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J A Gutierrez

Publications and source records attributed to J A Gutierrez.

At least 37 records · Page 2Linked to original sources
PubMed1998

Genetic and biochemical analysis of mutacin 1140, a lantibiotic from Streptococcus mutans.

Streptococcus mutans JH1000 and its derivatives were previously shown (J. D. Hillman, K. P. Johnson, and B. I. Yaphe, Infect. Immun. 44:141-144, 1984) to produce a low-molecular-weight, broad-spectrum bacteriocin-like inhibitory substance (BLIS). The thermosensitive vector pTV1-OK harboring Tn917 was used to isolate a BLIS-deficient mutant, DM25, and the mutated gene was recovered by shotgun cloning in Escherichia coli. Sequence analysis of insert DNA adjacent to Tn917 led to the identification of four open reading frames including two (lanA and lanB) which have substantial homology to the Staphylococcus epidermidis structural gene (epiA) and a modifying enzyme gene (epiB) for biosynthesis of the lantibiotic epidermin, respectively. Although the BLIS activity could not be recovered from broth cultures, high yields were obtained from a solid medium consisting of Todd-Hewitt broth containing 0.5% agarose that was stab inoculated with JH1140 (a spontaneous mutant of JH1000 that produces threefold-elevated amounts of activity). Agar could not substitute for agarose. Chloroform extraction of the spent medium produced a fraction which yielded two major bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The faster-migrating band was absent in chloroform extracts of the mutant, DM25. The amino acid sequence of this band was determined by Edman sequencing and mass spectroscopy. The results showed that it is a lantibiotic, which we have named mutacin 1140, and that the sequence corresponded to that deduced from the lanA sequence. We observed a number of similarities of mutacin 1140 to epidermin and an S. mutans lantibiotic, B-Ny266, but it appears to have significant differences in the positions of its thioether bridges. It also has other unique features with regard to its leader sequence and posttranslational modification. A proposed structure for mutacin 1140 is presented.

Amino Acid Sequence↗
PubMed1998

Mechanical distension modulates pulmonary alveolar epithelial phenotypic expression in vitro.

The pulmonary alveolar epithelium is composed of two distinct types of cells, type I and type II cells, both of which are critical for normal lung function. On the basis of experiments of both nature and in vivo studies, it has been hypothesized that expression of the type I or type II phenotype is influenced by mechanical factors. We have investigated the effects of mechanical distension on the expression of specific markers for the type I and type II cell phenotypes in cultured alveolar type II cells. Rat alveolar type II cells were tonically mechanically distended in culture. Cells were analyzed for a marker for the type I phenotype (rTI40, an integral membrane protein specific for type I cells) and for markers for the type II phenotype [surfactant protein (SP) A, SP-B, and SP-C] as well as for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Mechanical distension caused a 68 +/- 25% (n = 3) increase in mRNA content of rTI40 relative to undistended controls. In contrast, mechanical distension resulted in a decrease in mRNA content of SP-B to 35 +/- 19% (n = 3) and of SP-C to 20 +/- 6.7% (n = 3) of undistended controls. There was no effect on mRNA content of SP-A or GAPDH. The differences in mRNA content of SP-B and SP-C were found to be primarily due to changes at the transcriptional level by nuclear run-on assays. The effects on rTI40 appear to be due to posttranscriptional events. These data show that mechanical distension influences alveolar epithelial phenotypic expression in vitro, at least in part, at the transcriptional level.

Animals↗
PubMed1998

Expression of transforming growth factor-beta complex in arteriovenous malformations.

The factors responsible for the development of cerebral arteriovenous malformations (AVMs) are not well known. Patients with hereditary hemorrhagic telangiectasia (HHT) have cutaneous vascular dysplasia and a high propensity to develop systemic and cerebral AVMs. Transforming growth factor-beta (TGF-beta) complex has been implicated in HHT. The aim of this study was to evaluate the expression of TGF-beta 1, TGF-beta 2, TGF-beta 3, and their two receptors (R1 and R2) in AVMs and in normal brain vessels. Formalin-fixed, paraffin-embedded tissues from 20 patients with cerebral AVMs (including two patients with HHT) were sequentially sectioned into 6 microns sections. Similar sections from normal brain tissue were obtained from five patients without AVMs and no intracranial pathology, who had died from unrelated causes. The normal tissue sections included large intracranial arteries, small arteries, venous sinuses, cortical veins, and brain tissue containing arterioles, capillaries, and venules. All specimens underwent immunohistochemical analyses with polyclonal antibodies to the following antigens: TGF-beta 1, TGF-beta 2, TGF-beta 3, and R1 and R2. The immunoreactivity, when present, was consistently noted in endothelial cells and in the medial smooth muscle. The intensity of vessel wall immunostaining was graded on a scale from 0 to 3. The mean staining grades of normal vessels for TGF-beta 1, TGF-beta 2, TGF-beta 3, R1, and R2 were 0.6 (range 0-1), 3, 2.8 (range 2-3), 1.6 (range 0-2), and 3, respectively, whereas the mean staining grades of AVM vessels were 0.3 (range 0-1), 0.8 (range 0-1), 0.6 (range 0-1), 1.4 (range 0-2), and 0.9 (range 0-1), respectively. The study thus demonstrated that normal brain vessels (arteries, veins, small vessels) have strong (range 2.8-3) immunostaining for TGF-beta 2, TGF-beta 3, and R2, and that the AVM nidus vessels have a paucity (range 0.8-0.9) of staining for these factors. In AVM vessels that had zero immunoreactivity to the above three factors, the vessel wall was fibrocollagenous rather than muscular. Further studies to examine the TGF-beta complex behavior in AVMs are needed.

Antibodies↗
PubMed1997

Functional expression cloning and characterization of SFT, a stimulator of Fe transport.

A stimulator of Fe transport (SFT) was identified by functional expression cloning in Xenopus oocytes. SFT-mediated transport has properties defined for transferrin-independent Fe uptake, but its cytolocalization in recycling endosomes and the observed stimulation of transferrin-bound Fe assimilation indicate a key role in intracellular Fe membrane transport as well. SFT has six predicted transmembranous domains and a functionally important RExxE motif that resembles domains involved in yeast Fe transport and Fe-binding by ferritin L-chains. The observation that SFT oligomerizes, along with other structural and mechanistic features, suggests it may be a member of either the ATP-binding cassette or cation diffusion facilitator families. The 3' untranslated region of SFT contains a translation inhibitory element and inhibition of SFT expression in Xenopus oocytes was found to be relieved by coinjection of transcripts from other defined cDNAs that are also described in this report. SFT is the first component of the mammalian Fe membrane transport machinery to be identified.

Amino Acid Sequence↗
PubMed1997

Role of the citrate pathway in glutamate biosynthesis by Streptococcus mutans.

In work previously reported (J. A. Gutierrez, P. J. Crowley, D. P. Brown, J. D. Hillman, P. Youngman, and A. S. Bleiweis, J. Bacteriol. 178:4166-4175, 1996), a Tn917 transposon-generated mutant of Streptococcus mutans JH1005 unable to synthesize glutamate anaerobically was isolated and the insertion point of the transposon was determined to be in the icd gene encoding isocitrate dehydrogenase (ICDH). The intact icd gene of S. mutans has now been isolated from an S. mutans genomic plasmid library by complementation of an icd mutation in Escherichia coli host strain EB106. Genetic analysis of the complementing plasmid pJG400 revealed an open reading frame (ORF) of 1,182 nucleotides which encoded an enzyme of 393 amino acids with a predicted molecular mass of 43 kDa. The nucleotide sequence contained regions of high (60 to 72%) homology with icd genes from three other bacterial species. Immediately 5' of the icd gene, we discovered an ORF of 1,119 nucleotides in length, designated citZ, encoding a homolog of known citrate synthase genes from other bacteria. This ORF encoded a predicted protein of 372 amino acids with a molecular mass of 43 kDa. Furthermore, plasmid pJG400 was also able to complement a citrate synthase (gltA) mutation of E. coli W620. The enzyme activities of both ICDH, found to be NAD+ dependent, and citrate synthase were measured in cell extracts of wild-type S. mutans and E. coli mutants harboring plasmid pJG400. The region 5' from the citZ gene also revealed a partial ORF encoding 264 carboxy-terminal amino acids of a putative aconitase gene. The genetic and biochemical evidence indicates that S. mutans possesses the enzymes required to convert acetyl coenzyme A and oxalacetate to alpha-ketoglutarate, which is necessary for the synthesis of glutamic acid. Indeed, S. mutans JH1005 was shown to assimilate ammonia as a sole source of nitrogen in minimal medium devoid of organic nitrogen sources.

Aconitate Hydratase↗
PubMed1997

Genetic and physiologic analysis of a formyl-tetrahydrofolate synthetase mutant of Streptococcus mutans.

Previously we reported that transposon Tn917 mutagenesis of Streptococcus mutans JH1005 yielded an isolate detective in its normal ability to produce a mutacin (P. J. Crowley, J. D. Hillman, and A. S. Bleiweis, abstr. D55, p. 258 in Abstracts of the 95th General Meeting of the American Society for Microbiology 1995, 1995). In this report we describe the recovery of the mutated gene by shotgun cloning. Sequence analysis of insert DNA adjacent to Tn917 revealed homology to the gene encoding formyl-tetrahydrofolate synthetase (Fhs) from both prokaryotic and eukaryotic sources. In many bacteria, Fhs catalyzes the formation of 10-formyl-tetrahydrofolate, which is used directly in purine biosynthesis and formylation of Met-tRNA and indirectly in the biosynthesis of methionine, serine, glycine, and thymine. Analysis of the fhs mutant grown anaerobically in a minimal medium demonstrated that the mutant had an absolute dependency only for adenine, although addition of methionine was necessary for normal growth. Coincidently it was discovered that the mutant was sensitive to acidic pH; it grew more slowly than the parent strain on complex medium at pH 5. Complementation of the mutant with an integration vector harboring a copy of fhs restored its ability to grow in minimal medium and at acidic pH as well as to produce mutacin. This represents the first characterization of Fhs in Streptococcus.

Adenine↗
PubMed1997

Incomplete infarct and delayed neuronal death after transient middle cerebral artery occlusion in rats.

BACKGROUND AND PURPOSE: The clinical syndrome of transient ischemic attacks is accompanied in a significant percentage of patients by brain lesions or neuroimaging abnormalities whose structural counterparts have not been defined. The objective of this study was to analyze, in an experimental model of short-term (< 25 minutes) focal ischemia and long-term (< or = 28 days) reperfusion, the extent and nature of the structural abnormalities affecting neurons and glia located within the territory of the transiently occluded artery. METHODS: Adult Wistar rats (n = 121) had the origin of one middle cerebral artery (MCA) occluded with a nylon monofilament for periods of 10 to 25 minutes. Experiments of transient MCA occlusion were terminated at variable periods ranging from 1 day to 4 weeks. Control experiments consisted of (1) MCA occlusion without reperfusion (n = 7) lasting 7 to 14 days and (2) sham operations (n = 2) followed by 1- to 4-day survival. After in situ fixation, brain specimens were serially sectioned and subjected to detailed morphometric evaluations utilizing light and electron microscopes. The statistical method used to evaluate the results was based on ANOVA followed by Bonferroni's corrected t test and Student's t test comparisons. RESULTS: Brain lesions were not detectable in the sham-operated controls. All brains with permanent MCA occlusion (7 to 14 days) had large infarctions with abundant macrophage infiltration and early cavitation. Forty-five (37%) of the experiments involving transient MCA occlusion had no detectable brain lesions after 4 weeks. Selective neuronal necrosis was found in 76 of 121 rats (63%) with transient MCA occlusion. Neuronal necrosis always involved the striatum, and in 29% of the brains with ischemic injury, necrosis also included a short segment of the cortex. In the striatum, the length of the arterial occlusion was the main determinant of the number of necrotic neurons (20 minutes [22.6 +/- 19] is worse than 10 minutes [4.9 +/- 7]) (P < .0001). In the cortex, the length of reperfusion determined the number of necrotic neurons appearing in layer 3. Experiments with reperfusion of 4 to 7 days' duration yielded more necrotic neurons per microscopic field (2.02 +/- 3) than those lasting fewer days (0.04 +/- 0.1) (P < .05). The histological features of these lesions underwent continuous change until the end of the fourth week, at which time necrotic neurons were still visible both in the striatum and in the cortex. CONCLUSIONS: Arterial occlusions of short duration (< 25 minutes) produced, in 76 of 121 experiments (63%), brain lesions characterized by selective neuronal necrosis and various glial responses (or incomplete infarction). This lesion is entirely different from the pannecrosis/cavitation typical of an infarction that appears 3 to 4 days after a prolonged arterial occlusion. Delayed neuronal necrosis, secondary to a transient arterial occlusion or increasing numbers of necrotic neurons in experiments with variable periods of reperfusion, was a response observed only at a predictable segment of the frontoparietal cortex.

Animals↗
PubMed1997

MR of spinal meningeal melanocytoma.

We report an unusual case of meningeal melanocytoma, a rare pigmented lesion of the leptomeninges, that occurred within the leptomeninges of the thoracic spinal canal. The lesion showed increased signal intensity on T1-weighted MR images relative to gray matter, was isointense with gray matter on T2-weighted images, and enhanced mildly but homogeneously after administration of contrast material.

Aged↗
PubMed1997

Superoxide anions contribute to impaired regulation of blood pressure by nitric oxide during the development of cardiomyopathy.

Basal release of endothelium-derived nitric oxide (NO) has been shown to modulate vascular tone and arterial pressure, and may be altered in disease states. The present study was designed to evaluate the role of nitric oxide synthase (NOS) in the maintenance of mean arterial pressure (MAP) and heart rate (HR) in early and advanced stages of cardiomyopathy. MAP and HR were measured via a carotid arterial cannula in conscious, unrestrained male Golden Syrian and Syrian cardiomyopathic hamsters. Studies were performed in young hamsters (age, 60-90 days) at the early phase and old hamsters (age, 300-350 days) at the advanced phase of cardiomyopathy. N-Nitro-L-arginine (LNA; 0.3-30 micromol/kg i.a.), an inhibitor of NOS activity, produced a dose-dependent increase in MAP in YC (young control) and OC (old control) hamsters. The LNA-induced increase in MAP was significantly impaired in YM (young cardiomyopathic) and was abolished in OM (old cardiomyopathic) hamsters compared with control hamsters. Bradycardia in response to LNA was similar in all groups. The effects of LNA on MAP and HR were reversed by L-arginine (200 mg/kg i.a.). Phenylephrine (0.3-300 microg/kg i.a.), an alpha adrenoceptor agonist, produced a dose-dependent increase in MAP which was similar in C and M hamsters at both ages, which indicated that impaired pressor responses to LNA were not caused by a nonspecific alteration in vascular responsiveness of M hamsters. Additionally, L-arginine (100 or 300 mg/kg i.a.), the precursor to NO and sodium nitroprusside (0.3-300 microg/kg i.a.), an NO donor, produced similar effects on MAP and HR in all groups of hamsters. Endothelial NOS protein levels in aorta isolated from each group of hamsters were similar. In the presence of tiron (1000 mg/kg), a superoxide anion scavenger, the effects of LNA on MAP were significantly restored in OM compared with OC hamsters. These results indicate that the role of NO in regulation of MAP is reduced during the development of cardiomyopathy. This effect is not the result of a deficiency of L-arginine, a reduced sensitivity to exogenous NO or a decrease in vascular endothelial NOS protein in cardiomyopathic hamsters. However, scavenging of NO by superoxide anions may contribute to the diminished role of NO in regulation of blood pressure in the advanced stage of cardiomyopathy.

Animals↗
PubMed1996

Insertional mutagenesis and recovery of interrupted genes of Streptococcus mutans by using transposon Tn917: preliminary characterization of mutants displaying acid sensitivity and nutritional requirements.

New vectors were constructed for efficient transposon Tn917-mediated mutagenesis of poorly transformable strains of Streptococcus mutans(pTV1-OK) and subsequent recovery of interrupted genes in Escherichia coli (pT21delta2TetM). In this report, we demonstrate the utility of Tn917 mutagenesis of a poorly transformable strain of S. mutans (JH1005) by showing (i) the conditional replication of pTV1-OK, a repA(Ts) derivative of the broad-host-range plasmid pWVO1 harboring Tn9l7, in JH1005 at the permissive temperature (30 degrees C) versus that at the nonpermissive temperature (45 degrees C); (ii) transposition frequencies similar to those reported for Bacillus subtilis (10(-5) to 10(-4)) with efficient plasmid curing in 90 to 97% of the erythromycin-resistant survivors following a temperature shift to 42 to 45 degrees C; and (iii) the apparent randomness of Tn917 insertion as determined by Southern hybridization analysis and the ability to isolate nutritional mutants, mutants in acid tolerance, and mutants in bacteriocin production, at frequencies ranging from 0.1 to 0.7%. Recovery of transposon-interrupted genes was achieved by two methods: (i) marker rescue in E. coli with the recovery vector pTV21delta2TetM, a tetracycline-resistant and ampicillin-sensitive Tn9l7-pBR322 hybrid, and (ii) "shotgun" cloning of genomic libraries of Tn917 mutants into pUC19. Sequence analyses revealed insertions at five different genetic loci in sequences displaying homologies to Clostridium spp.fhs (66% identity), E. coli dfp (43% identity), and B. subtilis ylxM-ffh (58% identity), icd (citC [69% identity]), and argD (61% identity). Insertions in icd and argD caused nutritional requirements; the one in ylxM-ffh caused acid sensitivity, while those in fhs and dfp caused both acid sensitivity and nutritional requirements. This paper describes the construction of pTV1-OK and demonstrates that it can be efficiently employed to deliver Tn917 into S. mutans for genetic analyses with some degree of randomness and that insertions in the chromosome can be easily recovered for subsequent characterization. This represents the first published report of successful Tn9l7 mutagenesis in the genus Streptococcus.

Bacterial Proteins↗
PubMed1996

Cerebral white matter is highly vulnerable to ischemia.

BACKGROUND AND PURPOSE: The effects of ischemia on the cerebral white matter structure seldom have been studied possibly because white matter is generally considered less vulnerable to ischemia than gray matter. The objective of this study was to evaluate the early (< or = 24 hours) structural effects of experimental focal ischemia on the cerebral white matter of the rat as a preliminary step to investigating human conditions of unknown pathogenesis that are characterized by selective damage to the white matter. METHODS: Twenty-eight rats, including four controls, had a middle cerebral artery occluded with an intravascular filament for periods ranging between 0.5 and 24 hours. Brain samples from the subcortical white matter were examined with light and electron microscopic methods, and the abnormalities were quantified with an image-analysis system. RESULTS: As early as 30 minutes after the arterial occlusion, there was conspicuous swelling of oligodendrocytes and astrocytes; after 3 hours, large numbers of oligodendrocytes were lethally injured. These changes preceded by several hours the appearance of necrotic neurons in the cortex and basal ganglia. Vacuolation and pallor of the white matter were very marked after 24 hours and reflected the segmental swelling of myelinated axons, the formation of spaces between myelin sheaths and axolemma and astrocyte swelling. CONCLUSIONS: These results suggest that the cerebral white matter is highly vulnerable to the effects of focal ischemia. Pathological changes in oligodendrocytes and myelinated axons appear early and seem to be concomitant with, but independent of neuronal perikaryal injury. Modifications of this experimental model of focal ischemia could provide the means to test the hypothesis that selected types of human leukoencephalopathies have an ischemic origin.

Animals↗
PubMed1996

Severe transient hypoglycemia causes reversible change in the apparent diffusion coefficient of water.

BACKGROUND AND PURPOSE: The aim of this study was to determine the effects of temporary severe hypoglycemia on the apparent diffusion coefficient (ADC) acquired by diffusion-weighted MRI of brain water with the use of serial multislice ADC mapping in rats. Severe hypoglycemia reduces the extracellular space volume, as does ischemia. Demonstrating a reduction of ADC with hypoglycemia should increase our understanding of the mechanisms underlying ADC changes in ischemia and other conditions. METHODS: Fasted rats were given regular insulin (15 IU/kg IP). Rats were subjected to 15 minutes (n = 5) and 50 minutes (n = 5) of temporary severe hypoglycemia, causing a transiently isoelectric electroencephalogram (EEG). ADC mapping was performed every 30 seconds beginning at the onset of isoelectricity for 8.5 minutes. ADC maps were also obtained later during the isoelectric EEG period and 10, 20, 30, and 40 minutes after glucose infusion. Control images were obtained from a separate group of animals suffering cardiac arrest (n = 5). RESULTS: Abnormal ADC values were not observed before the onset of cerebral isoelectricity, except for isolated areas in the cortex and periventricular regions. Cortical ADC values globally declined at the onset of EEG isoelectricity. The ADC decline spread to subcortical regions within a few minutes. During the isoelectric period, significant declines of ADC values (27% to 45%) occurred in the entire brain. Glucose infusion normalized most of the ADC changes, even after a 50-minute period of isoelectricity. CONCLUSIONS: ADC mapping during hypoglycemia clearly demonstrates changes likely related to energy depletion. Most of these ADC declines were reversible. Hypoglycemia is a condition known to be associated with shrinkage of the extracellular space. These observations support the hypothesis that ADC reductions observed in ischemia are also related to shifts of water from the extracellular to the intracellular compartment.

Animals↗
PubMed1996

A model to improve the utilization of health and social services in Latino communities.

There are two salient issues beginning to impact drastically on health and social services in the nation: nativist anti-immigrant hysteria among the populace and President Clinton's national health care proposal. This article is neither an examination of the nativist anti-immigrant hysteria nor of the merits of the President's health care plan. Rather, the focus here is to propose a model to improve the utilization of health and social services to Latinos despite nativism and outcome of the final health care program.

California↗
PubMed1996

Molecular mechanisms of iron transport.

The homeostasis of iron transport and absorption represents a critical mechanism to maintain the necessary physiologic balance of this essential nutrient. Recent advances in our understanding of the molecular mechanisms involved in the transport of iron and its membrane translocation are reviewed. Translational and transcriptional control mechanisms that have been characterized to play a role in iron homeostasis are also discussed.

Animals↗
PubMed1996

[Role of the atrioventricular septum and mitro-aortic separation in the pathogenesis of fixed subaortic stenosis].

PURPOSE: To analyze, by cross-sectional echocardiography, morphological features of the atrial atrioventricular and ventricular septum potentially involved in the genesis of fixed subaortic stenosis. METHODS: Forty three children with fixed subaortic stenosis were compared with 86 normal children, matched by age, sex and body surface, and 43 children with congenital heart defects without fixed subaortic stenosis. RESULTS: The groups did not differ in as age, sex or body surface. The atrioventricular septum was significantly smaller in children with subaortic stenosis than in normal children or patients with other congenital heart diseases. The ventricular septum was significantly more aligned with the atrial septum in cases than in normal children and in patients with other congenital heart diseases. The odds ratio for the development of fixed subaortic stenosis was statistically significant in the presence of a short atrioventricular septum and with alignment of the ventricular and atrial septum, when analyzed in isolation or when controlled by perimembranous ventricular septal defect. CONCLUSION: It was concluded that in fixed subaortic stenosis the atrioventricular septum length is decreased and that this alteration may be a risk factor for its development.

Aortic Valve↗