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PubMed1985

Analysis of the Thy-1 pathway of T cell hybridoma activation using 17 rat monoclonal antibodies reactive with distinct Thy-1 epitopes.

Seventeen monoclonal anti-Thy-1 antibodies (mAb) derived from LOU/M rats immunized with mouse T cell clones were used to study the role of Thy-1 in antigen-independent T cell activation. These mAb identified Thy-1.2 or monomorphic determinants and immunoprecipitated a molecule of 25-28 kDa from detergent-solubilized, 125I-labeled T cell surface proteins. Competitive cross-inhibition binding assays demonstrated that these reagents defined 3 epitope groups including either Thy-1.2 (group A) or Thy-1 monomorphic (groups B and C) determinants. Experiments using high titered culture supernatants revealed that all 6 IgG mAb defining the epitope group C, and one IgG2c mAb directed at a determinant in group A were capable of stimulating the terpolymer-L-glutamic acid60-L-alanine33-Ltyrosine10 (GAT) plus I-Ad-reactive BALB/c T cell hybridoma T14-117.9 to produce interleukin 2 (IL2) in the absence of accessory cells. Cross-linking of cell-bound rat mAb by a BALB/c anti-rat kappa chain mAb, or the presence of B cell lymphomas in the culture resulted in an increase of the Thy-1-mediated IL2 responses of this hybridoma. Some mAb from group B required antibody doses exceeding 80 micrograms/ml in order to activate T cells, while others remained nonstimulatory at any dose tested. Striking synergy in mAb-mediated T cell activation was observed when nonmitogenic doses of mAb group groups A and C were mixed in the same culture. Analysis of a panel of GAT plus I-Ad-specific T cell hybridomas revealed that these cells markedly differed in the magnitude of their IL2 responses induced by a given amount of stimulating anti-Thy-1 mAb. Such reagents also stimulated normal thymocytes to express IL2 receptor on their surface. These studies show that the epitopic specificity and the amount of anti-Thy-1 mAb, and the susceptibility of the T cell examined represent important parameters for the triggering of the Thy-1 pathway of T cell activation.

Animals↗
PubMed2002

Gonadotrophins inhibit the expression of insulin-like growth factor binding protein-related protein-2 mRNA in cultured human granulosa-luteal cells.

Insulin-like growth factors (IGF) and IGF-binding proteins (IGFBP) have been shown to be involved in ovarian follicular growth/development and steroidogenesis. Recently, a number of low-affinity IGFBP-related proteins (IGFBP-rP) have been characterized. In this study, we investigated the expression of the gene for IGFBP-rP2 (also known as connective tissue growth factor, CTGF) in human granulosa cells in vitro and in vivo. Northern blot analysis demonstrated that IGFBP-rP2 mRNA is expressed in cultured human granulosa-luteal cells obtained from women undergoing an IVF programme. Accumulation of IGFBP-rP2 mRNA was dose-dependently down-regulated by FSH and LH after 24 h treatment (both P < 0.05) in cultured granulosa-luteal cells. The inhibitory effects of gonadotrophins were mimicked by treatment with the protein kinase A activator, (Bu)(2)cAMP. Protein kinase C inhibitor staurosporine reduced, whereas protein kinase C activator TPA (12-O-tetradecanoyl phorbol 13-acetate) increased, IGFBP-rP2 mRNA accumulation. These results suggest that the inhibitory effects of gonadotrophins on IGFBP-rP2 gene expression may involve signal transduction via both protein kinase A and C pathways. Immunohistochemical analysis revealed positive staining for IGFBP-rP2 in the granulosa and theca cells of normal human ovarian follicles. Corpus luteum and ovarian surface epithelial cells were also positively stained. Modulation of IGFBP-rP2 expression by gonadotrophic hormones may have a role in ovarian follicular development and in the ovulatory process.

Cells, Cultured↗
PubMed1999

Changes in signal transduction downstream from the granulocyte-macrophage colony-stimulating factor receptor during differentiation of primary hemopoietic cells.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multifunctional cytokine, having different effects on primitive hemopoietic cells and terminally differentiated end-cells of the myeloid lineage. Human primitive hemopoietic cells (CD34+) were obtained from the peripheral blood after mobilization and induced to proliferate and then differentiate with a combination of cytokines in vitro. Cells at different time points were then used to analyze the expression of the GM-CSF receptor and GM-CSF mediated activation of the JAK 2-STAT 5 and MAP kinase pathways. Scatchard analysis as measured by radioligand binding revealed that freshly purified CD34+ cells expressed 36+/-1 high affinity receptors per cell (mean +/- SE, n = 3) and the level of expression was not significantly different after 3 days in culture, but rose five- to tenfold by day 8. The day 0 CD34+ cells were hyporesponsive to GM-CSF, but by 3 days in culture the cells were still morphologically immature but were actively proliferating and exhibited maximal GM-CSF induced JAK 2-STAT 5 and MAP kinase activation at the optimal time point. Further culture of the CD34+ cells resulted in myeloid differentiation associated with prolongation of MAP kinase activation but not JAK 2-STAT 5 activation. These data indicate that the JAK 2-STAT 5 and MAP kinase pathways are independently regulated and that changes in these signaling pathways occur with differentiation.

Antigens, CD34↗
PubMed2004

Analysis of the PTCH1 signaling pathway in ovarian dermoids.

Dermoids belong to the group of developmental cysts and arise from germ cells. Studies on these tumors may therefore increase our understanding of normal germ cell development within different environments and cell lines derived from these lesions may also constitute an important vehicle for studying neoplasia and differentiation. Recently, we investigated the status of the PTCH1 locus in a large set of sporadic non-inflammatory, developmental cystic lesions. Our data showed allelic loss of microsatellite markers in close vicinity to the PTCH1 locus in both odontogenic keratocysts and dentigerous cysts as well as in ovarian dermoid cysts (ODC). In this study, we closely examined the status of the PTCH1 gene in ODCs. Although about 25% of cysts demonstrated LOH at the PTCH1 locus, no nonsense or missense mutations in the coding region of PTCH1 were detected in genomic DNA isolated from any of the ODCs examined by direct sequencing. Staining with PTCH1 and GLI1 antibodies showed that proteins were present in virtually all epithelial linings, with variable staining intensity not correlated with LOH and generally weaker for GLI1. However, cDNA microarray analysis performed on cell lines derived from ODCs did not show any significant alteration in the expression of the analyzed target genes of PTCH1 signaling in any of the cell lines examined, except for CyclinD1 (and several other genes generally not associated with PTCH1 signaling).

Cell Line, Tumor↗
PubMed2005

Gene profiling in muscle of severely burned children: age- and sex-dependent changes.

BACKGROUND: Thermal injury is associated with a pronounced catabolic response in skeletal muscle. This study identifies gene expression changes in skeletal muscle of thermally injured girls and boys using high-density oligonucleotide arrays. METHODS: Six burned children with a mean age of 8.3 +/- 1.3 years and TBSA burn size covering 51 +/- 6% admitted to our hospital with in 48 h of injury and six cleft lip and cleft palate patients were studied. Total RNA was isolated, in vitro transcribed, and hybridized to HG-U95 Av.2 Affymetrix arrays. Messenger RNA expression patterns of controls and burn patients were compared using Affymetrix GeneChip Analysis Suite 5.2 and dChip. RESULTS: Statistical analysis of the 12,625 genes on each array showed a significant increase in the expression of 77 genes in burn children and a decrease in 21 genes when compared to controls (P < 0.05). We found three genes in burned males and two genes in burned females with decreased expression in muscle compared to controls. Chromosomes 1, 2, 7, 12, and 16 showed genes with increased expression in muscle from burned children, while chromosomes 3, 7, 8, 19, and 22 had genes with decreased expression. Categories of genes affected were related to metabolism, proliferation, transcription/translation, immune response, stress response, angiogenesis, and signal transduction. CONCLUSIONS: Genes that are differentially expressed in skeletal muscle of burned children, but whose function in muscle is unknown, include those related to various transcription factors and those known to encode proteins involved in signaling pathways. Further analysis is required to achieve the ultimate goal of making functionally relevant conclusions about the molecular pathology of burn injury.

Adolescent↗
PubMed2001

Effect of Rho-kinase inhibition on vasoconstriction in the penile circulation.

A recent report from this laboratory (Chitaley K, Wingard C, Webb R, Branam H, Stopper V, Lewis R, and Mills T. Nature Medicine 7: 119-122, 2001) showed that inhibition of Rho-kinase increased the erectile response (intracavernosal pressure and mean arterial pressure) by a process that does not require nitric oxide or cGMP. The present study investigated whether vasoconstrictor agents, which are active in the penis, act via the Rho-kinase pathway. Western analysis revealed RhoA and Rho-kinase protein in the penis. Treatment with the selective Rho-kinase inhibitor Y-27632 significantly increased the magnitude of the erectile response. Intracavernous administration of endothelin-1 (ET-1; 50 pmol) or methoxamine (10 microg/kg) reduced the erectile response to autonomic stimulation. If Y-27632 was given before ET-1 or methoxamine, the vasoconstrictor effect was reduced, and intracavernosal pressure and mean arterial pressure remained elevated. However, when given after methoxamine, Y-27632 had a reduced vasodilatory effect, and Y-27632 had no vasodilatory effect when given after ET-1. These findings suggest that ET-1 and methoxamine increase Rho-kinase activity in the cavernous circulation and support the hypothesis that the vasoconstriction that maintains the penis in the nonerect state is mediated, in part, by the Rho-kinase pathway.

Amides↗
PubMedDate not supplied

Metabolism and turnover of cell surface-associated heparan sulfate proteoglycan and chondroitin sulfate proteoglycan in normal and cholesterol-enriched macrophages.

Analysis of sulfur-35-labeled proteoglycans indicated that cholesterol-enriched pigeon peritoneal macrophages synthesized 42% more 35S-labeled proteoglycan when compared with control macrophages during a 24-hour incubation. Proteoglycan turnover was subsequently studied in radiolabeled macrophage cultures after a 1-, 3-, 6-, 12-, or 24-hour chase with fresh media. During the chase, intracellular proteoglycan disappeared rapidly, whereas there was a small accumulation of 35S-labeled proteoglycan in the media that plateaued at about 6 hours and remained relatively constant thereafter. Pericellular heparan sulfate proteoglycan and chondroitin sulfate proteoglycan disappeared throughout the chase and did not appear to accumulate in the media or in the intracellular compartment. The rapid disappearance of intracellular proteoglycans along with the relative lack in metabolism of media proteoglycans indicated that the majority of pericellular proteoglycans were metabolized via an intracellular degradative pathway. Kinetic analysis of pericellular proteoglycans revealed the presence of a single pool of heparan sulfate proteoglycan (half-life [t1/2] = 6.9 hours) and a single pool of chondroitin sulfate proteoglycan (t1/2 = 11.5 hours) in control macrophage cultures. Cholesterol-enriched macrophage cultures also contained a single pool of pericellular heparan sulfate proteoglycan (t1/2 = 7.3 hours) but contained two pools of chondroitin sulfate proteoglycan (t1/2 = 0.8 hour and 25.9 hours).

Animals↗
PubMed1987

Drinking-induced alterations in reward pathways: an in vivo autoradiographic analysis.

An in vivo autoradiographic technique permitted the visualization of discrete neuroanatomical changes in opiate receptor binding as a result of 23-h water deprivation and drinking. Two groups of rats (n = 5) were placed on a 23-h water deprivation schedule for 10 days. On the last day, one group was given access to water for 15 min. These groups, plus a matched ad libitum water control group (n = 5), received an injection of 0.002 mg/kg [3H]diprenorphine ([3H]Dpr) through chronically implanted jugular catheters followed by preparation for opiate receptor autoradiography. Relative cerebral blood flow was estimated non-quantitatively by the injection of 75 microCi/kg iodo-[14C]antipyrene into 3 additional groups identically treated. Results indicated that water-deprivation stress increased [3H]Dpr binding in the claustrum, lateral hypothalamus, amygdala and ventral tegmental area while decreasing binding in the medial frontal cortex, lateral septum, dorsolateral thalamus and central gray. All effects of water deprivation were reversed in animals receiving water. Observations of changes in relative blood flow were shown to have no correlation with changes in opiate receptor binding. It appears that water deprivation stress causes a reduction in opioid release in areas along the mesotelencephalic dopamine pathway which may contribute to a drive state. Water intake may then reduce or otherwise alter the drive state through the release of opioids along these pathways, contributing to the perception of reward.

Animals↗
PubMed2004

Proteome analysis of Pseudomonas sp. K82 biodegradation pathways.

Pseudomonas sp. K82 is a soil bacterium that can degrade and use monocyclic aromatic compounds including aniline, 3-methylaniline, 4-methylaniline, benzoate and p-hydroxybenzoate as its sole carbon and energy sources. In order to understand the impact of these aromatic compounds on metabolic pathways in Pseudomonas sp. K82, proteomes obtained from cultures exposed to different substrates were displayed by two-dimensional gel electrophoresis and were compared to search for differentially induced metabolic enzymes. Column separations of active fractions were performed to identify major biodegradation enzymes. More than thirty proteins involved in biodegradation and other types of metabolism were identified by electrospray ionization-quadrupole time of flight mass spectrometry. The proteome analysis suggested that Pseudomonas sp. K82 has three main metabolic pathways to degrade these aromatic compounds and induces specific metabolic pathways for each compound. The catechol 2,3-dioxygenase (CD2,3) pathway was the major pathway and the catechol 1,2-dioxygenase (beta-ketoadipate) pathway was the secondary pathway induced by aniline (aniline analogues) exposure. On the other hand, the catechol 1,2-dioxygenase pathway was the major pathway induced by benzoate exposure. For the degradation of p-hydroxybenzoate, the protocatechuate 4,5-dioxygenase pathway was the major degradation pathway induced. The nuclear magnetic resonance analysis of substrates demonstrated that Pseudomonas sp. K82 metabolizes some aromatic compounds more rapidly than others (benzoate > p-hydroxybenzoate > aniline) and that when combined, p-hydroxybenzoate metabolism is repressed by the presence of benzoate or aniline. These results suggest that proteome analysis can be useful in the high throughput study of bacterial metabolic pathways, including that of biodegradation, and that inter-relationships exist with respect to the metabolic pathways of aromatic compounds in Pseudomonas sp. K82.

Aniline Compounds↗
PubMed2001

Cloning and characterization of an arginine vasotocin receptor from the euryhaline flounder Platichthys flesus.

A sequence coding for an arginine vasotocin (AVT) receptor has been identified by the screening of a hepatic cDNA library from the teleost Platichthys flesus. The 2701-bp receptor sequence is predicted to yield a 384-amino acid peptide, analysis of which indicates a seven-transmembrane spanning sequence typical of G-protein-coupled receptors with the N terminus on the outer surface of the cell membrane. Sequence analysis showed this sequence to have a high homology with the Catostomus commersoni AVT receptor (76%) and mammalian vasopressin V(1)-type receptor (62%), but only 55% homology with the C. commersoni isotocin receptor. A two-electrode voltage clamp was used to characterize the receptor expressed in Xenopus laevis oocytes. AVT induced an inward current which was dose dependent over the range 16.7 fmol to 5 pmol; isotocin was without effect over the same dose range. The mammalian vasopressin V(1)-type receptor agonist ([Phe(2), Orn(8)] oxytocin)() induced an inward current but was less potent than AVT, whereas the mammalian vasopressin V(2)-type receptor agonist ([Deamino(1), Val(4), D-Arg(8)] AVP) was without effect. Injection of oocytes with heparin or BAPTA suppressed the response to AVT, indicating receptor linkage to the phospholipase C-phosphatidylinositol pathway. Northern analysis demonstrated the presence of this AVT receptor mRNA in the brain, kidney, and gill of flounder.

Amino Acid Sequence↗
PubMed2003

Shaken and stirred: muscle structure and metabolism.

Muscles are ideal models with which to examine the relationship between structure and metabolism because they are some of the most highly structured cells, and are capable of the largest and most rapid metabolic transitions as well as the highest metabolic rates known. Studies of metabolism have traditionally been conducted within what can considered as the kinetic paradigm provided by 'solution biochemistry'; i.e. the rates of enzymatic reactions are studied in terms of their regulation by mass-action and allosteric effectors and, most recently, metabolic control analysis of pathways. This approach has served biology well and continues to be useful. Here, we consider the diffusion of small and large molecules in muscles and energy metabolism in the context of intracellular space. We find that in attempting to explain certain phenomena, a purely kinetic paradigm appears insufficient. Instead, phenomena such as the 'shuttling' of high-energy phosphate donors and acceptors and the binding of metabolic enzymes to intracellular structures or to each other are better understood when metabolic rates and their regulation are considered in the context of intracellular compartments, distances, gradients and diffusion. As in all of biology, however, complexity dominates, and to such a degree that one pathway may consist of several reactions that each behave according to different rules. 'Soluble' creatine kinase operates at or near equilibrium, while mitochondrial and myofibrillar creatine kinases directly channel substrate to (or from) the adenine nucleotide translocase and actomyosin-ATPase, their operation being thus displaced from equilibrium. Hexose 6-phosphate metabolism appears to obey the rules of solution biochemistry, e.g. phosphoglucoisomerase behaves as Haldane would have predicted in 1930. In contrast, given low steady-state substrate and product concentrations and high flux rates, a number of glycolytic reactions further downstream must be catalyzed by enzymes localized in close proximity to each other. Metabolites may be channeled within these complexes. When observed, mechanistic differences between species in the same steps or processes should not be surprising, considering how animals vary so much in structures, mechanical properties, mitochondrial contents and metabolic rates. This analysis suggests that declarations of the triumph of one mechanism or paradigm over all others, as well as calls for the abandonment of solution biochemistry, are unwarranted. Rather, metabolic biochemistry would seem better served by reconciling the old and the new.

Cytoplasm↗
PubMed1995

Shc binding to nerve growth factor receptor is mediated by the phosphotyrosine interaction domain.

Shc is an adaptor protein that contains two phosphotyrosine-binding domains, a Src homology 2 (SH2) domain and the newly described phosphotyrosine interaction (PI) domain. Shc interacts with several tyrosine-phosphorylated proteins and is itself tyrosine-phosphorylated in cells stimulated with a variety of growth factors and cytokines. Upon phosphorylation, Shc binds to the Grb2.Sos complex leading to the activation of the Ras signaling pathway. Mutational analysis of the nerve growth factor (NGF) receptor (TrkA) suggested that the binding of Shc to the activated receptor is required for NGF-induced neuronal differentiation of PC12 cells. Here we report that the PI domain of Shc directly binds to tyrosine 490 on the autophosphorylated NGF receptor. The PI domain specifically recognizes an I/LXN-PXpY motif (where p indicates phosphorylation) as determined by phosphopeptide competition assay. In addition, the PI domain is able to efficiently compete for binding of full-length Shc proteins to the NGF receptor. In PC12 cells, the Shc SH2 domain interacts with an unidentified tyrosine-phosphorylated protein of 115 kDa but not with the activated NGF receptor. The ability of Shc to interact with different tyrosine-phosphorylated proteins via its PI and SH2 domains may allow Shc to play a unique role in tyrosine kinase signal transduction pathways.

Adaptor Proteins, Signal Transducing↗
PubMed1992

Influence of synaptic identity on single-Ia-afferent connectivity and EPSP amplitude in the adult cat: homonymous versus heteronymous connections.

1. This study makes use of the pattern of synaptic connections between motoneurons and Ia afferents of triceps surae muscles in the cat to test the relative importance of synaptic identity, neuronal size, and neuronal topography as determinants of Ia-afferent connectivity and excitatory postsynaptic potential (EPSP) amplitude. 2. The synaptic actions of single-Ia medial gastrocnemius (MG) afferents were measured by intracellular recording in MG and lateral gastrocnemius (LG) motoneurons. The spike-triggered averaging technique was used to measure EPSPs generated by homonymous or heteronymous Ia afferents and motoneurons, i.e., neurons supplying the same or different muscles, respectively. In agreement with earlier studies, the pooled sample showed that the number of functional connections and the size of EPSPs were both significantly greater for homonymous than for heteronymous neurons. 3. Afferent conduction velocity, motoneuron conduction velocity, rheobase current, and position of the motoneuron relative to the spinal cord afferent entry were all correlated with EPSP amplitude, but the amplitude difference between homonymous and heteronymous connections remained significant after the statistical removal analysis of covariance (ANCOVA) of the contribution of these variables. Stepwise multiple-regression analysis showed that synaptic identity explained the greatest fraction of the variance in EPSP amplitude (9%), with significant but smaller fractions accounted for by rheobase current or motoneuron conduction velocity. 4. In a separate experiment, the monosynaptic affects from both homonymous and heteronymous single-Ia afferents were examined in each of 88 MG or LG motoneurons. The single-Ia afferents used in this portion of the study were sampled from both MG and LG muscles and selected for similar conduction velocities and spinal cord entry points.(ABSTRACT TRUNCATED AT 250 WORDS)

Afferent Pathways↗
PubMed2004

Dysfunction of p53 pathway in human colorectal cancer: analysis of p53 gene mutation and the expression of the p53-associated factors p14ARF, p33ING1, p21WAF1 and MDM2.

Mutations of p53 tumor suppressor gene increase with tumor progression in colorectal cancers. In this study, we examined the expressions of p33ING1, p14ARF, MDM2 and p21WAF1 mRNA in 25 advanced colorectal cancers by quantitative RT-PCR method, and compared the expression levels of p33ING1, p14ARF, p21WAF1 and MDM2 in relation to p53 status in the tumors. Fifteen of 25 colorectal cancers (60%) showed abnormal accumulation of p53 protein in the nucleus, and the remaining 10 colorectal cancers (40%) were negative for p53 immunostaining. We found a G --> T transition (nonsense mutation) at the first nucleotide of codon 298 (exon 8) in one p53-negative case, and a frame shift mutation on exon 7 in another p53-negative case. In remaining eight p53-negative cases, there was no mutation in the entire open reading frame of p53 cDNA. Interestingly, in eight cases with p53 wild-type gene, 6 cases (75%) showed a marked down-regulation of p14ARF mRNA, and three cases (37.5%) over-expressed MDM2 mRNA. Only one case with wild-type p53 gene showed normal level expression of p53 regulatory-factors (p33ING1, p14ARF, and MDM2). Thus, p53 tumor suppressor pathway was disrupted in 24 of 25 colorectal cancers (96%).

Aged↗
PubMed2006

Effects of the antidepressant mianserin in zebrafish: molecular markers of endocrine disruption.

Due to their environmental occurrence and intrinsic biological activity, human pharmaceuticals have received increasing attention from environmental and health agencies. Of particular, ecotoxicological concern are drugs that affect nervous- and endocrine-systems. Zebrafish genome-wide oligo arrays are used to collect mechanistic information on mianserin-induced changes in gene expression in zebrafish. Gene expression analysis in brain and gonad tissue clearly demonstrated the estrogenic activity of mianserin and its potency to disrupt normal endocrine (estrogenic) signaling, based on induction of molecular biomarkers of estrogenicity (e.g., vitellogenin1 and zona pellucida proteins). The possible mechanism underlying this estrogenic activity of mianserin is disturbance of the Hypothalamo-Pituitary-Gonadal (HPG) axis by direct interference of mianserin with the serotonergic and adrenergic systems in the brain of zebrafish. Taking into account the importance of the HPG-axis, and considering the concept of 'critical window of exposure', our results reveal the importance for more elaborate testing of endocrine disruptive effects of aquatic antidepressants at different lifestages and during longer exposure periods (e.g., life cycle studies). Although there is a low concordance between the gene expression results in this study and previous cDNA microarray hybridizations, the global mechanistic expression patterns are similar in both platforms. This argues in favor of pathway-driven analysis of gene expression results compared to gene-per-gene analysis.

Animals↗
PubMed2005

Comparative analysis of programmed cell death pathways in filamentous fungi.

BACKGROUND: Fungi can undergo autophagic- or apoptotic-type programmed cell death (PCD) on exposure to antifungal agents, developmental signals, and stress factors. Filamentous fungi can also exhibit a form of cell death called heterokaryon incompatibility (HI) triggered by fusion between two genetically incompatible individuals. With the availability of recently sequenced genomes of Aspergillus fumigatus and several related species, we were able to define putative components of fungi-specific death pathways and the ancestral core apoptotic machinery shared by all fungi and metazoa. RESULTS: Phylogenetic profiling of HI-associated proteins from four Aspergilli and seven other fungal species revealed lineage-specific protein families, orphan genes, and core genes conserved across all fungi and metazoa. The Aspergilli-specific domain architectures include NACHT family NTPases, which may function as key integrators of stress and nutrient availability signals. They are often found fused to putative effector domains such as Pfs, SesB/LipA, and a newly identified domain, HET-s/LopB. Many putative HI inducers and mediators are specific to filamentous fungi and not found in unicellular yeasts. In addition to their role in HI, several of them appear to be involved in regulation of cell cycle, development and sexual differentiation. Finally, the Aspergilli possess many putative downstream components of the mammalian apoptotic machinery including several proteins not found in the model yeast, Saccharomyces cerevisiae. CONCLUSION: Our analysis identified more than 100 putative PCD associated genes in the Aspergilli, which may help expand the range of currently available treatments for aspergillosis and other invasive fungal diseases. The list includes species-specific protein families as well as conserved core components of the ancestral PCD machinery shared by fungi and metazoa.

Amino Acid Sequence↗
PubMed1998

Differential roles of extracellular signal-regulated kinase-1/2 and p38(MAPK) in interleukin-1beta- and tumor necrosis factor-alpha-induced low density lipoprotein receptor expression in HepG2 cells.

The inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF), elevated in inflammatory, malignant, and infectious diseases, induce low density lipoprotein (LDL) receptor transcription in HepG2 cells, and such an induction can account for hypocholesterolemia associated with these states. However, the signaling mechanisms of cytokine-mediated LDL receptor induction are largely unexplored. In the present studies, we examined the potential involvement of different mitogen-activated protein kinase (MAPK) pathways. Northern analysis demonstrated that IL-1beta or TNF significantly increased LDL receptor transcript in HepG2 cells, whereas expression of another tightly regulated sterol-responsive squalene synthase gene was unaffected. IL-1beta treatment resulted in transient activation of three MAPK cascades, namely p46/54(JNK), p38(MAPK), and ERK-1/2, with maximal activation of 20-, 25-, and 3-fold, respectively, occurring 15-30 min after cytokine addition. PD98059, a specific inhibitor of MAPK kinase activity, inhibited IL-1beta-induced LDL receptor expression. In contrast, SB202190, a specific inhibitor of p38(MAPK), enhanced IL-1beta-induced LDL receptor expression, with a concomitant increase in ERK-1/2 activity. Similarly, TNF induced LDL receptor expression also required ERK-1/2 activation. Finally, sterols repressed IL-1beta induced receptor expression, without affecting ERK-1/2 activation. These results show that IL-1beta- or TNF-induced LDL receptor expression requires ERK-1/2 activation, that the p38(MAPK) pathway negatively regulates LDL receptor expression, and that sterols inhibit induction at a point downstream of ERK-1/2 in HepG2 cells.

Animals↗
PubMed2005

Potentiation of glucocorticoid activity in hypoxia through induction of the glucocorticoid receptor.

Tissue hypoxia is intimately associated with chronic inflammatory disease and may signal to the resolution of inflammatory processes. Glucocorticoid signaling through the glucocorticoid receptor (GR) represents a clinically important endogenous anti-inflammatory pathway. Microarray analysis reveals that the GR is transcriptionally up-regulated by hypoxia in human renal proximal tubular epithelial cells. Hypoxic up-regulation of the GR was confirmed at the level of promoter activity, mRNA, and protein expression. Furthermore, functional potentiation of glucocorticoid activity in hypoxia was observed as an enhancement of dexamethasone-induced glucocorticoid response element promoter activity and enhanced dexamethasone-mediated inhibition of IL-1beta-stimulated IL-8 expression and hypoxia-induced vascular endothelial growth factor expression. Knockdown of enhanced GR gene expression in hypoxia using specific GR small inhibitory RNA (siRNA) resulted in an attenuation of the enhanced glucocorticoid sensitivity. A role for the hypoxia-inducible transcription factor, HIF-1alpha, in the regulation of GR expression and the associated potentiation of glucocorticoid activity in hypoxia was also demonstrated. These results reveal a novel signaling aspect responsible for the incorporation of hypoxic and glucocorticoid stimuli, which we hypothesize to be an important co-operative pathway for the control of gene expression observed in complex tissue microenvironments in inflamed states.

Cell Hypoxia↗