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Biomedical subjects

A M Alvarez

Publications and source records attributed to A M Alvarez.

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

Epidermal growth factor impairs the cytochrome C/caspase-3 apoptotic pathway induced by transforming growth factor beta in rat fetal hepatocytes via a phosphoinositide 3-kinase-dependent pathway.

Transforming growth factor beta (TGF-beta)-mediated apoptosis is one of the major death processes in the liver. We have previously shown that epidermal growth factor (EGF) is an important survival signal for TGF-beta-induced apoptosis in fetal hepatocytes (Fabregat et al., FEBS Lett 1996;384:14-18). In this work we have studied the intracellular signaling implicated in the protective effect of EGF. We show here that EGF activates p42 and p44 mitogen-activated protein kinases (MAPK). However, mitogen extracellular kinase (MEK) inhibitors do not block the survival effect of EGF. EGF also activates phosphoinositide 3-kinase (PI 3-kinase) and protein kinase B (PKB/AKT) in these cells. The presence of PI 3-kinase inhibitors blocks the protective effect of EGF on cell viability, DNA fragmentation, and caspase-3 activity. We have found that TGF-beta disrupts the mitochondrial transmembrane potential (DeltaPsi(m))( )and activates the release of cytochrome c, this effect being blocked by EGF, via a PI 3-kinase-dependent pathway. A detailed study on bcl-2 superfamily gene expression shows that TGF-beta produces a decrease in the messenger RNA (mRNA) and protein levels of bcl-x(L), an antiapoptotic member of this family, capable of preventing cytochrome c release. EGF is able to maintain bcl-x(L) levels even in the presence of TGF-beta. PI 3-kinase inhibitors completely block the protective effect of EGF on TGF-beta-induced bcl-x(L )down-regulation. We conclude that PI 3-kinase mediates the survival effect of EGF on TGF-beta-induced death by acting upstream from the mitochondrial changes, i.e., preventing bcl-x(L) down-regulation, cytochrome c release, and activation of caspase-3.

Animals↗
PubMed2000

p38 mitogen-activated protein kinase mediates tumor necrosis factor-alpha-induced apoptosis in rat fetal brown adipocytes.

Tumor necrosis factor-alpha (TNFalpha) induces apoptosis and cell growth inhibition in primary rat fetal brown adipocytes. Here, we examine the role played by some members of the mitogen-activated protein kinase (MAPK) superfamily. TNFalpha activates extracellular regulated kinase-1/2 (ERK1/2) and p38MAPK. Inhibition of p38MAPK by either SB203580 or SB202190 highly reduces apoptosis induced by TNFalpha, whereas ERK inhibition potentiates it. Moreover, cotransfection of an active MKK3 mutant and p38MAPK induces apoptosis. p38MAPK inhibition also prevents TNFalpha-induced cell cycle arrest, whereas MEK1 inhibition enhances this effect, which correlates with changes in proliferating cell nuclear antigen expression, but not in cyclin D1. c-Jun and activating transcription factor-1 are potential downstream effectors of p38MAPK and ERKs upon TNFalpha treatment. Thus, TNFalpha-induced c-Jun messenger RNA expression requires ERKs activation, whereas p38MAPK inhibition enhances its expression. In addition, TNFalpha-induced activating transcription factor-1 phosphorylation is extensively decreased by SB203580. However, TNFalpha-induced NF-kappaB DNA-binding activity is independent of p38MAPK and ERK activation. On the other hand, C/EBP homology protein does not appear to mediate the actions of TNFalpha, because its expression is almost undetectable and even reduced by TNFalpha. Finally, although TNFalpha induces c-Jun N-terminal kinase (JNK) activation, transfection of a dominant negative of either JNK1 or JNK2 had no effect on TNFalpha-induced apoptosis. These results suggest that p38MAPK mediates TNFalpha-induced apoptosis and cell cycle arrest, whereas ERKs do the opposite, and JNKs play no role in this process of apoptosis.

Adipocytes↗
PubMed2000

[Evaluation of the effectiveness of a new vaccine against human leptospirosis in groups at risk].

In order to evaluate the effectiveness of a new vaccine against human leptospirosis, a prospective cohort study was done with persons in the Cuban province of Holguín who were at risk of becoming ill with leptospirosis. The study included 118,018 persons from 15 to 65 years old who were considered to face either permanent or temporary risk. The vaccinated cohort consisted of 101,137 persons. They received two vaccine doses, 6 weeks apart, of 0.50 mL via deep intramuscular injection into the deltoid muscle of the nondominant arm. The unvaccinated cohort consisted of 16,881 persons. Epidemiological surveillance began 21 days after the application of the second vaccine dose and continued for 1 year. The same criteria for suspected and confirmed cases were maintained throughout the study period. At the end of the surveillance period effectiveness was calculated as being higher than 97%. It is estimated that the vaccination program prevented eight out of ten cases that would have otherwise occurred. Vaccine reactogenicity was also measured in a subsample of 1,500 persons between 15 and 65 years old. The observed symptomatology was low. Slight pain at the injection site was the most frequent symptom (25%). The results of the study indicate the usefulness of the vaccine for disease prevention among people at risk, and its use is thus recommended.

Adolescent↗
PubMed1999

Apoptotic response to TGF-beta in fetal hepatocytes depends upon their state of differentiation.

Transforming growth factor-beta (TGF-beta1) induces death of fetal hepatocytes by an apoptotic mechanism. However, even when very high concentrations and/or long-term exposure to the cytokine is used, 40-50% of cells always survive. The process of cell survival is coincident with changes in morphology and phenotype, with cells showing a fibroblastic appearance and eliciting an epithelial-fibroblastic transition. Surviving cells continue responding to TGF-beta in terms of growth control. Expression of liver-specific genes is very low in these cells; this effect is due to the decrease in their rate of transcription as soon as 2 h after the addition of the factor. Surviving cells present a decreased DNA binding activity for liver-enriched transcription factors, an increased DNA binding activity for AP-1, and a high expression of protooncogenes. These cells are immature hepatocytes since in the presence of the appropriate signal (i.e., epidermal growth factor), they can differentiate, organizing in cell clusters and increasing both liver-specific mRNA expression and liver-enriched transcription factor activity. In accord with these results, TGF-beta, secreted at high concentrations during liver carcinogenesis, might induce death of normal cells while providing a selective advantage for the survival of cells that are "partially transformed" or "less differentiated" and unresponsive to the factor.

Animals↗
PubMed1999

Cocaine cytotoxicity in hepatocyte cultures from phenobarbital-induced rats: involvement of reactive oxygen species and expression of antioxidant defense systems.

The present study was designed to investigate whether cocaine modifies the production of reactive oxygen species, affects cellular enzyme-mediated antioxidant defense systems and, subsequently, promotes apoptosis and/or necrosis of hepatocytes. Primary cultures of hepatocytes isolated from phenobarbital-induced rats were exposed to cocaine (0-1000 microM) for 24 hr, and cell death (apoptosis or necrosis), antioxidant enzyme activities and mRNA levels, and peroxide generation were determined. Cocaine cytotoxicity by apoptosis was observed by detecting apoptotic nuclei using optic microscopy and by measurement of the hypodiploid peak (<2C) in DNA histograms obtained by flow cytometry. Necrosis was evidenced by lactate dehydrogenase (LDH) leakage, and peroxide production was quantified with 2',7'-dichlorodihydrofluorescein diacetate. Low concentrations of cocaine (less than 100 microM) resulted in an increase in dichlorofluorescein fluorescence, associated with an enhancement in apoptotic cell death and sharp decreases in the enzyme activities and RNAs of catalase and manganese-superoxide dismutase (Mn-SOD). The progressive decrease in peroxide production in cell cultures detected in the range of 250-1000 microM cocaine was associated with increases in LDH leakage and decreases in the percentage of apoptotic cells, accompanied by low levels in catalase and Mn-SOD enzyme activities and mRNAs, without apparent changes in apoptosis. These data indicate that oxygen radicals may contribute directly or indirectly to cocaine-induced apoptosis in cultured hepatocytes. We conclude that, in primary hepatocyte cultures, cocaine-induced cell death by necrosis was dependent on cocaine concentration, while cell death by apoptosis was parallel to peroxide concentration. The down-regulation of the gene expression of antioxidant enzyme systems should be one of the mechanisms involved in cocaine toxicity.

Animals↗
PubMed1999

Age-related changes on parameters of experimentally-induced liver injury and regeneration.

Age-dependent changes related to liver injury and regeneration were studied in rats aged 2, 12, and 30 months in a time period of 96 hr following a sublethal dose of thioacetamide (6.6 mmoles/kg body wt). Serum aspartate aminotransferase activity increased earlier in young rats, but the severity of injury was higher in those aged 12 months when compared to young and to old. Microsomal hepatocyte FAD monooxygenase activity was induced earlier in 2-month-old rats following intoxication and the increase was significantly lower both in the youngest and in the oldest groups when compared to adults. As a parameter of hepatocellular postnecrotic regeneration, DNA synthesis (2C --> 4C) was evaluated. The population of hepatocytes in S phase peaked more sharply and earlier in young rat hepatocytes, and was 8 to 12 times higher than the initial in hepatocytes from 2- and 12-month-old rats, while the rise was only 3 times in the oldest group. At 96 hr of intoxication the restoration towards normal in all these parameters was complete in young, incomplete in adult, and slightly detected in the oldest. Serum proliferative activity, assayed on mouse NIH 3T3 fibroblast cultures, increased preceding the necrosis and this increase was higher in 2- and 12-month-old (171% and 224%, respectively), while in the oldest the increase was only 110%. This mitogenic activity decreased in all groups during necrosis, showing a second peak, nondetectable in rats aged 30 months, parallel to regeneration. Serum TNFalpha level was absent in untreated animals and increased markedly following intoxication, the highest values being recorded at 72 hr of intoxication in serum from rats aged 12 months (347 +/- 30 pg/ml) and the lowest at 30 months (4.1 +/- 0.3 pg/ml). The serum ability to induce nitric oxide synthase activity on peritoneal macrophages ex vivo showed significant time- and age-dependent changes in nitric oxide release: a decrease throughout necrosis and an increase during regeneration. We conclude that the main age-related changes in the sequenced process of liver injury and regeneration are the delayed response in the development of cell killing and regeneration and the decreased regenerative ability, which significantly delays the restoration of liver function.

3T3 Cells↗
PubMed1999

Nitric oxide induces tyrosine nitration and release of cytochrome c preceding an increase of mitochondrial transmembrane potential in macrophages.

Treatment of elicited peritoneal macrophages or the macrophage cell line RAW 264.7 with high concentrations of nitric oxide donors is followed by apoptotic cell death. Analysis of the changes in the mitochondrial transmembrane potential (DeltaPsi(m)) with specific fluorescent probes showed a rapid and persistent increase of DeltaPsi(m), a potential that usually decreases in cells undergoing apoptosis through mitochondrial-dependent mechanisms. Using confocal microscopy, the release of cytochrome c from the mitochondria to the cytosol was characterized as an early event preceding the rise of DeltaPsi(m). The cytochrome c from cells treated with nitric oxide donors was modified chemically, probably through the formation of nitrotyrosine residues, suggesting the synthesis of peroxynitrite in the mitochondria. These results indicate that nitric oxide-dependent apoptosis in macrophages occurs in the presence of a sustained increase of DeltaPsi(m), and that the chemical modification and release of cytochrome c from the mitochondria precede the changes of DeltaPsi(m).-Hortelano, S., Alvarez, A. M., Boscá, L. Nitric oxide induces tyrosine nitration and release of cytochrome c preceding an increase of mitochondrial transmembrane potential in macrophages.

Animals↗
PubMed1999

Defining the burden of pneumonia in children preventable by vaccination against Haemophilus influenzae type b.

OBJECTIVES: To determine the burden of pneumonia requiring hospitalization in infants and young children preventable by vaccination against Haemophilus influenzae type b (Hib). DESIGN: Vaccination centers in Santiago, Chile, were randomly selected to administer PRP-T, an Hib conjugate vaccine, combined with diphtheria-tetanus toxoids-pertussis (DTP) vaccine or DTP alone. SUBJECTS: Infants who received > or =2 doses of DTP or DTP and Hib conjugate vaccine combined. MAIN OUTCOME MEASURES: Pneumonia episodes leading to hospitalization accompanied by indicators of likely bacterial infection including radiologic evidence of alveolar consolidation or pleural effusion, an elevated erythrocyte sedimentation rate (> or =40 mm/h) or bronchial breath sounds on auscultation. RESULTS: In participants age 4 to 23 months, PRP-T reduced the incidence of pneumonia associated with alveolar consolidation or pleural effusion by 22% (95% confidence interval, -7 to 43) from 5.0 to 3.9 episodes per 1000 children per year. When the pneumonia case definition included any of the following, alveolar consolidation, pleural effusion, erythrocyte sedimentation rate > or =40 mm/h or bronchial breath sounds, PRP-T provided 26% protection (95% confidence interval, 7 to 44) and prevented 2.5 episodes per 1000 children per year. CONCLUSIONS: Hib vaccine provides substantial protection against nonbacteremic pneumonia, particularly those cases with alveolar consolidation, pleural effusion or other signs of likely bacterial infection. Hib vaccination prevented approximately 5 times as many nonbacteremic pneumonia cases in infants as meningitis cases, thus indicating that the largest part of the effect of Hib vaccination might be undetectable by routine culture methods.

Chile↗
PubMed1999

Uracil/tegafur plus oral calcium folinate in advanced breast cancer.

Uracil and tegafur (in a molar ratio of 4:1 [UFT]) has proven activity against breast cancer and is delivered in an easy-to-administer oral formulation. Orzel, which combines UFT with the oral biomodulator, calcium folinate, may provide even greater antitumor efficacy against breast cancer. Here, we describe the preliminary results of this phase II trial investigating the feasibility of 250 mg/m2/day of UFT plus 45 mg/day of oral calcium folinate administered to highly pretreated patients with advanced breast cancer. The results indicate a highly tolerable regimen and an overall response rate of 27.8% in a group of poor-prognosis patients. These findings warrant continued investigation.

Administration, Oral↗
PubMed1999

Studies of chromaffin granule functioning by flow cytometry: transport of fluorescent epsilon-ATP and granular size increase induced by ATP.

Flow cytometry techniques, usually employed to characterize cellular populations, are reported here to be a valuable tool to approach the study of subcellular organelle functioning. Chromaffin granules rendered fluorescent by using an antibody against their membrane protein, synaptophysin, are detectable by flow cytometry. Moreover, these storage granules are able to transport the fluorescent ATP analogue, epsilon-ATP (1,N6-ethenoadenosine 5'-triphosphate), and the resulting granular fluorescence increase can also be followed by this technique. The saturation studies show a non-hyperbolic kinetic behaviour, with a two step curve. The K0.5 values were 0.26 and 2.5 mM and Hill numbers 1 and 6 respectively. In addition, an unexpected granular size increase, which was dependent on the epsilon-ATP concentration, occurred together with the fluorescence increase. Other nucleotide triphosphate substrates of V-ATPase, such as ATP or GTP, but not the non-hydrolyzable analogue ATP gamma S (adenosine 5'-O-(3-thiotriphosphate), mimic this effect, which exhibited sigmoidal saturation curves with K0.5 values of 1.8 and 3.1 mM for ATP and epsilon-ATP respectively. The V-ATPase inhibitors, suramin, EGTA or EDTA significantly reduced the granular size increase in the presence of ATP. Extragranular addition of noradrenaline has no effect by itself on the granular size, but significantly reduced the granular size increase induced by ATP. This effect was reversed by the amine transport inhibitor reserpine. The granular size increase induced by ATP was more effective in the presence of Cl- than Br- or I-. Moreover, no increase occurred in the presence of F- or acetate. The Cl- channel blockers were poorly effective, and only 2-(phenylamino)-benzoic acid (DPC) exhibited an effect on the ATP-induced granular size increase.

Adrenal Glands↗
PubMed1998

Transforming growth factor-beta (TGF-beta) and EGF promote cord-like structures that indicate terminal differentiation of fetal hepatocytes in primary culture.

When fetal hepatocytes were cultured in the presence of transforming growth factor-beta (TGF-beta 1) and epidermal growth factor (EGF), some morphological changes were observed. Under these conditions, cells migrated, from typical clusters that hepatocytes adopt in culture, to form elongated, cord-like structures similar to the hepatic acinus organization. Immunocytochemical analysis of these cells revealed high levels of albumin and cytokeratin 18, phenotypic markers of parenchymal hepatocytes. Although some of the cells in the cord-like structures presented a cortical ring distribution of F-actin filaments, the cord also presented thick peripheral bundles and cells of the tips showed thin stress fibers oriented to the cell edges, typical of a migratory phenotype. In addition to these morphological effects, flow cytometric analysis of the cells revealed a larger size, granularity and intracellular lipid content (as a parameter related to liver metabolic function), in TGF-beta + EGF-treated hepatocytes. Western blot analysis of the albumin levels revealed that both expression and secretion of albumin were increased in EGF + TGF-beta-treated cells. Finally, all these changes were coincident with an enhancement in the DNA-binding activity for hepatocyte nuclear factors (HNF1, HNF3, and HNF4), as revealed in gel-shift experiments with nuclear extracts. We conclude that a cooperative action between TGF-beta and EGF might modulate terminal maturation of fetal hepatocytes.

Actin Cytoskeleton↗
PubMed1998

Influence of aminoguanidine on parameters of liver injury and regeneration induced in rats by a necrogenic dose of thioacetamide.

1. When aminoguanidine, a nucleophilic hydrazine compound, was administered to rats (50 mg kg(-1) body wt) 30 min before a necrogenic dose of thioacetamide (500 mg kg(-1) body wt), significant changes related to liver injury and hepatocellular regeneration were observed. 2. The extent of necrosis was noticeably less pronounced, as detected by the peak of serum aspartate aminotransferase activity. Depletion of hepatic glutathione (GSH) and the increase in malondialdehyde concentration as markers of oxidative stress, produced by thioacetamide metabolism, were significantly diminished. However, the activity of microsomal FAD monooxygenase, the system responsible for thioacetamide oxidation, did not show significant alterations. Antioxidant enzyme systems involved in the glutathione redox cycle, such as glutathione reductase and glutathione peroxidase activities, slightly decreased following aminoguanidine pretreatment. 3. Primary cultures of peritoneal macrophages from control rats, when incubated in the presence of serum collected following thioacetamide intoxication, showed a significant decrease in nitric oxide (NO) release at 24 h, that was more pronounced in the group pretreated with aminoguanidine. However, the sharp and progressive increase in macrophage NO release, when incubated in the presence of serum obtained at 48, 72 and 96 h, were increased by aminoguanidine-pretreatment. 4. The cell population involved in DNA synthesis sharply increased in both groups at 48 h of intoxication, although the values at 0, 24, 72 and 96 h were markedly higher in the group pre-treated with aminoguanidine. Polyploidy at 72 and 96 h of intoxication was delayed by the effect of aminoguanidine and a progressive increase in the hypodiploid hepatocyte population, which reached 16% of the total at 96 h, was observed. 5. These results indicate that a single dose of aminoguanidine before thioacetamide administration, markedly diminished the severity of the liver injury by decreasing oxidative stress and lipoperoxidation, but hepatocellular regeneration was apparently unaffected probably due to an enhanced mitogenic activity.

Animals↗
PubMed1998

The effect of non-genotoxic carcinogens, phenobarbital and clofibrate, on the relationship between reactive oxygen species, antioxidant enzyme expression and apoptosis.

Phenobarbital and clofibrate, two non-genotoxic carcinogens, have been investigated regarding the relationship between reactive oxygen species, antioxidant enzyme expression and apoptosis in primary cultures of rat hepatocytes. Low toxicity concentrations, 200 and 100 microg/ml for phenobarbital and clofibrate respectively, were used to examine their effect on spontaneous or transforming growth factor beta1 (TGFbeta1)-induced apoptosis and on the expression of antioxidant defence enzymes (superoxide dismutases and catalase). The increased incidence of apoptotic nuclei was visualized in TGFbeta1-treated cultures with the fluorescent dye Hoechst 33258 and was quantified under all experimental conditions by measurement of the hypodiploid peak in DNA histograms obtained by flow cytometry. Both substances, when added separately to hepatocyte cultures and incubated for 24 and 48 h, significantly diminished spontaneous apoptosis and exhibited a slight suppression of TGFbeta1-induced apoptosis. Endogenous peroxide production by hepatocytes increased with TGFbeta1, phenobarbital or clofibrate and the increase was greater with phenobarbital and in the presence of TGFbeta1 with both drugs. Gene expression of catalase and Mn- and Cu,Zn superoxide dismutases (SOD) was evaluated by northern blot analysis of hepatocytes incubated in the presence of phenobarbital or clofibrate with or without TGFbeta1 and the following differences were detected: phenobarbital induced a significant decrease in both dismutases (to 56%, P < 0.05, and 55%, P < 0.05, for Mn- and Cu,Zn-SOD respectively) and a 2-fold increase (P < 0.01) in catalase; clofibrate induced a slight decrease in both SODs and a 4-fold increase (P < 0.05) in catalase; TGFbeta1 significantly decreased to 37% (P < 0.05) expression of catalase while not significantly affecting expression of both SODs. We conclude that inhibition of spontaneous apoptosis induced by either phenobarbital or clofibrate is accompanied by increases in the endogenous levels of peroxides and by significant induction of catalase gene expression. Furthermore, the lack of effect of both compounds on TGFbeta1-induced apoptosis could be a consequence of the inability of these two compounds to counteract the depressing effect of TGFbeta1 on expression of catalase.

Animals↗
PubMed1998

Epidemiology of invasive pneumococcal infections in infants and young children in Metropolitan Santiago, Chile, a newly industrializing country.

AIM: To study the epidemiology of invasive pneumococcal infections in infants and young children in Santiago, Chile, as a representative pediatric population in a newly industrializing country where pneumococcal conjugate vaccines may be used in the future. METHODS: A 5-year retrospective laboratory-based review (1989 to 1993) was followed by a 3-year prospective laboratory and hospital surveillance study in two of the six health administrative areas of Santiago to detect all hospitalized cases of invasive pneumococcal disease (defined as Streptococcus pneumoniae isolated from blood, cerebrospinal fluid or another normally sterile site) among infants and children (0 to 23 months of age in the retrospective and 0 to 59 months of age in the prospective study). RESULTS: During the 5-year retrospective survey the incidence of invasive pneumococcal disease was 90.6 cases per 10(5) infants 0 to 11 months old and 18.5 cases per 10(5) toddlers 12 to 23 months old. Similar rates (60.2 per 10(5) infants and 18.1 per 10(5) toddlers) were recorded during the 3 years of prospective surveillance. Among the 110 cases in children 0 to 59 months of age detected during the 3-year prospective surveillance, 2 clinical forms, pneumonia and meningitis, accounted for 87.2% of all cases; 13 of the 49 pneumonia patients (26%) had empyema as a complication. Notably 40 of the 110 cases (36.4%) occurred before 6 months of age (63.4% of the 63 infant cases). Serotypes 1, 14, 5 and 6B were the most prevalent. Overall 76 and 69%, respectively, of S. pneumoniae isolates were antigenic types that would be covered by the 11- or 9-valent conjugate vaccines under development. CONCLUSIONS: Invasive pneumococcal infections in Santiago, Chile, exhibit an epidemiologic pattern intermediate between that of developing and industrialized countries. The high burden of disease in early infancy dictates that an accelerated immunization schedule (beginning in the perinatal period) or maternal immunization with pneumococcal vaccines should be explored.

Age Factors↗
PubMed1998

HLA-DPbeta residue 69 plays a crucial role in allorecognition.

To investigate the contribution to allorecognition of the individual polymorphic positions Glu 69 and Val 36 from the DPB1*02012 allele, DPB1*02012 cDNA was subjected to site-directed mutagenesis and alleles expressing Lys at 69 and Ala at 36 were generated. The lymphoblastoid cell line (LCL) 45.EM1, a previously generated mutant B-LCL which expresses normal levels of DPA mRNA but is not able to transcribe DPB, was transfected with wild-type or mutant DPB1*02012 cDNAs. The ability of two HLA-DPw2 alloreactive CD4+ cytotoxic T-lymphocyte (CTL) clones to lyse the panel of DPB1*02012 wild-type and site-directed mutant B-cell lines was tested. Both CTL clones (8.3 and 8.9) lysed the B-LCL 45.1, which is haploid for HLA and expresses wild-type DPB1*02012, and transfectants expressing Ala at 36 instead of Val, indicating that this polymorphic residue is not critical for T-cell recognition. However, the change of Glu to Lys at 69 prevented recognition by clones 8.3 and 8.9. These data demonstrate that the residue at peptide-binding position 69 is crucial for T-cell receptor recognition and suggest the requirement for a negatively charged residue at this position for allostimulation of these T-cell clones. The side chain of DPbeta-69 is predicted to point into the peptide-binding groove, and the existence of positive(Lys) or negative (Glu) residues probably leads to substantial differences in the allo- or auto-DP-bound peptides or to differences in the conformation of the peptide-MHC complex, which would therefore be responsible for specific DPw2 allorecognition. The binding of a panel of monomorphic and polymorphic anti-HLA-DP monoclonal antibodies (mAbs) to these transfectants was also tested by flow cytometry. The changes at Glu 69 and Val 36 did not affect recognition by any of the monomorphic antibodies tested. However, the binding pattern of some of the polymorphic mAbs was clearly modified. Therefore, even though it is not crucial for T-cell allorecognition, polymorphic residue 36 must be involved in epitopes recognized by some polymorphic anti-DP antibodies, while residue 69 of the DPB molecule is crucial both for T-cell allorecognition and recognition by some mAbs.

Alleles↗
PubMed1998

p42/p44 mitogen-activated protein kinases activation is required for the insulin-like growth factor-I/insulin induced proliferation, but inhibits differentiation, in rat fetal brown adipocytes.

Insulin-like growth factor I (IGF-I)/insulin induced cytosolic p42/p44 mitogen-activated protein kinase (MAPK) activation in a time-dependent manner in fetal brown adipocytes, reaching a maximum at 5 min. Concurrently, nuclear p42/p44 MAPKs were also activated by IGF-I and insulin. This cytosolic and nuclear MAPK activation was totally prevented by pretreatment with the MAPK kinase (MEK1) inhibitor, PD98059. These results indicate that MEK mediates the IGF-I/insulin-induced p42/ p44 MAPK activation. IGF-I and insulin also increased the number of cells in the S + G2/M phases of the cell cycle, PCNA levels, and DNA synthesis at 24 h. This IGF-I/insulin-induced proliferation was completely blunted by the presence of MEK1 inhibitor. In contrast, inhibition of MEK1 potentiated the IGF-I-induced uncoupling protein (UCP-1) and the insulin-induced fatty acid synthase mRNAs expression after short and long-term treatments. Moreover, transient expression of a transfected active MEK construct (R4F) decreased IGF-I-induced UCP-1 and insulin-induced fatty acid synthase mRNA expression. These results demonstrate that p42/p44 MAPKs are essential intermediates for the IGF-I/insulin-induced mitogenesis, but may have a negative role in the regulation of adipocytic and thermogenic differentiation in brown adipocytes.

Adipose Tissue, Brown↗
PubMed1997

TNF-alpha induces apoptosis in rat fetal brown adipocytes in primary culture.

The effect of TNF-alpha on cell death in rat fetal brown adipocytes maintained in primary culture was determined. TNF-alpha inhibited proliferation and induced apoptosis in these cells. Most of the cells undergoing apoptosis after TNF-alpha treatment did not express PCNA, suggesting an induction of apoptosis by TNF-alpha in non-proliferative cells. IGF-I but not EGF prevented TNF-alpha-induced apoptosis.

Adipocytes↗