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C Garcia

Publications and source records attributed to C Garcia.

At least 19 recordsLinked to original sources
PubMed1995

Solution structure of the ribosome-binding domain of E. coli translation initiation factor IF3. Homology with the U1A protein of the eukaryotic spliceosome.

Initiation of translation in prokaryotes requires the formation of a complex between the messenger RNA, the 30 S ribosomal subunit and the initiator tRNA(fMet). Initiation factor IF3 binds to the 30 S ribosomal subunit and proof-reads the initiation complex, thereby ensuring the accuracy of this step. IF3 also plays a pleiotropic role in the regulation of translation, as a result of differential influences exerted on the levels of the initiation of translation of genes or groups of genes. IF3 is composed of two independent domain or roughly identical sizes. We have expressed and purified the C-terminal domain of E. coli IF3 and shown that it retains both the 30 S particle binding and 70 S ribosome dissociating activities of the native protein. We have obtained 1H and 15N NMR resonance assignments and its 3D solution structure was calculated using 551 restraints. It is composed of a mixed beta-sheet backed by two alpha-helices. It shows a striking resemblance to the U1A small nuclear ribonucleoprotein structure, which binds to the U1 snRNA in the eukaryotic spliceosome. This suggests a convergent evolution process for these two proteins that are associated with ribonucleoproteic complexes.

Amino Acid Sequence↗
PubMed1995

1H and 15N resonance assignments and structure of the N-terminal domain of Escherichia coli initiation factor 3.

Initiation factor IF3 from Escherichia coli is composed of two domains connected by a hydrophilic peptide. In this study, the N-terminal domain (residues 7-83) has been overexpressed, 15N labelled and purified. NMR assignments for this domain have been obtained by two-dimensional and three-dimensional heteronuclear and homonuclear spectroscopy. Using distance geometry and simulated annealing, a three-dimensional solution structure was calculated using 506 NOE and 56 dihedral angle restraints. The resulting structure is composed of a five-stranded antiparallel beta sheet surrounded by two alpha helices. Since the heteronuclear 1H-15N correlation spectrum of the N-terminal domain of IF3 is an almost exact subset of that of the native protein, the assignments obtained and the structure calculated should be directly transposable to the full-length protein.

Amino Acid Sequence↗
PubMed1995

Hypermutation generating the sheep immunoglobulin repertoire is an antigen-independent process.

Somatic hypermutation of light chain V genes during development of B cells in sheep ileal Peyer's patches was studied in three experimental conditions: in sterile fragments of the ileum surgically isolated from the gut during fetal life, in germ-free sheep, and in animals thymectomized during early fetal life. The somatic mutation pattern was found identical to control tissues in all three experiments. The same age-dependent amount of mutations, a higher than theoretical R/S ratio in complementarity-determining regions (CDRs), and a similar clustering of mutations in CDRs were observed. The mechanism, as estimated from the silent mutation pattern, appears to target mutations to CDRs; moreover, the major V lambda genes have a specific codon usage with a high purine content at the first two bases of the codons and a low content at the third position, which, together with a specific targeting of mutations to purines, favors replacement mutations in CDRs.

Animals↗
PubMed1995

Description of an in vitro platelet function analyzer--PFA-100.

A new in vitro system for the detection of platelet dysfunction, PFA-100, has been developed. It provides a quantitative measure of platelet function in anticoagulated whole blood. The system comprises a microprocessor-controlled instrument and a disposable test cartridge containing a biologically active membrane. The instrument aspirates a blood sample under constant vacuum from the sample reservoir through a capillary and a microscopic aperture cut into the membrane. The membrane is coated with collagen and epinephrine or adenosine 5'-diphosphate. The presence of these biochemical stimuli, and the high shear rates generated under the standardized flow conditions, result in platelet attachment, activation, and aggregation, slowly building a stable platelet plug at the aperture. The time required to obtain full occlusion of the aperture is reported as the "closure time." We have found that impairment of von Willebrand factor, or inhibition of platelet receptors glycoprotein Ib or IIb/IIIa with monoclonal antibodies or peptides, resulted in abnormal closure times. An antifibrinogen antibody, in contrast, failed to show any effect. The test appears to be sensitive to platelet adherence and aggregation abnormalities. The PFA-100 system has potential applications in routine evaluation of platelet function in the clinical setting because of its accuracy, ease of operation, and rapid turnaround of results.

Bleeding Time↗
PubMed1995

Sequence and domain organization of scruin, an actin-cross-linking protein in the acrosomal process of Limulus sperm.

The acrosomal process of Limulus sperm is an 80-microns long finger of membrane supported by a crystalline bundle of actin filaments. The filaments in this bundle are crosslinked by a 102-kD protein, scruin present in a 1:1 molar ratio with actin. Recent image reconstruction of scruin decorated actin filaments at 13-A resolution shows that scruin is organized into two equally sized domains bound to separate actin subunits in the same filament. We have cloned and sequenced the gene for scruin from a Limulus testes cDNA library. The deduced amino acid sequence of scruin reflects the domain organization of scruin: it consists of a tandem pair of homologous domains joined by a linker region. The domain organization of scruin is confirmed by limited proteolysis of the purified acrosomal process. Three different proteases cleave the native protein in a 5-kD Protease-sensitive region in the middle of the molecule to generate an NH2-terminal 47-kD and a COOH-terminal 56-kD protease-resistant domains. Although the protein sequence of scruin has no homology to any known actin-binding protein, it has similarities to several proteins, including four open reading frames of unknown function in poxviruses, as well as kelch, a Drosophila protein localized to actin-rich ring canals. All proteins that show homologies to scruin are characterized by the presence of an approximately 50-amino acid residue motif that is repeated between two and seven times. Crystallographic studies reveal this motif represents a four beta-stranded fold that is characteristic of the "superbarrel" structural fold found in the sialidase family of proteins. These results suggest that the two domains of scruin seen in EM reconstructions are superbarrel folds, and they present the possibility that other members of this family may also bind actin.

Acrosome↗
PubMed1995

Gastric mucosal pH and oxygen delivery and oxygen consumption indices in the assessment of adequacy of resuscitation after trauma: a prospective, randomized study.

OBJECTIVE: To compare gastric mucosal pH (pHi) and global oxygen variables [Oxygen Delivery Index (DO2I) and Oxygen Consumption Index (VO2I)] as indicators of adequacy of resuscitation after major trauma. METHODS: Twenty-seven patients were prospectively randomized into two groups: group 1 (n = 11), normalization and maintenance of pHi at or above 7.30; and group 2 (n = 16), maintaining a DO2I of 600 and a VO2I of > 150. The groups had statistically similar injury severity scores, lactate, and base deficit. RESULTS: The goals of therapy were achieved within 24 hours of admission in 10 of the 11 patients in group 1 and in 15 of the 16 patients in group 2. One patient (9.1%) in group 1 died. This patient had transient stabilization of pHi to 7.3 and subsequently had persistent mucosal acidosis. Of the 10 patients with pHi > 7.3 at 24 hours, 9 survived. In group 2, 5 (31.3%) died. Four of the 5 nonsurvivors had achieved DO2I and VO2I goals, but had pHi < 7.3 at 24 hours. A comparison of time taken for optimization of DO2I, VO2I, lactate, base excess, and pHi showed pHi and lactate as the variables different in survivors and nonsurvivors. Six of the 8 patients who developed multiple organ dysfunction syndrome had pHi < 7.3 at 24 hours. Persistently low pHi was the first sign of bacteremia (3 patients), small bowel gangrene or pregangrene (2 patients), intestinal anastomotic leak (2 patients), intra-abdominal hypertension (4 patients), and intra-abdominal abscess (5 patients). It was the first finding in all the nonsurvivors at least 72 hours before death. CONCLUSIONS: pHi may be an important marker to assess the adequacy of resuscitation. pHi monitoring may provide early warning for systemic complications in the postresuscitation period.

Adolescent↗
PubMed1995

Effect of SR-49059, a vasopressin V1a antagonist, on human vascular smooth muscle cells.

The effects of SR-49059, a new nonpeptide and selective arginine vasopressin (AVP) V1a antagonist, were investigated in binding and functional studies on cultured human aortic vascular smooth muscle cells (VSMC). Characterization of human vascular V1a receptors, using a specific V1a radioiodinated ligand, showed that [125I]-linear AVP antagonist binding to human VSMC membranes was time dependent, reversible, and saturable. A single population of high-affinity binding sites (apparent equilibrium dissociation constant = 15 +/- 6 pM; maximum binding density = 36 +/- 5 fmol/mg protein, i.e., approximately 3,000 sites/cell) with the expected V1a profile was identified. Exposure of these cells to AVP dose-dependently produced cytosolic free [Ca2+] increase [AVP concentration required to obtain a half-maximal response (EC50) = 23 +/- 9 nM] and proliferation (EC50 = 3.2 +/- 0.5 nM). SR-49059 strongly and stereospecifically inhibited [125I]-linear AVP antagonist binding to VSMC V1a receptors [inhibition constant (Ki) = 1.4 +/- 0.3 nM], AVP-evoked Ca2+ increase [concentration of inhibitor required to obtain 50% inhibition of specific binding (IC50) = 0.41 +/- 0.06 nM], and the mitogenic effects induced by 100 nM AVP (IC50 = 0.83 +/- 0.04 nM). OPC-21268, another nonpeptide V1a antagonist, was more than two orders of magnitude less potent than SR-49059 in these models. However, the consistent affinity (Ki = 138 +/- 21 nM) and activity found with OPC-21268 on human VSMC in comparison with the inactivity already observed for other human V1a receptors (liver, platelets, adrenals, and uterus) strongly suggested the existence of human AVP V1a-receptor subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)

Aorta↗
PubMed1995

Genetic and biochemical normalization in female carriers of Duchenne muscular dystrophy: evidence for failure of dystrophin production in dystrophin-competent myonuclei.

We studied 19 symptomatic female carriers of the Duchenne muscular dystrophy (DMD) gene. Most of these dystrophinopathy patients had had an erroneous or ambiguous diagnosis prior to dystrophin immunofluorescence testing. We assessed clinical severity by a standardized protocol, measured X-chromosome inactivation patterns in blood and muscle DNA, and quantitated the dystrophin protein content of muscle. We found that patients could be separated into two groups: those showing equal numbers of normal and mutant dystrophin genes in peripheral blood DNA ("random" X-inactivation), and those showing preferential use of the mutant dystrophin gene ("skewed" X-inactivation). In the random X-inactivation carriers, the clinical phenotype ranged from asymptomatic to mild disability, the dystrophin content of muscle was > 60% of normal, and there were only minor histopathologic changes. In the skewed X-inactivation patients, clinical manifestations ranged from mild to severe, but the patients with mild disease were young (5 to 10 years old). The low levels of dystrophin (< 30% on average) and the severe symptoms of the older patients suggested a poor prognosis for those with skewed X-inactivation, and they all showed morphologic changes of dystrophy. The random inactivation patients showed evidence of biochemical "normalization," with higher dystrophin content in muscle than predicted by the number of normal dystrophin genes. Seventy-nine percent of skewed X-inactivation patients (11/14) showed genetic "normalization," with proportionally more dystrophin-positive nuclei in muscle than in blood. In 65% of the skewed X-inactivation patients, dystrophin was not produced by dystrophin-positive nuclei; an average of 20% of myofiber nuclei were genetically dystrophin-positive but did not produce stable dystrophin. Biochemical normalization seems to be the main mechanism for rescue of fibers from dystrophin deficiency in the random X-inactivation patients. In the skewed X-inactivation patients, genetic normalization is active, but production failure of dystrophin by dystrophin-normal nuclei may counteract any effect of biochemical normalization. In the skewed X-inactivation patients, the remodeling of the muscle through cycles of degeneration and regeneration led to threefold increase in the number of dystrophin-competent nuclei in muscle myofibers (3.3 +/- 4.6), while dystrophin content was on the average 1.5-fold less then expected (-1.54 +/- 3.38). Our results permit more accurate prognistic assessment of isolated female dystrophinopathy patients and provide important data with which to estimate the potential effect of gene delivery (gene therapy) in DMD.

Adult↗
PubMed1995

[Myocardial revascularization in left coronary trunk lesion in patients over 65 years old].

PURPOSE: To study the short and long-term follow-up of patients with left main coronary artery disease (LMCAD) and age over 65 years, by comparing the results with patients under 65 years-old. METHODS: Twenty-two patients with LMCAD and mean age of 69 +/- 3.5 years (group I) were underwent isolated coronary artery bypass grafting (CABG) and compared to 31 patients with LMCAD, mean age of 54 +/- 7 years (group II), who also underwent isolated CABG. The life-table Kaplan-Meyer method was used to estimate the post-operative survival. The chi-square and Student "t" test were used when necessary. RESULTS: Despite higher operative mortality in group I (9.1% x 3.2%), the difference was statistically not significant. The operative morbidity was similar in both groups. Actuarial survival at 4 years was 85% in group I and 95% in group II. Actuarial survival free of cardiac events was 69% in group II and 75% in group II. CONCLUSION: The CABG is well tolerated and had low morbidity and acceptable mortality in old patients with LMCAD. The long-term survival in these patients was very similar to the younger patients.

Actuarial Analysis↗
PubMed1995

Flow cytometric DNA analysis of fine-needle aspirates of prostatic benign lesions.

Fine-needle aspiration samples from 54 consecutive patients suffering from prostatic benign lesions were analyzed for both cytological diagnosis and DNA cell content, by means of standard cytological criteria and flow cytometry, respectively. Of these patients, 13 displayed prostatitis: three being classified as acute prostatitis, 7 as chronic and 3 as granulomatosous prostatitis. All cases had a diploid DNA cell content, with a mean percentage of S-phase cells of 1.8 +/- 2.7%. Although no major differences were detected between patients with and without prostatitis, the former group had a slightly higher proportion of S-phase cells (1.4 +/- 2.2% vs 2.7 +/- 3.7%). Moreover, within those patients with prostatitis, acute prostatitis cases showed the highest proliferation rates although differences did not reach statistical significance.

Biopsy, Needle↗
PubMed1995

[Juvenile polyposis in 2 patients of the same family].

Two cases of juvenile familiar polyposis (one 40-year-old male and his 14-year-old daughter) are presented. The girl presented intermittent rectal bleeding since the age of 10 with growth retardation and chronic anemia. The child was treated by colectomy with more than 100 juvenile polyps without adenomatous alterations being observed in the surgical specimen. Juvenile gastric polyps were also endoscopically observed. The father underwent sigmoidectomy for a adenomatous polyp of this localization. Later review of the surgical specimen demonstrated a juvenile polyp with zones of adenomatous alterations. The diagnostic difficulties of this rare entity and the therapeutic options available are discussed.

Adenomatous Polyposis Coli↗
PubMed1994

Steroid receptor-mediated modulation of CD4+CD62L+ cell homing. Implications for drug abusers.

Naive human T cells home to peripheral lymph nodes via the leukocyte endothelial cell adhesion molecule-1 (LECAM-1, l-selectin, CD62L, Leu8 antigen) they express. We enriched populations of CD4+CD62L+ cells (attachment of Leu8+ T cells to flasks coated with anti-mouse IgG (AIS); Leu8+ T cells, 82.3% pure (+/- 2.3%), enriched for CD4+ cells by incubation over flasks coated with anti-CD4 antibody--this 3-4-day procedure yields an 88 +/- 1.4% recovery. Cells were treated with dexamethasone in vitro for 24-48 h, and monitored by flow cytometry. We found severe toxicity by this steroid at high concentration (10(-6) M: 35% decrease in CD62L+ T cells, 22% drop specifically in CD4+CD62L+ cells), suggesting the onset of receptor-mediated apoptotic events. The toxicity was dose dependent (5% and 7% drop in CD62L+ T and CD4+CD62L+ cells, respectively, at 10(-9) M, the concentration found in plasma 10 h following the administration of 1 mg dexamethasone). One mg of dexamethasone given to normal subjects leads to a 15-20% decrease in circulating CD4+CD62L+ cells at 10 h. This tends to be correlated with a drop in the number of glucocorticoid cytosolic receptors. Thus, steroids seem to modulate CD4+CD62L+ cell homing by means of receptor-mediated mechanisms.

Adult↗
PubMed1994

The disulfide linkages and glycosylation sites of the human natriuretic peptide receptor-C homodimer.

The natriuretic peptide receptor-C (NPR-C) constitutes greater than 95% of the natriuretic peptide binding sites in vivo. This cell surface glycoprotein is a disulfide-linked homodimer with a subunit molecular weight of 68,000. Two sources and types of ANP affinity-purified human NPR-C were used to map disulfide linkages and glycosylation sites of this receptor by mass spectrometry: the extracellular domain obtained by papain cleavage of a receptor-IgG fusion protein expressed in Chinese hamster ovary cells, and a baculovirus/Sf9-expressed cytoplasmic truncation mutant in which 34 of 37 cytoplasmic domain amino acids were deleted. Two intramolecular disulfide bonded loops were found in the 435 amino acid extracellular domain (C63-C91, C168-C216). The juxtamembrane residues C428 and C431 are involved in homodimer formation, confirmed by site-directed mutagenesis of full-length NPR. Three of the four potential Asn-linked glycosylation sites are occupied: N41 (complex), N248 (high mannose), and N349 (complex; partial occupancy). These data describe the intra- and intermolecular linkages in NPR-C, providing a model for the homologous guanylyl cyclase receptors, NPR-A and NPR-B; both of the cyclase receptors likely contain the first amino-terminal 29 amino acid loop, but only NPR-A possesses the second 49 amino acid loop in common with NPR-C.

Amino Acid Sequence↗
PubMed1994

The N-terminal half of initiation factor IF3 is folded as a stable independent domain.

Initiation factor IF3 plays an essential role in the initiation of protein translation by binding to the 30S ribosomal subunit and selecting a proper tRNA(fMet)/initiation codon complex. The domain structure of IF3 from Escherichia coli has been investigated by limited proteolysis followed by mass spectrometry and protein sequencing of the resulting peptides. This analysis revealed a highly segmented structure with two independent domains connected by a charged linker peptide, highly susceptible to proteolytic cleavage. The N-terminal domain is very stable and comparison of its 2-D NMR spectrum with that of intact IF3 revealed that it retains its three-dimensional fold.

Amino Acid Sequence↗
PubMed1994

Phenotypic characteristics of expressed tyrosine hydroxylase protein in the adult rat nucleus tractus solitarius: plasticity revealed by RU24722 treatment.

The phenotypic characteristics of expressed tyrosine hydroxylase protein have been precisely analysed in the rat nucleus tractus solitarius, which contains the majority of A2 noradrenergic and C2 adrenergic neurons of the medulla oblongata. This study was based upon quantitative analysis of immunohistochemical and immunoradioautographic staining of tyrosine hydroxylase protein in serial coronal sections. In control rats, there were few tyrosine hydroxylase-expressing cell bodies which express less than 2% of the immunoradiolabeled tyrosine hydroxylase protein measured in the structure. These cell bodies were scattered throughout an extensive immunopositive neuropile, which precisely delimited the topological space of the nucleus tractus solitarius quantiatively reconstructed using a polar coordinate system. The quantification of tyrosine hydroxylase tissue concentration from immunoradioautograms allowed us to subdivide the structure into two distinct regions. The posterior region of the nucleus tractus solitarius, which mainly corresponds to the A2 cell group, contains a relatively high tissue concentration of tyrosine hydroxylase protein (18.56 +/- 0.154 units per mg of tissue). The anterior region, which mainly corresponds to the C2 cell group, exhibits a relatively low concentration (12.09 +/- 0.81) of this protein. Three days after an intraperitoneal injection of RU24722, there was a strong increase (90 +/- 17%) in tyrosine hydroxylase protein content only in the anterior region of the nucleus tractus solitarius. This increase was associated with a dramatic elevation (142 +/- 20%) in the number of tyrosine hydroxylase-expressing cell bodies. The additional cell bodies were mainly located inside the initial perikarya-containing area.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Development of the chick thymus microenvironment: a study by lectin histochemistry.

The microenvironment of the chick thymus has been examined during development using lectin histochemistry. We have assayed WGA, Con A, RCA-I and TPA on thymic sections from 13, 15, 17 and 19 d chick embryos and 0, 5, 10 and 15 d chicks. All lectins were immunoperoxidase and colloidal gold-conjugated for transmission electron microscope observations. WGA labelled both the cortical and medullary thymic stroma at all the stages analysed. An intense reaction to WGA was observed in the subcortical region from stage 18 embryos to 5 d chicks. On the other hand, WGA did not stain medullary areas of the chick thymus. Con A lectin detected several cell clusters of stromal cells and thymocytes in cortical regions. These clusters could represent a lymphostromal complex with which Con A receptors are associated, probably in relation to cell adhesion. The residues detected by RCA were distributed both in stromal cells and thymocytes of the developing chick thymus. There was an increase of the reaction to RCA between the 19 d embryos and the 5 d chicks. This increase might be interpreted in terms of the secretion of thymic humoral factors at these stages. The thymic stromal cells stained with immunoperoxidase conjugated-TPA showed a reticular pattern in the medulla. There is a possibility that the fucosyl residues may be expressed in the Ia antigen as has previously been suggested in other species.

Animals↗