Biomedical subjects

X Zhou

Publications and source records attributed to X Zhou.

At least 433 records · Page 24Linked to original sources

PubMed1998

Effect of insulin-like growth factors (IGFs) and IGF-binding proteins on in vitro sperm motility.

OBJECTIVE: Previous studies have shown that exogenous growth hormone (GH) produces increases in sperm motility when given to subfertile men. Previous studies have also demonstrated the presence of IGFs and IGFBPs in seminal plasma. We have therefore investigated the effects of insulin-like growth factor-I (IGF-I), IGF-II, IGF-binding protein 2 (IGFBP-2) and intact IGFBP-3 on in vitro sperm motility. DESIGN AND METHODS: Using computer-aided sperm analysis, we investigated the effects of IGFs and IGFBPs on the in vitro sperm motility parameters: curvilinear velocity (CV), progressive velocity (PV), linearity (Ln), straightness (St), amplitude of lateral head movement (ALH), and beat frequency (BF). Washed motile sperm selected by the 'swim-up' method, from normozoospermic samples, were incubated at 37 degrees C in 5% CO2 in air with IGF-I, IGF-II, IGFBP-2, IGFBP-3, or control Earle's media, and were examined at time 0 and after 60 min incubation. Changes in motility parameters after 60 min incubation were compared with controls by analysis of variance (ANOVA). RESULTS: Compared to controls, statistically significant changes occurred at time 60 min after incubation: IGF-I decreased CV and ALH significantly (P < 0.05), but IGFBP-3 increased Ln, St, BF, and decreased ALH significantly (P < 0.05). In contrast, IGF-II, IGFBP-2, and a combination of IGF-I/IGFBP-3, had no significant effects. CONCLUSIONS: IGF-I and IGFBP-3 have differing and opposing effects on in vitro sperm motility parameters and thus may have a role in modulating in vivo sperm motility.

Cell Separation↗

PubMed1998

Heat shock transcription factor-1 regulates heat shock protein-72 expression in human keratinocytes exposed to ultraviolet B light.

In response to ultraviolet radiation (UVR), skin keratinocytes increase expression of heat shock proteins that can protect cells from stress-induced damage. This heat shock response is known to be transcriptionally regulated in eukaryotic cells exposed to certain forms of environmental stress. In the skin, absorption of ultraviolet B light occurs primarily in the epidermis, and therefore, using primary cultures of normal human epidermal keratinocytes, we have examined whether transcriptional activation of the hsp72 gene occurs following UVB irradiation. Cultured keratinocytes were exposed to UVB (290-320 nm, 300 J per m2) and then incubated at 37 degrees C for various intervals before harvesting. Immediately following UV exposure, the heat shock transcription factor 1 (HSF1) dissociated from HSP72-HSF1 complexes, underwent trimerization and phosphorylation, and demonstrated DNA binding activity to the heat shock element in the promoter region of the hsp72 gene. UVB also increased hsp72 mRNA, with peak levels observed 1-3 h post-UVR. HSP72 protein was constitutively expressed in keratinocytes, and its expression was increased by UVB, with maximum levels at 6 h post-UVR. The stress response may be extremely important in the protection of human skin from UVB radiation, and modulation of heat shock protein expression and/or function offers a potential therapeutic target in the prevention of photoaging and skin cancer.

Cells, Cultured↗

PubMed1998

Clinical performance of the AMDL DR-70 immunoassay kit for cancer detection.

A clinical study using DR-70 immunoassay for the detection of 13 different cancers have been conducted with 277 healthy subjects and 136 cancer patients. The test results showed that the DR-70 immunoassay kit was capable of detecting cancers with high degree of specificity and sensitivity. At 95% specificity level, the sensitivity of the assay was 87.8%, 92.6%, 65.2% and 66.7%, respectively for lung, stomach, breast and rectum cancers. Furthermore the test kits were shown to be stable and performed reproducibly.

Adolescent↗

PubMed1998

Constructing and counting phylogenetic invariants.

The method of invariants is an approach to the problem of reconstructing the phylogenetic tree of a collection of m taxa using nucleotide sequence data. Models for the respective probabilities of the 4m possible vectors of bases at a given site will have unknown parameters that describe the random mechanism by which substitution occurs along the branches of a putative phylogenetic tree. An invariant is a polynomial in these probabilities that, for a given phylogeny, is zero for all choices of the substitution mechanism parameters. If the invariant is typically non-zero for another phylogenetic tree, then estimates of the invariant can be used as evidence to support one phylogeny over another. Previous work of Evans and Speed showed that, for certain commonly used substitution models, the problem of finding a minimal generating set for the ideal of invariants can be reduced to the linear algebra problem of finding a basis for a certain lattice (that is, a free Z-module). They also conjectured that the cardinality of such a generating set can be computed using a simple "degrees of freedom" formula. We verify this conjecture. Along the way, we explain in detail how the observations of Evans and Speed lead to a simple, computationally feasible algorithm for constructing a minimal generating set.

Algorithms↗

PubMed1998

Influence of nitric oxide on vascular, metabolic, and contractile responses to dobutamine in in situ canine hearts.

UNLABELLED: The left anterior descending coronary arteries of 30 anesthetized, open-chest dogs were perfused via an extracorporeal circuit. Coronary blood flow (CBF), myocardial oxygen consumption (MVO2), and segmental shortening (SS) were measured. Studies were performed with coronary perfusion pressure (CPP) or CBF constant. With CPP constant, effects of intracoronary (IC) infusions of dobutamine (2.5, 5.0, or 10.0 microg/min) were evaluated alone (control) and after inhibition of nitric oxide (NO) synthase with NG-nitro-L-arginine methyl ester (L-NAME). With CBF constant, a NO donor (sodium nitroprusside [SNP] 80 microg/min IC) or nitroglycerin [NTG] 40 microg/min IC) or a releaser of endogenous NO (acetylcholine [ACh]; 20 microg/ min IC) was infused along with dobutamine. Increases in CBF during dobutamine and isoproterenol were compared before and after blockade of beta1-adrenergic receptors with atenolol. Dobutamine caused proportional, dose-dependent increases in CBF, MVO2, and SS, which were not altered by L-NAME. Administration of the NO donors or ACh during dobutamine markedly decreased CPP, but only ACh also reduced SS and MVO2. These latter effects persisted after L-NAME. Atenolol blunted increases in CBF by dobutamine more than those by isoproterenol. We conclude that endogenous NO did not modulate the coronary vasodilation or the increases in myocardial contractility and MVO2 during dobutamine. In addition, neither SNP nor NTG altered myocardial contractility or MVO2 in dobutamine-stimulated myocardium, whereas ACh had a negative inotropic effect in dobutamine-stimulated myocardium that was independent of NO. IMPLICATIONS: Endogenous nitric oxide (NO) did not modulate increases in coronary blood flow, myocardial contractility, or myocardial oxygen consumption during intracoronary infusions of dobutamine. The NO donors sodium nitroprusside and nitroglycerin had no effect on contractility or oxygen consumption in dobutamine-stimulated myocardium. Acetylcholine had negative inotropic effect in dobutamine-stimulated myocardium that was independent of NO.

Animals↗

PubMed1998

Is calcium a coronary vasoconstrictor in vivo?

BACKGROUND: Calcium produces constriction in isolated coronary vessels and in the coronary circulation of isolated hearts, but the importance of this mechanism in vivo remains controversial. METHODS: The left anterior descending coronary arteries of 20 anesthetized dogs whose chests had been opened were perfused at 80 mmHg. Myocardial segmental shortening was measured with ultrasonic crystals and coronary blood flow with a Doppler flow transducer. The coronary arteriovenous oxygen difference was determined and used to calculate myocardial oxygen consumption and the myocardial oxygen extraction ratio. The myocardial oxygen extraction ratio served as an index of effectiveness of metabolic vasodilation. Data were obtained during intracoronary infusions of CaCl2 (5, 10, and 15 mg/min) and compared with those during intracoronary infusions of dobutamine (2.5, 5.0, and 10.0 microg/min). RESULTS: CaCl2 caused dose-dependent increases in segmental shortening, accompanied by proportional increases in myocardial oxygen consumption. Although CaCl2 also increased coronary blood flow, these increases were less than proportional to those in myocardial oxygen consumption, and therefore the myocardial oxygen extraction ratio increased. Dobutamine caused dose-dependent increases in segmental shortening and myocardial oxygen consumption that were similar in magnitude to those caused by CaCl2. In contrast to CaCl2, however, the accompanying increases in coronary blood flow were proportional to the increases in myocardial oxygen consumption, with the result that the myocardial oxygen extraction ratio remained constant. CONCLUSIONS: Calcium has a coronary vasoconstricting effect and a positive inotropic effect in vivo. This vasoconstricting effect impairs coupling of coronary blood flow to the augmented myocardial oxygen demand by metabolic vascular control mechanisms. Dobutamine is an inotropic agent with no apparent direct action on coronary resistance vessels in vivo.

Animals↗

PubMed1998

Isoflurane-induced dilation of porcine coronary arterioles is mediated by ATP-sensitive potassium channels.

BACKGROUND: Isoflurane causes increases in coronary blood flow in vivo, which are mediated by the adenosine triphosphate (ATP)-sensitive potassium channels, but the role of the arterioles (resistance vessels) in these responses is controversial. METHODS: Medium porcine coronary arterioles (internal diameter, 172 +/- 51 [SD] microm) were placed in a chamber supplied with Kreb's buffer, pressurized (40 mmHg), and preconstricted with acetylcholine (10(-8)-10(-6) M). Vascular diameter (VD) was assessed using an optical density video-detection system. Isoflurane (in 95% oxygen and 5% carbon dioxide) was added to buffer using a membrane oxygenator supplied by a calibrated vaporizer. In series 1 (n = 14), 2% isoflurane was administered according to an abrupt (ISO-A) and gradual (ISO-G) protocol. In series 2 (n = 13) and 3 (n = 6), ISO-A (1.5%) was assessed before and after glibenclamide (an ATP-sensitive potassium channel antagonist) or 8-phenyltheophylline (a nonselective adenosine receptor antagonist), respectively. In series 4 (n = 5), validation studies were performed using sodium nitroprusside and adenosine diphosphate to verify that the vascular smooth muscle and endothelium of the vessels were functionally intact. In series 5 (n = 6), ISO-A (0.75 and 1.5%) was compared during preconstriction with acetylcholine and the thromboxane analog U46619 (10(-6) M). RESULTS: ISO-G caused essentially concentration-dependent increases in VD. At 2% isoflurane, the increases in VD were greater during ISO-A than ISO-G. Glibenclamide, but not 8-phenyltheophylline, attenuated isoflurane-induced increases in VD. Both sodium nitroprusside and adenosine diphosphate caused dose-dependent increases in VD. Isoflurane caused equivalent concentration-dependent increases in VD during acetylcholine and U46619. CONCLUSIONS: Isoflurane is a concentration-dependent dilator of porcine coronary arterioles preconstricted with acetylcholine or U46619. This effect is blunted by gradual administration, suggesting that the vessels may adapt to the relaxing effects of isoflurane. Isoflurane-induced dilation of coronary arterioles is mediated by the ATP-sensitive potassium channels but not by the adenosine receptors.

Anesthetics, Inhalation↗

PubMed1998

Tetramethylpyrazine, a Chinese drug, blocks coronary vasoconstriction by endothelin-1 and decreases plasma endothelin-1 levels in experimental animals.

The purpose of this study was to investigate effects of tetramethylpyrazine (TMP), a Chinese plant-derived medicine, on coronary vasoconstriction and related electrocardiographic and histologic changes caused by endothelin-1 (ET-1), and on plasma ET-1 levels. ET-1 (75 pmol) was administered into the left coronary artery (LCA) in anesthetized closed-chest dogs with and without prior infusion of TMP (80 mg/kg). Coronary arterial diameter (CAD) was determined by coronary arteriography (CAG). Blood pressure and electrocardiogram (ECG) were monitored continuously. Histologic damage in tissues was ascertained microscopically. Plasma ET-1 and 6-keto-PGF1 levels were determined by RIA 90 min after i.v. injection of TMP (25 mg/kg; n = 5) in rabbits. Intracoronary injection of ET-1 resulted in a significant vasoconstriction of the entire vascular bed of the LCA, with a decrease in CAD of 35.9 +/- 5.7% (n = 5; p < 0.01) and ischemic changes on ECG and in tissues of endocardium, myocardium, coronary endothelial cells, and capillary vessels. Pretreatment with TMP produced a significant increase in CAD by 38.5 +/- 7.8% (n = 5; p < 0.01) and greatly suppressed the vasoconstriction produced by ET-1. The myocardial tissue damage estimated from the ratio of ischemic area for the entire area after ET-1 injection (35.6%) was completely abolished by TMP (0.6%). In addition, TMP injection induced a significant decrease in plasma ET-1 levels and an increase in 6-keto-PGF1 levels in rabbits. The Chinese medicine TMP could be a useful therapeutic agent in ischemic heart disease by suppressing coronary vasoconstriction and ischemic changes in the tissues produced by ET-1.

6-Ketoprostaglandin F1 alpha↗

PubMed1998

Chemotherapy-induced apoptosis in melanoma cells is p53 dependent.

Metastatic melanomas are often resistant to chemotherapy. To study whether the p53 mutational status affects chemosensitivity, we compared the responses to chemotherapy of four melanoma cell lines containing the wild-type p53 and four cell lines carrying the mutant p53. Cisplatin, at 10 microM, virtually killed all the cells in the wild-type p53 cell lines, while 57-95% of the cells in the mutant p53 cell lines survived (P = 0.005). After treatment with 100 nM of vincristine, on average 18% of the wild-type p53 melanoma cells survived compared with 55% of the mutant p53 cells (P = 0.04). After treatment with 40 nM, 200 nM or 1 microM of camptothecin the survival rates were, on average, 16%, 8% and 4% for the wild-type p53 melanoma cells, compared with 89%, 67% and 38% for the mutant p53 cells, respectively (P = 0.00004, P = 0.003 and P = 0.04, respectively). The anticancer agents were not toxic to normal melanocytes at doses inducing cytotoxicity in wild-type p53 melanoma cells. The main mechanism of cytotoxicity appears to be drug-induced apoptosis. Cisplatin, camptothecin and vincristine all induced apoptosis in wild-type p53 melanoma cells, but not in mutant p53 cells. Our results suggest that chemotherapy-induced apoptosis in melanoma cells is p53 dependent, and mutation of the p53 gene is an indicator of drug resistance in melanoma.

Antineoplastic Agents↗

PubMed1998

Types of variation in DNA-A among isolates of East African cassava mosaic virus from Kenya, Malawi and Tanzania.

Complete nucleotide sequences of the DNA-A-like molecules of three East African cassava mosaic virus (EACMV) isolates from Kenya (-K, 2801 nt) and Malawi (-MH and -MK, both 2804 nt) were determined. These sequences were compared with that published for a Tanzanian isolate (-T, 2801 nt) and the partial sequence of a third Malawian isolate. Intergenic region sequences of all isolates, and deduced amino acid sequences of their AC1 (Rep) proteins, each formed a tightly related cluster that was distinct from the comparable components of other begomoviruses. Other complementary-sense genes (AC2, AC3, AC4) differed between EACMV isolates in a way consistent with the accumulation of point mutations. In contrast, virus-sense genes (CP, AV2) of isolates -MH and -MK differed (substantially for AV2) from those of other EACMV isolates but somewhat resembled those of tomato yellow leaf curl virus-Israel, suggesting they had been acquired by recombination with an unidentified begomovirus.

Capsid↗

PubMed1998

Four DNA-A variants among Pakistani isolates of cotton leaf curl virus and their affinities to DNA-A of geminivirus isolates from okra.

Complete DNA-A sequences of nine Pakistani geminivirus isolates from leaf curl-affected cotton (CLCuV-PK) or from okra, and the partial sequences of several additional isolates were determined. Sequences of isolates from cotton were of four types. Isolates from leaf curl-affected okra had virtually the same sequences as those from cotton. Isolates from yellow vein mosaic-affected okra were of two types (OYVMV types 201 and 301), both distinct from but closely related to the virus isolates from cotton. Of these six types, two types of CLCuV-PK are the most closely related but another (CLCuV-PK type 72b) is the most distinct. Of the encoded proteins, coat protein (CP) is the most strongly conserved (92-100% amino acid sequence identity), and AC4 protein the most variable (41-87%). The 5' and 3' halves of the intergenic region of some isolates had different affinities and occurred in seven combinations, suggesting that recombination had occurred and that the origin of replication was a favoured recombination site. Similarly, the first 1520 nt of CLCuV-PK type 804a DNA resembled those of OYVMV type 301 DNA but the remaining 1224 nt were very different. The AC1 (Rep) gene and 5' part of the intergenic region of CLCuV-PK type 72b closely resembled those of OYVMV type 301, whereas the rest of the sequence did not. The cotton leaf curl epidemic in Pakistan is caused by several distinct variants, with recombination events involving OYVMV and other unspecified geminiviruses having probably been involved in their evolution.

Base Sequence↗

PubMed1998

In vitro packaging of adeno-associated virus DNA.

We have developed an in vitro procedure for packaging of recombinant adeno-associated virus (AAV). By using AAV replicative-form DNA as the substrate, it is possible to synthesize an infectious AAV particle in vitro that can be used to transfer a marker gene to mammalian cells. The packaging procedure requires the presence of both the AAV Rep and capsid proteins. Two kinds of in vitro products can be formed which facilitate DNA transfer. Both are resistant to heat and have a density in cesium chloride gradients that is indistinguishable from that of the in vivo-synthesized wild-type virus. This indicates that the particles formed have the appropriate protein-to-DNA ratio and a structure that shares the heat resistance of mature AAV particles. The two types of particles can be distinguished by their sensitivity to chloroform and DNase I treatment. The chloroform-resistant product is, by several criteria, an authentic AAV particle. In addition to having the correct density and being resistant to treatment with chloroform, DNase I, and heat, this particle is efficiently synthesized only if the AAV genome contains intact terminal repeats, which are known to be required for AAV packaging. It is also precipitated by a monoclonal antibody that recognizes mature virus particles but not bound by an antibody that recognizes monomeric or denatured capsid proteins. The chloroform-resistant species is not made when aphidicolin is present in the reaction mixture, suggesting that active DNA replication is required for in vitro packaging. In contrast, the chloroform-sensitive product has several features that suggest it is an incompletely assembled virus particle. It is sensitive to DNase I, does not require the presence of AAV terminal repeats, and is capable of transferring DNA that is theoretically too large to package. Sucrose gradient centrifugation of the in vitro-synthesized products reveals that the particles have sedimentation values between 60S and 110S, which is consistent with partially assembled and mature AAV particles. The in vitro packaging procedure should be useful for studying the mechanism by which a human icosahedral DNA virus particle is assembled, and it may be useful for producing recombinant AAV for gene therapy. The chloroform-sensitive particle may also be useful for transferring DNA that is too large to be packaged in mature recombinant AAV.

Adenosine Triphosphate↗

PubMed1998

Transmembrane potential changes caused by monophasic and biphasic shocks.

Transmembrane potential change (DeltaVm) during shocks was recorded by a double-barrel microelectrode in 12 isolated guinea pig papillary muscles. After 10 S1 stimuli, square-wave S2 shocks of both polarities were given consisting of 10-ms monophasic and 10/10-ms and 5/5-ms biphasic waveforms that created potential gradients from 1.1 +/- 0.3 to 11.9 +/- 0.4 V/cm. S2 shocks were applied with 30, 60- to 70-, and 90- to 130-ms S1-S2 coupling intervals so that they occurred during the plateau, late portion of the plateau, and phase 3 of the action potential, respectively. Some shocks were given across as well as along the fiber orientation. The shocks caused hyperpolarization with one polarity and depolarization with the opposite polarity. The ratio of the magnitude of hyperpolarization to that of depolarization at the three S1-S2 coupling intervals was 1.5 +/- 0.3, 1.1 +/- 0.2, and 0.5 +/- 0.2, respectively. DeltaVm during the shock was significantly greater for the monophasic than for the two biphasic shocks. The prolongation of total repolarizing time (TRT) was significantly greater for monophasic (119.8 +/- 19.1%) and 10/10-ms biphasic (120.5 +/- 18.2%) than for 5/5-ms biphasic (113.0 +/- 12.9%) waveforms. The dispersion of the normalized TRT between instances of hyperpolarization and depolarization caused by the two shock polarities was 7.4 +/- 7.1% for monophasic, 3.0 +/- 4.1% for 10/10-ms biphasic, and 2.8 +/- 3.1% for 5/5-ms biphasic shocks (P < 0.05 for monophasic vs. biphasic). Shock fields along fibers produced a larger DeltaVm and prolongation of TRT than those across fibers. We conclude that 1) a change in shock polarity causes an asymmetrical change in membrane polarization depending on shock timing; 2) the 5/5-ms biphasic waveform causes the smallest DeltaVm, prolongs repolarization the least, and causes the smallest polarity-dependent dispersion; and 3) the changes in transmembrane potential and repolarization are influenced by fiber orientation.

Animals↗

PubMed1998

Endothelin-1-induced mesangial cell contraction involves activation of protein kinase C-alpha, -delta, and -epsilon.

In endothelin-1 (ET-1)-stimulated mesangial cells, to identify the independent roles of calcium and protein kinase C (PKC) causing contraction, the changes in planar surface area in response to ET-1, ionomycin, or phorbol 12-myristate 13-acetate (PMA) were compared. ET-1, PMA, and ionomycin reduced planar area to 49 +/- 3%, 56 +/- 3%, and 78 +/- 2% of basal (means +/- SE, n = 40-50 cells), respectively. ET-1 or ionomycin increased cytosolic calcium from 80 +/- 7 to 220 +/- 30 nM or 97 +/- 10 to 192 +/- 10 nM, respectively. The myosin light chain kinase inhibitor, ML-7, blunted ET-1- but not PMA-stimulated contraction (82 +/- 3% and 48 +/- 6% of time 0, respectively). Cells pretreated with 10 microM chelerythrine for 1 h or PMA for 24 h failed to contract to either ET-1 or PMA. To identify the specific PKC isoform response to ET-1, cytosolic, membrane, and particulate fractions of mesangial cell lysates were immunoblotted with PKC isoform-specific polyclonal antibodies. ET-1 increased membrane PKC-alpha, -delta, and -epsilon to 173 +/- 30%, 162 +/- 26%, and 166 +/- 11% of basal (P < 0.05 vs. basal), respectively, and decreased PKC-delta and PKC-epsilon in the cytosol to 56 +/- 11% and 37 +/- 6% of basal, respectively (P < 0.05). ET-1 increased particulate PKC-delta and PKC-epsilon to 172 +/- 15% and 187 +/- 33% of basal (P < 0.05), respectively. PKC-alpha in the cytosol and particulate fractions was not altered by ET-1, but translocation to the nucleus and cell periphery was observed by confocal immunofluorescence imaging. Ionomycin did not change PKC isoform distribution. PKC-zeta was expressed but unaltered by ET-1. Therefore, mesangial cell ET-1-stimulated contraction not only involves a calcium-dependent pathway but also includes the activation of one or more PKC-alpha, -delta, and -epsilon, but not PKC-zeta.

Animals↗

PubMed1998

Study of serum leptin in cafeteria-diet-overfed rats. Influence of diet, insulin and corticosterone.

In a group of 15 male Wistar rats overfed with cafeteria foods (delivering a mean fat percentage of 60%) during 5 months from the age of 8 weeks and in a control group of 15 rats fed with a standard chow for the same period, serum leptin, insulin and corticosterone were measured by RIA and body composition was determined by dual-energy X-ray absorptiometry. Significantly higher fasting serum concentrations of leptin, insulin and corticosterone were found in the cafeteria-diet group. Fasting leptin concentrations were significantly higher in rats with a body fat percentage of more than 25% compared to the others, irrespective of the type of feeding. The log serum leptin correlated positively with body fat percentage and fasting insulin concentration but not with corticosterone concentration. Leptin concentration corrected for body fat mass was, however, comparable between the two diet groups, while the leptin/insulin ratio was lower in the cafeteria-diet group. In conclusion, chronic overfeeding resulting in an increased body fat percentage in rats is associated with hyperleptinemia, hyperinsulinemia and hypercorticism. Serum leptin levels appear to primarily track total body fat percentage and are unaffected by dietary fat manipulation in cafeteria-diet-induced obese rats.

Absorptiometry, Photon↗

PubMed1998

Pituitary growth hormone release and gene expression in cafeteria-diet-induced obese rats.

In human obesity as well as in rat obesity models a decrease in spontaneous and stimulated GH secretion has been a constant finding. The presence of a decreased pituitary GH synthesis in diet-induced obese male rats was investigated and its possible relationship with obesity-related changes in peripheral hormones was analyzed. Cafeteria-diet-overfed obese male Wistar rats with body fat percentage above 30% had a significantly decreased pituitary GH mRNA transcript level assessed by both Northern blot and in situ hybridization, and a lower pituitary GH protein level as demonstrated by immunocytochemistry. The GH transcript level correlated negatively with the serum leptin and positively with the IGF-I concentration. No differences in circulating tri-iodothyronine, non-fasting insulin and corticosterone levels were found between overfed and control rats. GH release by cultured pituitary cells from overfed rats was comparable to that by cells prepared from control rats. In contrast, incubation of normal pituitary cells with serum from overfed rats for 3 days gave a significantly lower GH release than after incubation with serum from non-obese rats. In conclusion, cafeteria-diet-induced obese male Wistar rats have a decreased pituitary GH gene expression and a modifiable GH release in in vitro experiments. A possible role for peripheral circulating factors, like leptin and IGF-I, in decreasing the pituitary GH synthesis and release in obese rats is discussed.

Animals↗

PubMed1998

Chelation of intracellular calcium prevents mesangial cell proliferative responsiveness.

Mesangial cell transformation into a proliferative phenotype, observed in many glomerular diseases, occurs in response to growth factors and cytokines. This study tests the hypothesis that intracellular calcium is necessary for stimulation of mesangial cell proliferative responsiveness to a variety of growth factors. Furthermore, these experiments tested whether nonspecific calcium entry via a calcium ionophore was sufficient to elicit the same response. Rat primary mesangial cells (passages 5 to 10) were growth-arrested for 48 h in 0.5% fetal bovine serum (FBS), then stimulated with 0.1 microM endothelin-1, 1.9 microM platelet-derived growth factor (PDGF)-BB, 0.5% FBS, or 0.1 microM ionomycin, with or without the intracellular calcium chelator 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid (BAPTA). Calcium signaling was measured in Fura-2-loaded cells on coverslips by dual-wavelength spectrofluorometry and in Fluo-3-loaded cells by confocal fluorescence laser microscopy. [3H]-Thymidine incorporation was measured after 12 to 24 h of stimulation with each test agent. Expression of c-fos mRNA was analyzed by Northern blot. All test agents stimulated a significant increase in cytosolic and nuclear calcium, which were both effectively inhibited with BAPTA. All agents stimulated a significant increase in [3H]-thymidine incorporation and enhanced c-fos mRNA expression (no detectable c-fos mRNA was observed in quiescent cells). BAPTA prevented the enhanced [3H]-thymidine incorporation stimulated by endothelin-1 and PDGF, and partial inhibition of FBS-stimulated incorporation with BAPTA was observed. BAPTA inhibited c-fos expression observed in response to these agents. Phorbol ester induction of c-fos mRNA in the absence of raised cytosolic or nuclear calcium was also suppressed by BAPTA. Cell viability as measured by thiazolyl blue and trypan blue was not altered by BAPTA. It is concluded that normal regulation of intracellular calcium is necessary for mesangial cell proliferative responsiveness.

Animals↗

PubMed1998

Chloride transport by the rabbit cortical collecting duct: dependence on H,K-ATPase.

The rabbit cortical collecting duct (CCD) exhibits the capacity for active chloride absorption when basolateral Na-K-ATPase is inhibited by ouabain. The present studies examine the contribution of H,K-ATPase to this ouabain-insensitive Cl absorption and to related ion fluxes. Rabbits were fed a KCl-rich diet with no measurable Na for 4 to 13 d before isolation of the CCD for microperfusion. Application of peritubular ouabain (0.1 mM) significantly increased (P < 0.001) net luminal absorptive chloride flux (J(N)Cl) without an effect on lumen-to-bath isotopic 36Cl flux (J(lb)Cl). The H,K-ATPase inhibitor Sch 28080 (1 to 10 microM) abolished ouabain-insensitive J(N)Cl, but transepithelial voltage (V(T)) was not significantly affected. The contribution of H,K-ATPase activity on active Cl flux (J(A)Cl) and passive Cl flux (J(P)Cl) was also assessed. Ouabain significantly increased J(A)Cl and Sch 28080 inhibited J(A)Cl, but J(P)Cl was not affected by Sch 28080. To assess the contribution of changes in net bicarbonate flux (JtCO2) to changes in J(N)Cl, JtCO2 was measured under identical conditions as for J(N)Cl. Ouabain significantly increased JtCO2, and this ouabain-insensitive bicarbonate flux was inhibited by Sch 28080 without significantly affecting V(T). To assess the possibility that the CCD may possess mechanisms for neutral salt absorption, lumen-to-bath 86Rb efflux (K(Rb)), and 22Na efflux (K(Na)) were also measured. Ouabain significantly increased K(Rb), and Sch 28080 inhibited this ouabain-insensitive K(Rb). Furthermore, Sch 28080 and A80915a (a structurally distinct H,K-ATPase inhibitor) significantly inhibited K(Na) in the presence of 1 mM luminal amiloride. These observations suggest that, in addition to potassium, sodium can be transported via the H,K-ATPase. Although the CCD contains more than one cell population, the data could be fitted very well to the function of the B-type intercalated cell. A cell model is proposed for the hypothesis that ouabain-insensitive chloride absorption is mediated by the parallel operation of an apical H,K-ATPase with an apical Cl-HCO3 exchanger and that the H,K-ATPase can function, under certain conditions, as a mechanism of Na absorption.

Animals↗