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Biomedical subjects

L DaSilva

Publications and source records attributed to L DaSilva.

18 recordsLinked to original sources
PubMed2004

A biochanin-enriched isoflavone from red clover lowers LDL cholesterol in men.

OBJECTIVE: To determine whether the two major isoflavones in red clover differ in their effect on low-density lipoprotein cholesterol (LDL-C). DESIGN: A randomised, placebo-controlled, double-blind trial; two parallel groups taking one of the two isoflavones within which treatment and placebo were administered in a crossover design. SETTING: Free-living volunteers. SUBJECTS: A total of 46 middle-aged men and 34 postmenopausal women. INTERVENTION: Two mixtures of red clover isoflavones enriched in either biochanin (n=40) or formononetin (n=40) were compared. Placebo and active treatment (40 mg/day) were administered for 6 weeks each in a crossover design within the two parallel groups. MAIN OUTCOME MEASURES: Plasma lipids were measured twice at the end of each period. RESULTS: Baseline LDL-C concentrations did not differ significantly between men (n=46) and women (n=34), nor between those randomised to biochanin or formononetin. Interaction between time and treatments, biochanin, formononetin and corresponding placebos (two-way ANOVA) on LDL-C showed a significant effect of biochanin treatment alone. The biochanin effect was confined to men; median LDL-C was 3.61 (3.05-4.14) mmol/l with biochanin and 3.99 (3.16-4.29) mmol/l with the corresponding placebo (RM ANOVA with Dunnett's adjustment P<0.05). The difference between placebo and biochanin effects on LDL-C was 9.5%. No other lipid was affected and women failed to respond significantly to treatment. CONCLUSION: Isolated isoflavones from red clover enriched in biochanin (genistein precursor) but not in formononetin (daidzein precursor), lowered LDL-C in men. This may partly explain the previous failure to demonstrate cholesterol-lowering effects with mixed isoflavones studied predominantly in women. SPONSORSHIP: Novogen Ltd, North Ryde NSW, Australia, provided partial support including provision of tablets and outside monitoring.

Aged↗
PubMed2002

Cryopreservation of reduced cytochrome C for determination of N-formyl-methionyl-leucyl-phenylalanine-stimulated superoxide anion production in human whole blood.

Various methods are available for measuring the production of reactive oxygen species by phagocytes, but they are limited in their use by the need for their immediate application, cell isolation and of cell-activation by unphysiological stimuli. In addition, after measurement of reactive oxygen metabolites using oxidizing agents, the reduced compounds formed have to be determined during or immediately after their formation. In the present study, an improved cytochrome C assay was investigated which allowed measurements of superoxide anions in whole blood samples following activation of phagocytes by physiological stimuli such as the bacterial tripeptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). The fMLP-stimulated production of superoxide anion (O(2)(-)) showed a sigmoidal-shaped fMLP dose-response curve, and constant O(2)(-) production rates (nmol.1(-1)x10(6) granulocytes) could be determined reliably up to a blood granulocyte concentration of 1 x 10(4) x microl(-1). To allow the determination of reduced cytochrome C later after its formation, the effect of long-term storage at -20 degrees C on the stability of reduced cytochrome C was tested up to 16 weeks. The results obtained show that the determination of reduced cytochrome C in whole blood represents a simple and reliable method. Most importantly, O(2)(-)-reduced cytochrome C can be frozen and stored without any alterations, at least up to 2 weeks. Thus the method seems to be superior to other methods of detection, especially when the experimental conditions do not allow immediate spectrophotometry (e.g. mountain medicine, space medicine). Under such conditions the present assay would allow reliable measurement of reduced cytochrome C, even after weeks of cryopreservation.

Cell Separation↗
PubMed2000

Selective disruption of interleukin 4 autocrine-regulated loop by a tyrosine kinase inhibitor restricts activity of T-helper 2 cells.

Interleukin (IL) 4 is a potent immunomodulatory cytokine secreted by T-helper 2 (Th2) cells and Th2 mast cells that promotes the commitment of cells. However, unregulated production and release of IL-4 can exacerbate allergic reactions and increase susceptibility to infectious organisms and viruses. Here, we present evidence that AG-490, a Janus tyrosine kinase (JAK) 2-JAK3 inhibitor, effectively blocked IL-4 gene expression and secretion in the Th2 cell line D10 that was not occurring after anti-CD3 antibody stimulation, whereas AG-490 had no inhibitory effect on production of other Th2 cytokines or cytokines synthesized by the corresponding Th1 cell line clone 29. AG-490 potently inhibited IL-4-mediated proliferation of both D10 and the IL-4-dependent cell line CT.4S. Moreover, AG-490 markedly inhibited IL-4 activation of JAK3 and blocked the downstream activation of signal transducer and activator of transcription 6, as judged by tyrosine phosphorylation, DNA binding, and transcription assays. In contrast, AG-490 did not affect tumor necrosis factor alpha activation of NF-kappaB at similar concentrations of drug. These data suggest that tyrosine kinase inhibitors that inhibit JAK3 may have previously unrecognized and selective clinical potential as immunotherapeutic drugs to treat Th2-mediated diseases driven by IL-4. (Blood. 2000;95:3816-3822)

Animals↗
PubMed2000

Activation of human T lymphocytes is inhibited by peroxisome proliferator-activated receptor gamma (PPARgamma) agonists. PPARgamma co-association with transcription factor NFAT.

T lymphocyte activation is highlighted by the induction of interleukin-2 (IL-2) gene expression, which governs much of the early lymphocyte proliferation responses. Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. PPARgamma mRNA expression was found in human peripheral blood T lymphocytes, raising the possibility of PPARgamma involvement in the regulation of T cell function. Here we show that PPARgamma ligands, troglitazone and 15-deoxy-Delta(12,14) prostaglandin J(2), but not PPARalpha agonist Wy14643, inhibited IL-2 production and phytohemagglutinin-inducible proliferation in human peripheral blood T-cells in a dose-dependent manner. This inhibitory effect on IL-2 was restricted to the PPARgamma2-expressing, not the PPARgamma-lacking, subpopulation of transfected Jurkat cells. The activated PPARgamma physically associates with transcriptional factor NFAT regulating the IL-2 promoter, blocking NFAT DNA binding and transcriptional activity. This interaction with T-cell-specific transcription factors indicates an important immunomodulatory role for PPARgamma in T lymphocytes and could suggest a previously unrecognized clinical potential for PPARgamma ligands as immunotherapeutic drugs to treat T-cell-mediated diseases by targeting IL-2 gene expression.

Base Sequence↗
PubMed1998

Molecular cloning of FKHRL1P2, a member of the developmentally regulated fork head domain transcription factor family.

Here we report the expression of a fork head domain protein in human T helper cells. We cloned and characterized a fork head cDNA from human T helper cell mRNA using differential display RT-PCR. The cDNA contains a 546-nucleotide (nt) open reading frame (ORF) that codes for the carboxyl-terminal 180 amino acids (aa) of the recently identified fkhrl1 gene. This ORF does not contain the characteristic DNA-binding domain found in members of the forkhead protein family. In-vitro transcription/translation of this cDNA expressed a protein of approximately 20 kDa. We have generated antibodies that specifically immunoprecipitated the in-vitro-translated 20-kDa protein. This antibody also recognizes in human T lymphocytes a 70-kDa protein corresponding in size to that predicted for the fkhrl1 gene product. The mRNA levels for fkhrl1 is elevated in T helper-induced lymphocytes in comparison to PHA-stimulated T lymphocytes. Further characterization of FKHRL1 and its related family members should shed light on the transcriptional mechanisms of this fork head gene subfamily and their role in T helper cell differentiation and regulation of cell growth.

Amino Acid Sequence↗
PubMed1998

Mechanisms of cytokine signal transduction: IL-2, IL-4 and prolactin as hematopoietin receptor models.

Cytokines, hormones and hematopoietic growth factors transduce biological signals across the cell membrane via a highly conserved family of single membrane-spanning receptors. The intracellular signal transducing machinery responsible for mediating these responses has remained largely unknown. However, recent identification of a homologous class of tyrosine kinases, Janus Kinases (JAKs), and a related family of transcription factors, signal transducers and activators of transcription (STATs), has shed new light on the molecular mechanisms responsible for mediating hematopoietin signaling and immune response. Current research efforts within the field of cytokine signaling have now shifted to understanding how these molecules are activated by hematopoietic receptors, positively and negatively regulated by kinases and phosphatases, and how they impact on gene transcription to ultimately coordinate cell homeostasis, proliferation and differentiation. This article will review some of our results identifying the involvement of JAKs, STATs, and secondary effector molecules activated following engagement of hematopoietic receptors for IL-2, IL-4, and prolactin. Here, we provide evidence for the ingenious ability of cytokine receptors to selectively recruit and activate these proteins among a repertoire of possible alternative biochemical messengers as a means to affect unique and general cell responses.

Animals↗
PubMed1997

Immunocompromised tumor-bearing mice show a selective loss of STAT5a/b expression in T and B lymphocytes.

Impaired immune responses are frequently observed in tumor-bearing hosts during progression of tumor growth, but the molecular basis of these functional defects remains unclear. To investigate tumor-induced immunosuppression, we first established that lymphocytes from mice bearing s.c. mammary adenocarcinoma (TS/A) tumors were severely impaired in their ability to generate cellular and humoral Ag-specific responses. Lymphocytes from these mice were then screened for abnormalities in the expression of signal transducing proteins known to be involved in the regulation of cellular immunity. Interestingly, purified T and B cells isolated from immunocompromised tumor-bearing mice displayed a marked decrease in the transcription activators STAT5a and -b at the protein level and to a lesser extent at the mRNA level. By contrast, no change in the expression of STAT1, -3, and -6 or of the TCR itself were detected. The correlation in the loss of T and B cell function with the selective decrease in STAT5a/b expression suggests that regulation of the STAT5 signaling pathway may provide a molecular mechanism for modulating the immune system.

Animals↗
PubMed1997

Two discrete regions of interleukin-2 (IL2) receptor beta independently mediate IL2 activation of a PD98059/rapamycin/wortmannin-insensitive Stat5a/b serine kinase.

Many cytokines, hormones, and growth factors activate Janus kinases to tyrosine phosphorylate select members of the Stat transcription factors. For full transcriptional activation, Stat1 and Stat3 also require phosphorylation of a conserved serine residue within a mitogen-activated protein kinase phosphorylation consensus site. On the other hand, two recently identified and highly homologous Stat5a and Stat5b proteins lack this putative mitogen-activated protein kinase phosphorylation site. The present study set out to establish whether Stat5a and Stat5b are under the control of an interleukin-2 (IL2)-activated Stat5 serine kinase. We now report that IL2 stimulated marked phosphorylation of serine and tyrosine residues of both Stat5a and Stat5b in human T lymphocytes and in several IL2-responsive lymphocytic cell lines. No Stat5a/b phosphothreonine was detected. Phosphoamino acid analysis also revealed that Stat5a/b phosphotyrosine levels were maximized within 1-5 min of IL2 stimulation, whereas serine phosphorylation kinetics were slower. Interestingly, IL2-induced serine phosphorylation of Stat5a differed quantitatively and temporally from that of Stat5b with Stat5a serine phosphorylation leveling off after 10 min and the more pronounced Stat5b response continuing to rise for at least 60 min of IL2 stimulation. Furthermore, we identified two discrete domains of IL2 receptor beta (IL2Rbeta) that could independently restore the ability of a truncated IL2Rbeta mutant to mediate Stat5a/b phosphorylation and DNA binding to the gamma-activated site of the beta-casein gene promoter. These observations demonstrated that there is no strict requirement for one particular IL2Rbeta region for Stat5 phosphorylation. Finally, we established that the IL2-activated Stat5a/b serine kinase is insensitive to several selective inhibitors of known IL2-stimulated kinases including MEK1/MEK2 (PD98059), mTOR (rapamycin), and phosphatidylinositol 3-kinase (wortmannin) as determined by phosphoamino acid and DNA binding analysis, thus suggesting that a yet-to-be-identified serine kinase mediates Stat5a/b activation.

Androstadienes↗
PubMed1996

Prolactin recruits STAT1, STAT3 and STAT5 independent of conserved receptor tyrosines TYR402, TYR479, TYR515 and TYR580.

The present study of prolactin (PRL) receptor-mediated recruitment of signal transducers and activators of transcription (STATs) demonstrates that PRL activates STAT3, in addition to STAT1 and STAT5 as previously reported, and that STAT1, STAT3 and STAT5 are mediators of PRL effects in cells whether of lymphoid, myeloid or mammary epithelial origin. Furthermore, receptor mutants M240 and T280 that do not mediate PRL-induced JAK2 activation and cell proliferation, are also unable to mediate STAT activation, supporting the proposed model of JAK2 as the initial effector protein used by PRL receptors. On the other hand, tyrosine phosphorylation analysis and electrophoretic mobility shift assays showed that receptor mutant G328, which lacks four of the five conserved cytoplasmic tyrosine residues of PRL receptors, retained the ability to activate JAK2 and STAT1, STAT3 and STAT5. These results support the notion that phosphotyrosyl residues other than those of the receptor, i.e., JAK2, are involved in recruiting STAT proteins to the activated PRL receptor complex.

Animals↗
PubMed1994

Growth signaling and JAK2 association mediated by membrane-proximal cytoplasmic regions of prolactin receptors.

Prolactin (PRL) has recently been demonstrated to induce tyrosine phosphorylation and activation of the cytoplasmic tyrosine kinase JAK2 in PRL-dependent Nb2 lymphoma cells. The present study represents an initial effort to identify cytoplasmic regions of the PRL receptor (PRLR) that are critical to growth signal generation and JAK2 activation. Variably truncated rat PRLRs were stably expressed in the murine 32D cell line. PRL-induced proliferation was mediated by the full-length receptor and the mutant G328, which contains the membrane-proximal Homology Boxes 1 and 2 characteristic of hematopoietin receptors. In contrast, mutant receptors lacking either Box 2 or both Boxes 1 and 2 were not capable of transmitting a growth signal. The mitogenic capacity of the PRLR variants correlated with JAK2 association and activation, as well as with induction of mRNA levels for the growth-related gene ornithine decarboxylase. We conclude that mitogenic competence of rat PRLRs resides within the first 94 amino acids of the cytoplasmic domain. The data suggest that Homology Box 1/Box 2 region is critical to JAK2 phosphorylation and association with the PRLR. These findings will serve as a basis for more detailed molecular characterization of PRLR domains essential for JAK2 interaction and growth signal generation.

Amino Acid Sequence↗
PubMed1990

Equine infectious anemia virus tat: insights into the structure, function, and evolution of lentivirus trans-activator proteins.

Equine infectious anemia virus (EIAV) contains a tat gene which is closely related to the trans-activator genes of the human and simian immunodeficiency viruses. Nucleotide sequence analysis of EIAV cDNA clones revealed that the tat mRNA is composed of three exons; the first two encode Tat and the third may encode a Rev protein. Interestingly, EIAV Tat translation is initiated at a non-AUG codon in exon 1 of the mRNA, perhaps allowing an additional level of gene regulation. The deduced amino acid sequence of EIAV tat, combined with functional analyses of tat cDNAs in transfected cells, has provided some unique insights into the domain structure of Tat. EIAV Tat has a C-terminal basic domain and a highly conserved 16-amino-acid core domain, but not the cysteine-rich region, that are present in the primate immunodeficiency virus Tat proteins. Thus, EIAV encodes a relatively simple version of this kind of trans activator.

Amino Acid Sequence↗
PubMed1990

Structure and expression of the equine infectious anemia virus transcriptional trans-activator (tat).

Equine infectious anemia virus (EIAV) encodes a tat gene which is closely related to the trans-activators encoded by the human and simian immunodeficiency viruses. Nucleotide sequence analysis of EIAV cDNA clones revealed that the tat message is composed of three exons; the first two encode tat and the third may encode rev.. Interestingly, EIAV tat translation is initiated at a non-AUG codon in the first exon of the message, perhaps allowing an additional level of gene regulation. The deduced amino acid sequence of EIAV tat, combined with functional analyses of tat cDNAs in transfected cells, have provided some unique insights into the domain structure of this protein. EIAV Tat has a C-terminal basic domain, a highly conserved 16 amino acid core domain, but not the cysteine-rich region, that is present in the primate immunodeficiency virus Tat proteins. Thus EIAV encodes a relatively simple version of this kind of trans-activator.

Amino Acid Sequence↗