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Biomedical subjects

G Fink

Publications and source records attributed to G Fink.

At least 19 recordsLinked to original sources

MAP kinase and cAMP filamentation signaling pathways converge on the unusually large promoter of the yeast FLO11 gene.

In Saccharomyces cerevisiae, two major signal transduction pathways, the Kss1 MAPK pathway and the cAMP-regulated pathway, are critical for the differentiation of round yeast form cells to multicellular, invasive pseudohyphae. Here we report that these parallel pathways converge on the promoter of a gene, FLO11, which encodes a cell surface protein required for pseudohyphal formation. The FLO11 promoter is unusually large, containing at least four upstream activation sequences (UASs) and nine repression elements which together span at least 2.8 kb. Several lines of evidence indicate that the MAPK and cAMP signals are received by distinct transcription factors and promoter elements. First, regulation via the MAPK pathway requires the transcription factors Ste12p/Tec1p, whereas cAMP-mediated activation requires a distinct factor, Flo8p. Secondly, mutations in either pathway block FLO11 transcription. Overexpression of STE12 can suppress the loss of FLO8, and overexpression of FLO8 can suppress the loss of STE12. Finally, multiple distinct promoter regions of the FLO11 promoter are required for its activation by either Flo8p or Ste12p/ Tec1p. Thus, like the promoters of the key developmental genes, HO and IME1, the FLO11 promoter is large and complex, endowing it with the ability to integrate multiple inputs.

Calcium-Calmodulin-Dependent Protein Kinases

Serotonin transporter (SERT) mRNA and binding site densities in male rat brain affected by sex steroids.

Estrogen increases serotonin transporter (SERT) mRNA and binding sites in female rat brain. In order to determine whether changes in SERT are gender- and steroid-specific we have now carried out studies on adult male Wistar rats which were either intact or castrated (under halothane anesthesia) and injected with arachis oil, estradiol benzoate (EB), testosterone propionate (TP) or the non-aromatizable androgen, 5alpha-dihydrotestosterone (5alpha-DHT). The number of SERT mRNA-expressing cells in the dorsal raphe (DR) nucleus was decreased by castration and increased by treatment (for approximately 32 h) with EB or TP, but not 5alpha-DHT. Sex steroids had no effect on the number of SERT mRNA-expressing cells in the median raphe nucleus. The density of SERT sites, assessed by autoradiography of [3H]paroxetine binding, was significantly reduced in arcuate nucleus and median raphe after castration, and increased in arcuate, basolateral amygdala and ventromedial hypothalamic nucleus by treatment with EB or TP, but not 5alpha-DHT. Estradiol, but not testosterone or 5alpha-DHT reduced the density of SERT sites in midbrain central grey. These data show that testosterone as well as estrogen affects SERT expression in male brain, and that the action of testosterone probably depends upon its enzymatic conversion, by aromatase, to estradiol. Our findings may have implications for sex steroid control of mood and behavior, and the action of neurotoxic derivatives of amphetamine, such as 3, 4-methylenedioxymethamphetamine, in the human.

Animals

Serotonergic 5-HT2A receptors important for the oestradiol-induced surge of luteinising hormone-releasing hormone in the rat.

Serotonin (5-HT) plays a role in mediating the oestradiol-induced surge of luteinising hormone (LH), but so far the 5-HT receptor subtype involved has not been identified. Our previous in-situ hybridization and pharmacological studies suggest that the action of 5-HT involves the 5-HT2A receptor. The aim of the present study was to investigate this possibility by the direct approach of determining whether 5-HT2A receptor antagonists block the oestradiol-induced surge of luteinising hormone releasing hormone (LHRH). Adult female Wistar rats, which had shown at least two consecutive 4-day oestrous cycles, were ovariectomised under halothane anaesthesia in the morning of dioestrus and injected with vehicle (arachis oil) alone or oestradiol benzoate (OB). At 12.00 h of the next day, presumptive pro-oestrus, the animals were injected intraperitoneally with one of three 5-HT2A antagonists, a selective 5-HT reuptake inhibitor (fluoxetine), or the appropriate vehicles; hypophysial portal blood was then collected under alphaxalone anaesthesia between 15.00 and 19.00 h. The amount of LHRH released into hypophysial portal blood during consecutive 30-min periods was determined by radioimmunoassay. As expected, oestradiol, but not oil, triggered a surge of LHRH in hypophysial portal blood with a peak at about 16.00 h of presumptive pro-oestrus. This oestradiol-induced surge of LHRH was blocked by ketanserin, ritanserin and the highly selective 5-HT2A receptor antagonist, RP62203, but not by fluoxetine. These results provide the first direct evidence that the 5-HT2A receptor plays an important role in the oestradiol-induced surge of LHRH.

Anesthesia

Ras signaling is required for serum-induced hyphal differentiation in Candida albicans.

Serum induces Candida albicans to make a rapid morphological change from the yeast cell form to hyphae. Contrary to the previous reports, we found that serum albumin does not play a critical role in this morphological change. Instead, a filtrate (molecular mass, <1 kDa) devoid of serum albumin induces hyphae. To study genes controlling this response, we have isolated the RAS1 gene from C. albicans by complementation. The Candida Ras1 protein, like Ras1 and Ras2 of Saccharomyces cerevisiae, has a long C-terminal extension. Although RAS1 appears to be the only RAS gene present in the C. albicans genome, strains homozygous for a deletion of RAS1 (ras1-2/ras1-3) are viable. The Candida ras1-2/ras1-3 mutant fails to form germ tubes and hyphae in response to serum or to a serum filtrate but does form pseudohyphae. Moreover, strains expressing the dominant active RAS1(V13) allele manifest enhanced hyphal growth, whereas those expressing a dominant negative RAS1(A16) allele show reduced hyphal growth. These data show that low-molecular-weight molecules in serum induce hyphal differentiation in C. albicans through a Ras-mediated signal transduction pathway.

Amino Acid Sequence

The occurrence, recrudescence, and worsening of asthma in a population of young adults: impact of varying types of occupation.

OBJECTIVE: To describe the rates of exacerbation of existing asthma and incidence of new disease in Israeli men during military service. DESIGN: All 17-year-old Israeli nationals are obliged by law to appear at the Israel Defense Forces (IDF) recruiting office for medical examination. The medical history of army recruits was noted during the 30-month period after their induction into the IDF, and medical examinations were performed by pulmonary specialists in all suspected cases of asthma. The duty status of the soldiers in combat units (CUs), maintenance units (MUs), and clerical tasks was related to their asthma status. RESULTS: Of a total of 59,058 recruits, 1.0% developed asthma during the 30 months of this study; of those in CUs, 1.2% developed asthma; of those in MUs, 0.8% developed asthma; and of those performing clerical tasks, 0.6% developed asthma. The relative risk for developing or worsening of asthma was related to both the preexisting asthma status of the recruit and the environment in which he carried out his military service. The annual incidence of occupational-related asthma in MUs was found to be 800/million: five to six times the rates reported elsewhere. CONCLUSIONS: Service in CUs was associated with an increased frequency of exacerbation of asthma among recruits with previous disease and with the appearance of disease de novo. "Normal" conscripts with a history of childhood asthma are at a higher risk of developing overt asthma when compared to subjects with no such history. We found a 25% relative excess of incident cases of asthma in soldiers posted in MUs compared to those performing clerical tasks [(0.8 to 0.6%)/0.8%]. This difference is probably attributed to the difference in occupational hazards in these categories. Further studies are needed to determine if this represents the elicitation of underlying preexisting airway lability by new work demands or other environmental conditions, or if this represents a new development of airway lability because of specific immune or nonimmune factors.

Adolescent

Ca2+/calcineurin-inhibited adenylyl cyclase, highly abundant in forebrain regions, is important for learning and memory.

Activation of cAMP synthesis by intracellular Ca2+ is thought to be the main mode of cAMP generation in the brain. Accordingly, the Ca2+-activated adenylyl cyclases I and VIII are expressed prominently in forebrain neurons. The present study shows that the novel adenylyl cyclase type IX is inhibited by Ca2+ and that this effect is blocked selectively by inhibitors of calcineurin such as FK506 and cyclosporin A. Moreover, adenylyl cyclase IX is inhibited by the same range of intracellular free Ca2+ concentrations that stimulate adenylyl cyclase I. Adenylyl cyclase IX is expressed prominently in the forebrain. Substantial arrays of neurons positive for AC9 mRNA were found in the olfactory lobe, in limbic and neocortical areas, in the striatum, and in the cerebellar system. These data show that the initiation of the cAMP signal by adenylyl cyclase may be controlled by Ca2+/calcineurin and thus provide evidence for a novel mode of tuning the cAMP signal by protein phosphorylation/dephosphorylation cascades.

Adenylyl Cyclase Inhibitors

Testosterone as well as estrogen increases serotonin2A receptor mRNA and binding site densities in the male rat brain.

Our previous findings in female rats suggest that the potent effects of sex steroids on mood and mental state may be mediated, in part, by the effect of estrogen on the 5-hydroxytryptamine2A receptor (5-HT2AR) in brain. The aim of the present study was to determine the effect of acute (approximately 32h) sex steroid manipulation on central 5-HT2AR in the adult male Wistar rat. Castration (under halothane anesthesia) decreased while testosterone or estrogen, but not 5alpha-dihydrotestosterone (5alpha-DHT), increased significantly the 5-HT2AR mRNA content in dorsal raphe nucleus and the density of 5-HT2AR binding sites in frontal, cingulate and primary olfactory cortex and nucleus accumbens. The lack of effect of 5alpha-DHT, a potent androgen which cannot be converted to estrogen, suggests that the action of testosterone depends upon its conversion to estrogen by aromatase. This may also explain why estrogen, but not testosterone or 5alpha-DHT, increased the density of 5-HT2AR binding sites in the caudate-putamen, a brain region where aromatase is scarce. These findings are discussed in relation to the possible role of the 5-HT2AR in depression, schizophrenia and Alzheimer's Disease.

Animals

Ethene-Norbornene Copolymerization Using Homogenous Metallocene and Half-Sandwich Catalysts: Kinetics and Relationships between Catalyst Structure and Polymer Structure. 1. Kinetics of the Ethene-Norbornene Copolymerization Using the

The kinetics of the ethene-norbornene copolymerization using the [(isopropylidene)(eta5-inden-1-ylidene-eta5-cyclopentadienyl)]zirconium dichloride (iPr[IndCp]ZrCl2)/methylaluminoxane catalyst has been investigated at 70 degreesC in a concentrated solution of norbornene in toluene and an ethene pressure ranging from 4 to 60 bar (58-870 psi). The ethene reaction rate has been measured during the copolymerization process at varying reactant concentrations. The reaction orders and rate constants were determined and compared to the corresponding values of the ethene and norbornene homopolymerizations. It was found that kEhomo > kEco and kNhomo < kNco. The copolymerization parameters are r1 = 0.9 and r2 = 0.05.

Journal Article

Ethene-Norbornene Copolymerization Using Homogenous Metallocene and Half-Sandwich Catalysts: Kinetics and Relationships between Catalyst Structure and Polymer Structure. 2. Comparative Study of Different Metallocene- and Half-Sandwich/Methylaluminoxane Catalysts and Analysis of the Copolymers by 13C Nuclear Magnetic Resonance Spectroscopy.

The kinetics of ethene-norbornene copolymerizations using the metallocenes iPr[(3-R-Cp)Ind]ZrCl2 (with R = methyl or tert-butyl), MeCH[Cp]2ZrCl2, iPr[(3-R-Cp)Flu]ZrCl2 (with R = H, methyl, isopropyl, or tert-butyl), and Me2Si[(3-tert-butyl-Cp)Flu]ZrCl2 and half-sandwich catalysts Me2Si[Me4CpNtBu]TiCl2, Me2Si[Me4CpNtBu]ZrCl2, Me2Si[FluNtBu]ZrCl2, R-(+)-Me2Si[Me4CpNCH(CH3)-1-naphthyl]TiCl2, and C2H4[Me4CpNMe2]Cr(eta1,eta1-C4H8) together with methylaluminoxane as cocatalyst, have been investigated at 70 degreesC in a concentrated solution of norbornene in toluene and under an ethene pressure ranging from 4 to 60 bar (58-870 psi). The ethene reaction rates were measured during the copolymerization process at various ethene concentrations and the ethene reaction orders were determined. In some cases fractional ethene reaction orders higher than 1 were found, indicating a complex mechanism. The microstructure of the copolymers were analyzed by 13C NMR spectroscopy. The highest norbornene contents were achieved using metallocenes with sterically less demanding ligands such as MeCH[Cp]2ZrCl2. Unexpectedly, low norbornene contents (<50 mol %) were achieved with the half-sandwich catalysts. Depending on the catalyst structure, the microstructure of the copolymers consists of mainly isolated norbornene units, alternating monomer sequences or short norbornene microblocks with a maximum length of two or three. Additionally, the tacticity of the norbornene microblocks could be controlled by the catalyst structure. A mechanistic model, based on chain migratory insertion, is presented to explain the different copolymer structures through nonbonding steric interactions between monomer, growing polymer chain, and ligand system. On the basis of this model, penultimate effects (Markov statistic second order) caused by the last two inserted monomer units can be assumed.

Journal Article

Ethene-Norbornene Copolymerization with Homogeneous Metallocene and Half-Sandwich Catalysts: Kinetics and Relationships between Catalyst Structure and Polymer Structure. 3. Copolymerization Parameters and Copolymerization Diagrams.

The copolymerization parameters of the ethene-norbornene copolymerization using various metallocene and half-sandwich catalysts were determined on the basis of composition data with linear graphical methods: Aside from r1 and r2, values for r11 and r21 could be determined in the case of alternating copolymerizations. In this connection, r21 differs significantly from catalyst to catalyst and reflects the different tendency of each catalyst to produce copolymers with an alternating monomer sequence. The maximum norbornene contents in the copolymer could be achieved by use of sterically less hindered metallocene catalysts. As an exception to this, the half-sandwich catalysts produced copolymers with surprisingly low norbornene contents.

Journal Article

Ethene-Norbornene Copolymerization with Homogeneous Metallocene and Half-Sandwich Catalysts: Kinetics and Relationships between Catalyst Structure and Polymer Structure. 4. Development of Molecular Weights.

The molecular weights of ethene-norbornene copolymers, produced with various metallocene- and amidocyclopentadienyl-methylaluminoxane (MAO) catalysts, have been determined by high-temperature gel-permeation chromatography: with one exception, increasing molecular weights were found with an increasing norbornene content in the copolymer. This observation is due to the fact that the beta-hydride elimination process is not possible in the case of norbornene, because of special steric conditions of the cis-2,3-exo inserted norbornene; other possible termination reactions are discussed, considering the specialties of the norbornene. The effect of the catalyst structure on the molecular weights is discussed in terms of steric and electronic factors of the different ligands: sterically less hindered ligands produce ethene-norbornene copolymers with relatively low molecular weights; ligands with a larger extension and electron-pushing effect such as the fluorenyl ligand, induced the formation of high molecular weights. Additional alkyl groups at the indenyl or fluorenyl ligand increased the molecular weights additionally.

Journal Article

Rhodopsin-family receptors associate with small G proteins to activate phospholipase D.

G-protein-coupled receptors of the rhodopsin family transduce many important neural and endocrine signals. These receptors activate heterotrimeric G proteins and in many cases also cause activation of phospholipase D, an enzyme that can be controlled by the small G proteins ARF and RhoA. Here we show that the activation of phospholipase D that is induced by many, but not all, Ca2+-mobilizing G-protein-coupled receptors is sensitive to inhibitors of ARF and of RhoA. Receptors of this type were co-immunoprecipitated with ARF or RhoA on exposure to agonists, and the effects of GTP analogues on ligand binding to the receptor changed to a profile that is characteristic of small G proteins. These receptors contain the amino-acid sequence AsnProXXTyr in their seventh transmembrane domain, whereas receptors capable of activating phospholipase D without involving ARF contain the sequence AspProXXTyr. Mutation of this latter sequence to AsnProXXTyr in the gonadotropin-releasing hormone receptor conferred sensitivity to an inhibitor of ARF, and the reciprocal mutation in the 5-HT2A receptor for 5-hydroxy-tryptamine reduced its sensitivity to the inhibitor. Receptors carrying the AsnProXXTyr motif thus seem to form functional complexes with ARF and RhoA.

ADP-Ribosylation Factors

Significance of prominent atrial-wave and diastasis deflection in radionuclide diastolic volume curve during exercise in detection of coronary artery disease.

BACKGROUND: Exercise testing with multigated acquisition technetium radionuclide cineangiography (MUGA) is a useful modality that can discriminate systolic and diastolic performance in patients with ischemic heart disease. However, some patients may have abnormal left ventricular filling dynamics with normal regional and global systolic function. HYPOTHESIS: The purpose of the study was to assess exercise-induced diastolic dysfunction as expressed by a prominent atrial (A) wave or diastasis deflection at the left ventricular volume curve, in patients with different degrees of ischemic heart disease. METHODS: In all, 32 men and 7 women aged 35-70 years (mean 54 +/- 8.6 years) underwent MUGA at rest and during exercise for analysis of the radionuclide volume curve. Within 6 weeks, thallium-201 scintigraphy and coronary angiography were performed and the patients were categorized into three groups: (1) disease-free (n = 10), (2) single-vessel disease (> 50% stenosis) (n = 19), and (3) double-vessel disease or more (n = 10). A waves or diastasis deflections were compared among the groups. RESULTS: Significant differences (p < 0.01) were noted in A-wave deflection relative to peak diastolic volume curve during exercise (Aexe/T) between Group 1 and Groups 2 and 3. Group 1 manifested only a mild rise in A-wave deflection from rest (20.20 +/- 8.49%) to exercise (25.85 +/- 8.49%), whereas Groups 2 and 3 exhibited a significant increase from 25.89 +/- 9.55% and 28.40 +/- 12.6%, respectively, to 60.21 +/- 22.5% and 63.0 +/- 22.86%, respectively. Group 2 had a significantly (p < 0.05) higher maximal heart rate than Group 3. CONCLUSIONS: The addition of prominent A-wave or diastasis deflection to a normal systolic response during exercise testing with multigated radionuclide cineangiography might be a sensitive marker of coronary artery disease. The A wave represents diastolic dysfunction of the left ventricle, considered an early event in the ischemic cascade.

Adult

TNF but not IL-1 decreases pancreatic acinar cell survival without affecting exocrine function: a study in the perfused human pancreas.

Substantial quantities of interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) are produced within the pancreatic parenchyma during acute pancreatitis. Recent evidence suggests that IL-1 beta and TNF-alpha propagate acute pancreatitis and intensify the resulting pancreatic acinar cell death. This study examines the direct effect of IL-1 beta and TNF-alpha on pancreatic acinar cells. Human pancreata (n = 6), harvested during organ procurement, were perfused ex vivo through the splenic artery using a sterile, oxygenated colloid solution. Each pancreas was perfused with either recombinant human IL-1 beta or TNF-alpha for 2 h and subsequently with the cholecystokinin analogue caerulein (positive control). Venous effluent was collected continuously and amylase and lipase were determined at 15-min intervals. Pancreatic histology was graded at baseline and following cytokine and caerulein perfusion. To examine the long-term effects of these cytokines on acinar cell viability, additional in vitro studies utilized the AR42J acinar cell line which was exposed to either IL-1 beta or TNF-alpha with survival determined daily by MTT assay. Perfusion of the human pancreas with either IL-1 beta or TNF-alpha did not alter amylase, lipase, or histology. Caerulein did induce pancreatitis as measured by increased amylase, lipase, and pancreatic histology. Survival of pancreatic acinar cells decreased when they were incubated with TNF-alpha but not IL-1 beta. Although present in large amounts within the pancreas during acute pancreatitis, IL-1 beta and TNF-alpha have no direct effect on acinar cell viability or exocrine function acutely nor do they induce pancreatitis. When present for more than 24 h, however, TNF-alpha but not IL-1 beta has a dramatic effect on acinar cell survival.

Acute Disease

Pulmonary function threshold for distinguishing ventilatory- and nonventilatory-limited patients with airflow obstruction.

Patients with chronic obstructive pulmonary disease (COPD) may demonstrate great variability between results on the pulmonary function test (PFT) compared to those on the cardiopulmonary exercise test (CPXT). The purpose of this study was to correlate PFT and CPXT indices and to identify PFT threshold values for predicting exercise capacity in patients with airflow limitation. Fifty-seven patients (48 men and 9 women) of mean age 66.4 +/- 4.8 years with COPD and 40 age-matched control patients underwent PFT and CPXT. Based on the CPXT results, the patients were divided into ventilatory-limited (VL) and nonventilatory-limited (NVL), and the findings were correlated with the PFT indices. Linear regression analysis was used to determine the relationship between dyspnea index (VEmax/MVV) and forced expiratory volume in one second (FEV1). The cutoff value for VL was FEV1 < 38% and for NVL FEV1 > 68%. The prominent limiting symptom (61%) in the VL group was dyspnea sensation, with leg discomfort presenting in only 14%; corresponding rates in the NVL group were 38% and 31%. We conclude that the FEV1 is a reliable index for distinguishing VL from NVL COPD patients during CPXT at two extremes: below 38% of the predicted value (VL) and above 68% of the predicted value (NVL).

Aged

Sex steroid control of mood, mental state and memory.

1. Sex steroid hormones exert profound effects on mood and mental state. Thus, in women, oestrogen is thought to protect against depression and delay the onset of schizophrenia and Alzheimer's disease. 2. Our studies in the female rat show that oestradiol, in its positive feedback mode for gonadotrophin release, increases the expression of genes for the 5-hydroxytryptamine 5-HT2A receptor and the serotonin transporter (SERT) in the dorsal raphe nucleus and the density of 5-HT2A receptor and SERT sites in regions of the forebrain that, in the human, are concerned with cognition, mental state, emotion and memory. 3. In the male rat, castration decreases while oestrogen and testosterone, but not 5 alpha-dihydrotestosterone (5 alpha-DHT), increase the density of 5-HT2A receptors in forebrain. The fact that 5 alpha-DHT has no effect suggests that the action of testosterone depends on its conversion to oestradiol by aromatase. 4. In intact rats, the density of 5-HT2A receptors in cerebral cortex is significantly higher in pro-oestrous female than in male and dioestrous female rats, showing that the spontaneous, preovulatory surge of oestradiol that reaches a peak at 12.00 h of pro-oestrus also increases the density of 5-HT2A receptors in cortex. 5. Oestrogen and testosterone (by way of its conversion to oestrogen) also stimulate the expression of the arginine vasopressin gene in the bed nucleus of the stria terminalis of the rodent, a mechanism that plays a key role in olfactory memory. 6. These actions of sex steroid hormones are discussed in the context of genomic versus non-genomic mechanisms, the recent discovery that there are two oestradiol receptors with different distributions in brain, the significance of our findings for our understanding of the control of mood, mental state and memory and the mechanism by which oestrogen stimulation of the 5-HT2A receptor could delay the onset of Alzheimer's disease.

Affect

Assessment of functional results after percutaneous transluminal coronary angioplasty by cardiopulmonary exercise test.

Twenty-nine patients with documented coronary artery disease underwent cardiopulmonary exercise tests before and following a percutaneous transluminal coronary angioplasty (PTCA). The patients medication regimen and exercise protocols remained the same in both cases. Following PTCA, significant improvement (p < 0.001-0.0001) was noted in oxygen consumption (1,526.8 +/- 470.0 vs. 1,686.2 +/- 390 ml/min), oxygen pulse (12.40 +/- 2.73 vs. 13.44 +/- 2.9 ml/beat), oxygen pulse score (7.62 +/- 1.29 vs. 8.85 +/- 1.26 points) and in the ventilatory anaerobic threshold (993.1 +/- 177.6 vs. 1,089.8 +/- 150.9 ml/min) but not (p > 0.05) in maximal heart rate (128.7 +/- 16.9 vs. 132.0 +/- 17.2 beats/min). Thus, a cardiopulmonary exercise test is an effective method to assess functional results following PTCA.

Adult