a cDNA of dengue virus type 4 were cloned into transcription vectors. These sequences included the E, NS1, NS2A, NS2B, NS3 genes. RNA transcripts produced in vitro from these plasmids were used in hybridization assays to detect dengue viral sequences. With these RNA-probes we have been able to detect molecules of serotype-specific dengue 4 viral RNA. Moreover, the riboprobes detected viral sequences of other serotypes in the following order of sensitivity 4 > 2 > 3 > 1, and might be useful to differentiate serotypes.

Animals↗
PubMed1993

Changes in hypothalamic met-enkephalin levels of rats induced by painful stimulation and morphine treatment.

The possible influence of painful stimulation and morphine analgesia on hypothalamic met-enkephalin levels, and the possible correlation between this biochemical parameter and animal behaviour was studied. The results indicate that both painful stimulation and morphine treatment induce an increase in hypothalamic metenkephalin. On the other hand, naloxone did not cause any variation. In animals submitted to painful stimulus, morphine treatment induced a decrease in hypothalamic met-enkephalin levels. Finally, when both morphine and naloxone were administrated before nociceptive stimulation, much higher levels were measured. There was no correlation between the level of hypothalamic met-enkephalin and the pain rating.

Animals↗
PubMed1992

Membrane intermediates in the peptidoglycan metabolism of Escherichia coli: possible roles of PBP 1b and PBP 3.

The two membrane precursors (pentapeptide lipids I and II) of peptidoglycan are present in Escherichia coli at cell copy numbers no higher than 700 and 2,000 respectively. Conditions were determined for an optimal accumulation of pentapeptide lipid II from UDP-MurNAc-pentapeptide in a cell-free system and for its isolation and purification. When UDP-MurNAc-tripeptide was used in the accumulation reaction, tripeptide lipid II was formed, and it was isolated and purified. Both lipids II were compared as substrates in the in vitro polymerization by transglycosylation assayed with PBP 1b or PBP 3. With PBP 1b, tripeptide lipid II was used as efficiently as pentapeptide lipid II. It should be stressed that the in vitro PBP 1b activity accounts for at best to 2 to 3% of the in vivo synthesis. With PBP 3, no polymerization was observed with either substrate. Furthermore, tripeptide lipid II was detected in D-cycloserine-treated cells, and its possible in vivo use in peptidoglycan formation is discussed. In particular, it is speculated that the transglycosylase activity of PBP 1b could be coupled with the transpeptidase activity of PBP 3, using mainly tripeptide lipid II as precursor.

Bacterial Proteins↗
PubMed1991

Modulation of interleukin-1 and tumor necrosis factor expression by beta-adrenergic agonists in mouse ameboid microglial cells.

Brain macrophages (ameboid microglial cells) purified to homogeneity and cultured in vitro synthesize and release IL-1 and TNF upon stimulation with lipopolysaccharide (LPS). This induction can be measured at the levels of transcription and translation. In the present study we have analysed whether certain compounds normally present in the nervous tissue could regulate cytokine production by brain macrophages. We demonstrate that the beta-adrenergic agonist isoproterenol, at a concentration of 10(-7) M; inhibits the LPS-induced transcription and release of TNF alpha. At the same concentration, isoproterenol increases the accumulation of IL-1 alpha and IL-1 beta mRNAs. In spite of its strong effect on IL-1 mRNA accumulation, the adrenergic agonist did not enhance IL-1 activity produced by microglial cells. On the contrary, as is the case for TNF, the LPS-induced production of IL-1 was inhibited by isoproterenol. The effects of isoproterenol on cytokine production specifically involve the beta 2 and not the beta 1 adrenergic receptor. It thus appears (i) that the accumulation of mRNAs coding for TNF alpha on one hand and IL-1 alpha and beta on the other is regulated in two opposite ways by the stimulation of the beta 2-adrenergic receptor and (ii) that mRNA accumulation and cytokine production and secretion are not necessarily coupled.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1991

Occurrence and evolution of T3 fibroblast nuclei in healing tendons from growing chicks.

We applied immunocytochemical techniques including anti-triiodothyronine (T3) serum and microscopic analysis with colloid gold as the developer to identify the specific receptors of thyroid hormones located within the cell nucleus and between the heteroeuchromatin transition zone in 25 chicks. All chicks underwent tenotomy, followed by tenorrhaphy and immobilisation of the leg in plaster. There was a gradual decrease in the number of T3 receptors of collagen-forming fibroblasts as the tendons healed and their capacity to synthetize collagen diminished.

Animals↗
PubMed1990

The product of rab2, a small GTP binding protein, increases neuronal adhesion, and neurite growth in vitro.

The rab genes code for small GTP binding proteins that share with p21ras the ability to bind and hydrolyze GTP. They present significant sequence homologies with the products of YPT1 and SEC4, two small GTP binding proteins involved in the regulation of secretion in the yeast. Several rab genes are expressed in the developing and adult mouse brain. To test directly the possible involvement of these genes in neuronal differentiation, purified rab proteins produced in E. coli were introduced into neurons dissociated from E15 rat midbrain. The most striking effects were obtained with rab2 protein (rab2p). Compared with untreated cells, neurons loaded with rab2p presented an enhanced adhesion to the culture substratum. This phenomenon was visible 3 hr after seeding and was followed within 24 hr by a dramatic increase in neurite growth. Loading the same population of neurons with the products of four other rab genes either decreased neuronal adhesion and neurite growth or had no effect. These experiments suggest that the expression of rab2p plays an important role in neuronal differentiation.

Animals↗