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Biomedical subjects

J Gil

Publications and source records attributed to J Gil.

At least 37 records · Page 2Linked to original sources
PubMed2000

Increased levels of activated subsets of CD4 T cells add to the prognostic value of low CD4 T cell counts in a cohort of HIV-infected drug users.

OBJECTIVE: To identify subsets of CD4 T lymphocytes that can predict the development of AIDS and to assess whether increased levels of these cellular markers could provide additional independent prognostic information to the CD4 T cell count and plasma HIV-1-RNA levels. DESIGN AND METHODS: In a prospective study, a cohort of 85 HIV-positive intravenous drug users [clinical categories of the CDC classification A (n = 48) and B (n = 37)] were followed for a period of 37+/-13 months. Memory and activated CD4 and CD8 T cells were quantitated by three-colour flow cytometry at baseline and expressed as a percentage of total CD4 and CD8 lymphocytes. Clinical evaluations were performed at 6 month intervals. The relationships between these lymphocyte subsets and progression to AIDS were studied using Kaplan-Meier plots and proportional hazards regression models. RESULTS: After adjustment for the level of CD4 T cells and plasma HIV-1-RNA levels, the elevation in the subset CD4+CD38+DR+ was the marker within the functionally distinct subsets of CD4 T lymphocytes with additional prognostic value in bivariate Cox regression models. In multivariate models, increased percentages of CD4+CD38+DR+ T cells provided the strongest independent prognostic information for progression to AIDS (relative hazard, 1.07; P < 0.0001). CONCLUSION: Our results suggest that high levels of CD4+CD38+HLA-DR+ T cells reflect the increasing degree of CD4 T cell activation during the progression of HIV infection, and could be used together with the CD4 T cell and HIV-RNA levels to evaluate more accurately the progressive cellular immune impairment associated with the risk of progression to AIDS.

Adult↗
PubMed2000

Aspirin induces apoptosis through mitochondrial cytochrome c release.

Aspirin and other non-steroidal anti-inflammatory drugs induce apoptosis in many cell types. Although the involvement of caspases has been demonstrated, the mechanism leading to caspase activation remains unknown. We have studied the role of the mitochondrial pathway in aspirin-induced apoptosis. The apoptotic effect of aspirin was analyzed in different cell lines (Jurkat, MOLT-4, Raji and HL-60) showing induction of mitochondrial cytochrome c release and caspases 9, 3 and 8 processing. Furthermore, early aspirin-induced cytochrome c release was not affected by the caspase inhibitor Z-VAD x fmk and preceded loss of mitochondrial membrane potential. Therefore, aspirin-induced apoptosis involves caspase activation through cytochrome c release.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2000

The interferon-induced protein kinase (PKR), triggers apoptosis through FADD-mediated activation of caspase 8 in a manner independent of Fas and TNF-alpha receptors.

The interferon-induced dsRNA-dependent protein kinase (PKR) induces apoptosis of mammalian cells. Apoptosis induction by PKR involves phosphorylation of the translational factor eIF-2alpha and activation of the transcriptional factor NF-kappaB, but caspase pathways activated by PKR are not known. Upregulation of Fas mRNA by PKR has been suggested to play a role in PKR-induced apoptosis. To learn how PKR induces apoptosis, we have analysed the role of molecules in death receptor pathways. We showed the involvement of the FADD-caspase 8 pathway on PKR-induced apoptosis based on four experimental findings: upregulation of caspase 8 activity during PKR-induced apoptosis, blocking of PKR-induced apoptosis by the use of a chemical inhibitor of caspase 8, and inhibition of PKR-induced apoptosis by expression of both a FADD dominant negative or a viral FLIP molecule. Significantly, despite the PKR-mediated upregulation of Fas mRNA expression, the Fas receptor-ligand pathway is not needed for PKR-induced apoptosis. Antibodies that inhibit TNFalpha-TNFR1 or Fas-FasL interactions were not able to block PKR-induced apoptosis. Taken together, our observations establish the involvement of caspase 8 in PKR-induced apoptosis and suggest that death receptors other than Fas or TNFR1 or, alternatively, a novel mechanism involving FADD independently of death receptors, are responsible for PKR-induced apoptosis.

3T3 Cells↗
PubMed2000

Influence of centrifugation and different extenders on post-thaw sperm quality of ram semen.

Using a 2-step extension methodology to freeze ram semen, 2 freezing protocols (P1 and P2) and 3 extenders were evaluated in a split-sample experiment. The freezing protocols were tested in combination with Extenders A and B (Experiment 1), and B and C (Experiment 2). Protocol 1 included centrifugation before filling the straws to reconcentrate the diluted semen to a calculated sperm concentration of 800 x 10(6) cells/mL. Protocol 2 involved appropriate ejaculate extension to yield 800 x 10(6) cells/mL as in P1, albeit avoiding centrifugation. Extenders A and B were milk-based and were supplemented with 5% egg yolk and fructose. Extender B was clarified by centrifugation (twice at 3310 g/20 min). Extender C was based on TRIS-citrate-fructose supplemented with 20% egg yolk and clarified as described for Extender B. Final glycerol concentration was 7% for all 3 extenders. Post-thaw parameters studied were subjective motility, computer assisted sperm motility analysis (CASA), membrane integrity (SYBR-14/P1), and capacitation status (chlortetracycline assay, CTC). The overall sperm concentration (x 10(6)/straw) differed (P<0.001) between P1 (mean+/-SD, 138.1+/-14.8) and P2 (216.5+/-13.9). Despite centrifugation, P1 appeared to be less harmful for spermatozoa than P2, yielding higher percentages of subjective motility, linearity, membrane integrity and uncapacitated spermatozoa. Due to the difference in concentrations obtained between P1 and P2, the total calculated numbers of spermatozoa having desirable characteristics were higher in samples processed as P2. In Experiment 1, P1 resulted in lower calculated numbers x 10(6) in the Aldose of subjective motility (87.2+/-5.1 vs 125.3+/-5.1; P<0.05), linearity (70.6+/-4.3 vs 79.8+/-4.3; NS), intact-membrane (77.4+/-5 vs 108.5+/-5.1; P<0.001), and uncapacitated (36.5+/-2.5 vs 46.5+/-2.5; P<0.05) spermatozoa, than P2. In Experiment 2, calculated sperm numbers (x 10(6)/straw) were lower in P1 than in P2 for subjective motility (80.8+/-5.4 vs 92.0+/-5.4; NS), linearity (63.3+/-5.6 vs 73.1+/-5.6; NS), membrane integrity (77.7+/-3.6 vs 101.0+/-3.6; P<0.001), and uncapacitated spermatozoa (28.3+/-3.24 vs. 4.1+/-3.2; P<0.01). Extender B (clarified milk extender) was consistently better than Extender A (nonclarified milk extender) for all parameters studied, but the difference was only statistically significant for linearity after 1 h of incubation at 38 degrees C (44.0+/-2.4 vs 36.2+/-2.4; P<0.05). Extender B was also better than Extender C (TRIS-citrate-fructose) for percentage of uncapacitated (49.7+/-2.2 vs 34.4+/-2.3; P<0.001), subjective motile (57.5+/-2.7 vs 43.8+/-2.7; P<0.01), and linear motile (46.5+/-2.8 vs 33.7+/-2.8; P<0.01) spermatozoa, but not for membrane integrity (51.6+/-1.5 vs 51.7+/-1.5). It was concluded that exclusion of centrifugation, as in P2, yielded higher sperm numbers with desirable characteristics per straw. Clarification of milk-based extender (B) resulted in better post-thaw sperm quality, especially compared with TRIS-based extender (C).

Animals↗
PubMed2000

In vivo regulation of protein synthesis by phosphorylation of the alpha subunit of wheat eukaryotic initiation factor 2.

The regulation of protein synthesis is a critical component in the maintenance of cellular homeostasis. A major mechanism of translational control in response to diverse abiotic and biotic stress signals involves the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha). The pathway has been demonstrated in all eukaryotes except plants, although components of a putative plant pathway have been characterized. To evaluate the in vivo capability of plant eIF2alpha to participate in the translation pathway, we have used vaccinia virus recombinants that constitutively express wheat eIF2alpha and inducibly express the eIF2alpha dsRNA-stimulated protein kinase, PKR, in BSC-40 cells. Activation of PKR in cells expressing wild-type wheat eIF2alpha resulted in an inhibition of cellular and viral protein synthesis and an induction of cellular apoptosis correlating with phosphorylation of eIF2alpha on serine 51. Expression of a nonphosphorylatable mutant (51A) of plant eIF2alpha reversed the PKR-mediated translational block as well as the PKR-induced apoptosis. A direct interaction of the plant proteins with the mammalian translational initiation apparatus is supported by coimmunoprecipitation of wild-type plant eIF2alpha and the 51A mutant with mammalian eIF2gamma and the localization of the plant proteins in ribosome fractions. These findings suggest that plant eIF2alpha is capable of interacting with the guanine nucleotide exchange factor eIF2B within the context of the eIF2 holoenzyme and provide direct evidence for its ability to participate in phosphorylation-mediated translational control in vivo.

Amino Acid Sequence↗
PubMed2000

Down syndrome critical region gene 2: expression during mouse development and in human cell lines indicates a function related to cell proliferation.

The isolation of the genes located in chromosome 21 and the characterisation of their function are essential steps towards the understanding of the physiopathological mechanisms involved in Down syndrome. We have used two complementary approaches to characterise the function of the novel gene DSCR2 (Down Syndrome Critical Region gene 2): the isolation and characterisation of the mouse gene homologue to the human DSCR2 gene, and the analysis of the expression of the gene in different human cell lines. We have isolated and characterised a 1012 bp of a mouse cDNA having a high homology to the human DSCR2 gene. The predicted mouse dscr2 protein has an identity of 85.4% as compared to the human protein, indicating that the DSCR2 protein has been conserved during the evolution. However, the amino acid sequence is not homologous to other known proteins, or to known protein domains. The dscr2 gene is expressed throughout all the stages of the mouse embryo development. In the adult mouse the gene is expressed in testis, kidney, liver, brain, heart, skeletal muscle, and pancreas. The expression analysis of the DSCR2 gene in different human tumour derived cell lines indicates that the gene is expressed in all proliferating cell lines tested. The levels of the DSCR2 mRNA correlate with cellular growth of T98G and Jurkat cells in response to different treatments. The expression pattern throughout the foetal development together with the correlation observed with the cell cycle indicates a possible function for the DSCR2 gene related to cell proliferation.

Amino Acid Sequence↗
PubMed2000

Activation of NF-kappa B by the dsRNA-dependent protein kinase, PKR involves the I kappa B kinase complex.

Besides its known role as a translational controlling factor, the double stranded RNA-dependent protein kinase (PKR) is a key transcriptional regulator exerting antiviral and antitumoural activities. We have recently described that induction of NF-kappa B by PKR is involved in apoptosis commitment. To define how PKR mediates NF-kappa B activation by dsRNA, we have used two different approaches, one based on expression of PKR by a vaccinia virus (VV) recombinant and the other based on induction of endogenous PKR by poly I:C (pIC) treatment. We found that NF-kappa B complexes induced by PKR are composed primarily of p50-p65 heterodimers and also of c-rel-p50 heterodimers. As described for other stimuli, following pIC treatment, PKR phosphorylates the NF-kappa B inhibitor I kappa B alpha at serine 32 before degradation. Expression by VV recombinants of IKK1 or IKK2 dominant negative mutants together with PKR showed inhibition of PKR-induced NF-kappa B activation, as measured both by gel shift and luciferase reporter assays. Immunoprecipitation analysis revealed that PKR interacts with the IKK complex. Our findings demonstrate that physiological function(s) of PKR involve activation of the I kappa B kinase complex. Oncogene (2000) 19,1369 - 1378.

Biopolymers↗
PubMed2000

Sonographic diagnosis of a pseudoaneurysm in an intravenous drug abuser.

We report a case of a traumatic pseudoaneurysm and arteriovenous fistula in the groin of a drug abuser. Gray-scale and Doppler sonography were used to establish the diagnosis. Gray-scale sonography revealed an anechoic, ovoid, 7 x 4 cm mass posterior to the right superficial femoral artery, which was displaced anteriorly, with limited visualization of the deep femoral artery. Power Doppler sonography showed complete color filling of the mass, and turbulence was seen on conventional color Doppler sonography. The turbulence was confirmed with pulsed Doppler sonography of the mass. Pulsed Doppler imaging of the right external iliac vein also showed an arterialized spectral flow pattern.

Adult↗
PubMed2000

Maternal cocaine abuse and fetal renal arteries: a morphometric study.

The purpose of the present study was to evaluate the pathological findings and perform morphometric analysis of the renal arteries of fetuses exposed to cocaine in utero. The control group consisted of 22 stillborn fetuses of unknown etiology whose mothers' urine was negative for cocaine or any other vasoactive substances. The study group comprised 29 stillborn fetuses whose mothers' urine was positive only for cocaine on the day of admission. Sections of fetal kidneys (4 microm), stained with hematoxylin and eosin and periodic acid-Schiff reagent, were examined under light microscopy (x40) to identify interlobular arteries. Morphometric analysis of these arteries was performed using a self-assembled system with a touch-sensitive screen as an interactive peripheral. Their inner and outer circumferences were measured by outlining them on the screen with a stylus. The radius (r) was calculated from the measurement of the circumference (2.pi.r). The difference of the radii of the outer and inner circumferences was the thickness of the arterial wall. The interlobular arterial thickness was significantly greater (P<0.001) in the cocaine-exposed group (mean 15.46+/-5.8 microm, 2SD) compared with the normal (mean 9.03+/-3.96 microm, 2SD). There was a significantly (P<0.001) positive relation with advancing gestational ages in both groups. The circumferences of the lumen of the arteries showed a significant (P<0.05) relation with advancing gestational ages in the normal group only. In the cocaine-exposed group, the arterial lumen circumference (mean 167.88+/-17.58 microm, 2SD) was significantly (P<0.001) smaller than in the normal group (mean 227.73+/-6.82 microm, 2SD). Thus, maternal cocaine abuse is associated with thickening of the interlobular arterial wall of the fetal kidney and narrowing of the lumen.

Cocaine↗
PubMed2000

Subcellular ontogeny of brain pyroglutamyl peptidase I.

The present work studies pyroglutamyl-peptidase I activity in several subcellular fractions of the developing brain. In the synaptosomal fraction, soluble pGlu-peptidase I activity is low until postnatal Day 9 (with a peak at postnatal Day 2) and the activity increases at postnatal Day 15. In later stages there are not significant changes of synaptosomal pGlu-peptidase I activity. However, in the cytosolic fraction the activity is high from ED22 until postnatal Day 9, and afterwards decreases. The changes in the particulate fractions are generally less drastic.

2-Naphthylamine↗
PubMed2000

Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): mechanism of action.

Interferons are a family of cytokines that exerts antiviral, antitumor and immunomodulatory actions by inducing a complex set of proteins. One of the best known IFN-induced protein is the dsRNA-dependent protein kinase (PKR), that mediates both antiviral and anticellular activities. PKR inhibits translation initiation through the phosphorylation of the alpha subunit of the initiation factor eIF-2 (eIF-2 alpha) and also controls the activation of several transcription factors such as NF-kappa B, p53, or STATs. In addition, PKR mediates apoptosis induced by many different stimuli, such as treatment with LPS, TNF-alpha, viral infection, or serum starvation. The mechanism of apoptosis induction by PKR involves phosphorylation of eIF-2 alpha and activation of NF-kappa B. In this way, expression of different genes is regulated by PKR. Among the genes upregulated in response to PKR are Fas, Bax and p53. The pathway of PKR-induced apoptosis involves FADD activation of caspase 8 by a mechanism independent of Fas and TNFR. Since IFNs are used as drugs for different disorders such as viral infection and cancer, understanding the pathway of apoptosis induction triggered by PKR should be useful in the rational design of IFN therapies.

Apoptosis↗
PubMed2000

Identification of a new HLA-DRB4 allele (DRB4*01033) by PCR-SSP and direct sequencing.

In this report, we describe the identification of a novel DRB4*01 allele, DRB4*01033, found in two Spanish Caucasian individuals. The new allele was detected during routine HLA typing by an unusual pattern of amplification obtained by polymerase chain reaction using sequence-specific primers (PCR-SSP) that did not match with any of the previously described DRB4 alleles. In order to establish the polymorphism responsible for this pattern exons 2 and 3 of the DRB4 locus were amplified and directly sequenced. The new DRB4*01 allele is identical to DRB4*0103101 except for a single nucleotide substitution in codon 78 (TAC-->TAT). This nucleotide change does not cause an amino acid change as both triplets code for a tyrosine.

Alleles↗
PubMed2000

Prodigiosin from the supernatant of Serratia marcescens induces apoptosis in haematopoietic cancer cell lines.

The effects of supernatant from the bacterial strain Serratia marcescens 2170 (CS-2170) on the viability of different haematopoietic cancer cell lines (Jurkat, NSO, HL-60 and Ramos) and nonmalignant cells (NIH-3T3 and MDCK) was studied. We examined whether this cytotoxic effect was due to apoptosis, and we purified the molecule responsible for this effect and determined its chemical structure. Using an MTT assay we showed a rapid (4 h) decrease in the number of viable cells. This cytotoxic effect was due to apoptosis, according to the fragmentation pattern of DNA, Hoechst 33342 staining and FACS analysis of the phosphatidylserine externalization. This apoptosis was blocked by using the caspase inhibitor Z-VAD.fmk, indicating the involvement of caspases. Prodigiosin is a red pigment produced by various bacteria including S. marcescens. Using mutants of S. marcescens (OF, WF and 933) that do not synthesize prodigiosin, we further showed that prodigiosin is involved in this apoptosis. This evidence was corroborated by spectroscopic analysis of prodigiosin isolated from S. marcescens. These results indicate that prodigiosin, an immunosuppressor, induces apoptosis in haematopoietic cancer cells with no marked toxicity in nonmalignant cells, raising the possibility of its therapeutic use as an antineoplastic drug.

3T3 Cells↗
PubMed2000

Iterative thresholding for segmentation of cells from noisy images.

We introduce an iterative thresholding algorithm for the segmentation of cells from noisy cell images. The thresholding image, which is initially a constant, changes iteratively with both the previous segmentation and image local activity. Experimental results for both synthesized and real cell images are provided to demonstrate the performance of the algorithm.

Algorithms↗