Biomedical subjects

X He

Publications and source records attributed to X He.

At least 325 records · Page 18Linked to original sources

PubMed1996

Adenovirus-mediated expression of aquaporin-5 in epithelial cells.

A recombinant adenovirus coding for rat aquaporin-5 was constructed and plaque purified. The recombinant adenovirus (AdrAQP5) mediated the expression of aquaporin-5 in rat and human salivary cell lines and in dog kidney cells in vitro as demonstrated by Northern blot and Western blot analyses, and by confocal microscopy after immunofluorescent labeling. In kidney cells, expression of the transgene was optimal if cells were infected at their basolateral surface, a phenomenon associated with the distribution of integrin receptors on these cells. The expressed aquaporin-5 protein was functionally active because viral-mediated gene transfer resulted in a significant increase in the osmotically directed net fluid secretion rate across monolayers of kidney cells. AdrAQP5 should provide an efficient and useful means to impart facilitated water permeability to cells lacking such a pathway.

Adenoviridae↗

PubMed1996

The endothelial cell protein C receptor. Cell surface expression and direct ligand binding by the soluble receptor.

Expression of the endothelial cell protein C receptor (EPCR) gene in mammalian cells imparts the capacity to bind activated protein C (APC) or protein C. Immunochemical analysis of CCD41, apparently the murine homologue of EPCR, suggested centrosomal localization, raising questions about the location of the EPCR gene product and its role in protein C binding. In this study, we express a soluble form of EPCR, demonstrate EPCR expression on the cell surface, and direct binding between soluble EPCR and protein C/APC. Affinity purified polyclonal and a monoclonal antibody against EPCR bound to the cell surface of EPCR-transfected cells but not to control cells. A 49-kDa protein, a mass similar to soluble EPCR, was immunoprecipitated from the cell surface of endothelium and cells transfected with human EPCR but not from control cells. The FLAGtrade mark antibody and APC bound to cells expressing an EPCR construct containing the FLAGtrade mark epitope located in a putative extracellular domain, whereas an EPCR construct truncated just before the putative transmembrane domain produced only soluble EPCR antigen. Soluble EPCR inhibited APC binding to EPCR expressing cells in a concentration-dependent fashion, Kd (app) = 29 nM and bound to immobilized protein C in a Ca2+-dependent fashion. Thus, EPCR is a type 1 transmembrane protein that binds directly to APC.

Animals↗

PubMed1996

A single mouse glutathione synthetase gene encodes six mRNAs with different 5' ends.

To understand more about the role of glutathione (GSH) in metabolism, we have cloned both cDNA and genomic sequences for mouse glutathione synthetase (GSH syn), the enzyme that catalyzes the last step in the synthesis of glutathione. The mouse cDNA contains an open reading frame (ORF) of 474 aa and shares 64 and 95% deduced amino acid sequence identity with Xenopus cDNA and rat cDNA, respectively. The cDNA complements Schizosaccaromyces pombe strains deficient in GSH syn. The gene is a single-copy gene spanning approximately 30 kb and is composed of at least 15 exons. Steady-state RNA levels and enzyme activity levels are highest in kidney, about 3-fold lower in liver, and 8- to 10-fold lower in lung and brain. We have identified six different GSH syn RNAs: three, termed types A1, A2, and A3, have different 5' ends that localize to different sites in the gene, but appear to encode the same protein (474 aa). Types B, C1, and C2 all have unique 5' ends and type-specific ORFs, which are shorter than that for types A1, A2, and A3. In liver only type A1 GSH syn RNA is detectable, while in kidney 90% of GSH syn RNA is type A1 and types B and C account for about 10%.

Alternative Splicing↗

PubMed1996

Late induction of CREB/ATF binding and a concomitant increase in cAMP levels in T and B lymphocytes stimulated via the antigen receptor.

Transcription factors of the cAMP-responsive element (CRE) binding protein/activating transcription factor (CREB/ATF) family were implicated in the expression of T cell-specific genes and in the expression of oncogenic retroviruses associated with leukemia in T and B lymphocytes. To study the regulation of CREB/ATF transcription factors during lymphocyte activation, studies were pursued in primary cultures of resting murine splenic T and B lymphocytes stimulated via the Ag receptor. Using consensus/CRE and proliferating cell nuclear Ag (PCNA)/CRE as probes in the DNA binding assay, we showed that a marked induction of CRE binding is associated with activation of splenic T lymphocytes with anti-CD3 Ab. CRE binding was markedly induced after 48 h; it gradually declined at 72 h, but remained elevated above control levels after 120 h. Most significant, activation by anti-CD3 was associated with a marked induction of cAMP levels that preceded the onset of DNA synthesis and the induction of IL-2 secretion and reached a peak after 48 h (9.5- to 11-fold), concomitant with the peak in CRE binding. Rapamycin, a potent immunosuppressant, inhibited the induction of cAMP levels by anti-CD3 concomitant with inhibition of CRE binding activity and arrest of DNA synthesis. A marked induction in CRE binding after 48 h was also found in splenic B lymphocytes stimulated by LPS and anti-Ig and was correlated with a 3- to 4-fold increase in the intracellular levels of cAMP. Two inducible CRE complexes were found to bind to consensus/CRE and PCNA/CRE; the major complex contained primarily CREB homodimers and was constitutively expressed in resting lymphocytes. Conversely, stimulation of lymphocytes was associated with formation of a new, slow migrating CRE complex that demonstrated high inducibility in both consensus/CRE and PCNA/CRE. We show that this de novo inducible CRE complex contains CREB and ATF2, but not ATF1. Taken collectively, these results suggest that recruitment of CREB and ATF2 to the promoter of genes is tightly regulated during activation of T and B lymphocytes and implicate a cross-talk of cAMP and non-cAMP pathways in the regulation of transcriptional processes at late stages of activation in T and B lymphocytes stimulated via the Ag receptor.

Activating Transcription Factors↗

PubMed1996

High-performance liquid chromatography assay of rifapentine in human serum.

A high-performance liquid chromatographic method for the determination of rifapentine in human serum was developed. The method utilized a Spherisorb C18 column, ultraviolet detection (336 nm), rifampin as internal standard and a calibration curve (C = 7.010 As/Ain +/- 0.156, r = 0.999) with reproducibility studies which yield a coefficient of variation (C.V.) of intra-day and inter-day assays lower than 10%. The average recovery of rifapentine from serum in the concentration range of 0.5 to 30 micrograms/ml was 92.93 +/- 9.704%.

Antitubercular Agents↗

PubMed1996

Probing the coenzyme and substrate binding events of CDP-D-glucose 4,6-dehydratase: mechanistic implications.

NAD+-dependent nucleotidyl diphosphohexose 4,6-dehydratases which transform nucleotidyl diphosphohexoses into corresponding 4-keto-6-deoxy sugar derivatives are essential to the formation of all 6-deoxyhexoses. Studies of the CDP-D-glucose 4,6-dehydratase (Eod) from Yersinia had shown that this dimeric protein binds only 1 equiv of NAD+/mol of enzyme and, unlike other enzymes of the same class, displays a unique NAD+ requirement for full catalytic activity. Analysis of the primary sequence revealed an extended ADP-binding fold (GHTGFKG) which deviates from the common Rossman consensus (GXGXXG) and thus may have contributed to Eod's limited NAD+ affinity. In particular, the presence of His17 in the beta-turn region and that of Lys21 in a position typically occupied by a small hydrophobic residue may impose electronic or steric perturbations to this essential binding motif. To better understand the correlation between the binding properties and primary sequence, mutants (H17G and K21I) were constructed to provide enzymes containing an ADP binding region which more closely resembles the Rossman-type fold. Analysis of the cofactor and substrate binding characteristics of the wild-type and mutant enzymes helped define the presence of two binding sites for both CDP-d_glucose and NAD+ per enzyme molecule. While both mutants displayed enhanced NAD+ affinity, the H17G mutation resulted in an enzyme with slightly higher kcat and a 3-fold increase in catalytic efficiency (kcat/Km). The large anticooperativity found for NAD+ binding (K1=40.3 + or - 0.4 nM, K2=539.8 + or - 4.8 nM) may explain why the cofactor binding sites of wild-type Eod are only half-occupied. Further examination also revealed the purified Eod to contain sequestered NADH and that the affinity of Eod for NADH(K1=0.21 + or - 0.01 nM, K2= 7.46 + or -0.25 nM) is much higher than that for NAD+. Thus, it is possible that Eod's half-site saturation of NAD+ per enzyme dimer may also be attributed to a significant portion of the cofactor binding sites being occupied by NADH. Interestingly, the sequestered NADH is released upon binding with CDP-D-glucose. These results implicate a new kinetic mechanism for Eod catalysis.

Amino Acid Sequence↗

PubMed1996

Human alpha-tocopherol transfer protein: gene structure and mutations in familial vitamin E deficiency.

Familial vitamin E deficiency (AVED) causes ataxia and peripheral neuropathy that resembles Friedreich's ataxia. AVED is thought to be caused by a defect in the transport of vitamin E in liver cells, which is the probable function of alpha-tocopherol transfer protein (alphaTTP). We have cloned the cDNA and several genomic phage clones covering the entire human alphaTTP gene and determined the junctions between the five exons and four introns that composed the gene for human alphaTTP. Three mutations in three unrelated North American families with AVED were identified. Two mutations, 485delT and 513insTT, cause a frame shift and a premature stop codon and the third mutation 574G-->A would substitute Arg192 to His in alphaTTP. The 2 patients with a severe form of AVED were homozygous with 485delT and 513insTT, respectively, while the patient with a mild form of the disease was compound heterozygous with 513insTT and 574G-->A. These findings have identified the underlying genetic defect in AVED and have confirmed the role of alphaTTP in AVED.

Adolescent↗

PubMed1996

Increased responsiveness of rheumatoid factor-producing B cells in seronegative and seropositive rheumatoid arthritis.

OBJECTIVE: To compare the frequencies and responsiveness of rheumatoid factor (RF)-producing B cells in the peripheral blood of patients with seronegative and seropositive rheumatoid arthritis (RA). METHODS: Frequencies of IgM+, IgG+, and RF+ B cells were determined by limiting-dilution analysis of purified peripheral blood B cells from 6 patients with seropositive RA, 8 patients with seronegative RA, and 7 normal controls. B cell help was provided by cloned T helper cells, which were stimulated by either anti-CD3 or the bacterial superantigen staphylococcal enterotoxin D (SED). IgM and IgG antibodies and RF in culture supernatants were detected by enzyme-linked immunosorbent assay. RESULTS: In the presence of anti-CD3-stimulated T helper cells, 2-10% of B cells from normal individuals secreted IgM and IgG antibodies. The frequency of RF+ B cells was low and ranged from 1:182 to 1:885 (RF+: IgM+) B cells. In patients with seropositive RA, the numbers of Ig-producing B cells were reduced by a factor of 2, while the fraction of RF+ B cell precursors was expanded by more than 50-fold (7-20% of IgM+ B cells; P = 0.004). Patients with seronegative RA had higher frequencies of RF-producing B cells (1.5-6% of IgM+ B cells) than normal individuals (P = 0.002), but not to the same extent as seropositive patients (P = 0.002). Stimulation of B cells using SED preferentially induced RF+ B cells in normal controls and in patients with seronegative and seropositive RA. CONCLUSION: B cell precursors with the potential to secrete RF were detectable in high frequencies in normal individuals and in patients with seropositive and seronegative RA. In all donors, these B cells could be stimulated with the bacterial superantigen SED. In normal individuals, RF+ B cells remained nonresponsive to help provided by anti-CD3-activated T cells, but were responsive in RA patients. Seronegative and seropositive RA form a continuous spectrum of disease, with a higher number of RF-secreting B cells in the seropositive patients.

Adult↗

PubMed1996

Dopaminergic transmission in the rat retina: evidence for volume transmission.

The study was designed to determine whether dopaminergic neurotransmission in the retina can operate via volume transmission. In double immunolabelling experiments, a mismatch as well as a match was demonstrated in the rat retina between tyrosine hydroxylase (TH) and dopamine (DA) immunoreactive (ir) terminals and cell bodies and dopamine D2 receptor-like ir cell bodies and processes. The match regions were located in the inner nuclear and plexiform layers (D2 ir cell bodies plus processes). The mismatch regions were located in the ganglion cell layer, the outer plexiform layer, and the outer segment of the photoreceptor layer, where very few TH ir terminals can be found in relation to the D2 like ir processes. In similar experiments analyzing D1 receptor like ir processes versus TH ir nerve terminals, mainly a mismatch in their distribution could be demonstrated, with the D1 like ir processes present in the outer plexiform layer and the outer segment where a mismatch in D2 like receptors also exists. The demonstration of a mismatch between the localization of the TH terminal plexus and the dopamine D2 and D1 receptor subtypes in the outer plexiform layer, the outer segment and the ganglion cell layer (only D2 immunoreactivity (IR)) suggests that dopamine, mainly from the inner plexiform layer, may reach the D2 and D1 mismatch receptors via diffusion in the extracellular space. After injecting dopamine into the corpus vitreum, dopamine diffuses through the retina, and strong catecholamine (CA) fluorescence appears in the entire inner plexiform layer and the entire outer plexiform layer, representing the match and mismatch DA receptor areas, respectively. The DA is probably bound to D1 and D2 receptors in both plexiform layers, since the DA receptor antagonist chlorpromazine fully blocks the appearance of the DA fluorescence, while only a partial blockade is found after haloperidol treatment which mainly blocks D2 receptors. These results indicate that the amacrine and/or interplexiform DA cells, with sparse branches in the outer plexiform layer, can operate via volume transmission in the rat retina to influence the outer plexiform layer and the outer segment, as well as other layers of the rat retina such as the ganglion cell layer.

Animals↗

PubMed1996

Mutations in the glutathione synthetase gene cause 5-oxoprolinuria.

5-Oxoprolinuria (pyroglutamic aciduria) resulting from glutathione synthetase (GSS) deficiency is an inherited autosomal recessive disorder characterized, in its severe form, by massive urinary excretion of 5-oxoproline, metabolic acidosis, haemolytic anaemia and central nervous system damage. The metabolic defect results in low GSH levels presumably with feedback over-stimulation of gamma-glutamylcysteine synthesis and its subsequent conversion to 5-oxoproline. In this study, we cloned and characterized the human GSS gene and examined three families with four cases of well-documented 5-oxoprolinuria. We identified seven mutations at the GSS locus on six alleles: one splice site mutation, two deletions and four missense mutations. Bacterial expression and yeast complementation assays of the cDNAs encoded by these alleles demonstrated their functional defects. We also characterized a fifth case, an homozygous missense mutation in the gene in an individual affected by a milder-form of the GSS deficiency, which is apparently restricted to erythrocytes and only associated with haemolytic anaemia. Our data provide the first molecular genetic analysis of 5-oxoprolinuria and demonstrate that GSS deficiency with oxoprolinuria and GSS deficiency without 5-oxoprolinuria are caused by mutations in the same gene.

Amino Acid Metabolism, Inborn Errors↗

PubMed1996

Pulsatile flow in the human left coronary artery bifurcation: average conditions.

The localization of atherosclerosis in the coronary arteries may be governed by local hemodynamic features. In this study, the pulsatile hemodynamics of the left coronary artery bifurcation was numerically simulated using the spectral element method for realistic in vivo anatomic and physiologic conditions. The velocity profiles were found to be skewed in both the left anterior descending and the circumflex coronary arteries. Velocity skewing arose from the bifurcation as well as from the curvature of the artery over the myocardial surface. Arterial wall shear stress was significantly lower in the bifurcation region, including the side walls. The greatest oscillatory behavior was localized to the outer wall of the circumflex artery. The time-averaged mean wall shear stress varied from about 3 to 98 dynes/cm2 in the left coronary artery system. The highly localized distribution of low and oscillatory shear stress along the walls strongly correlates with the focal locations of atheroma in the human left coronary artery.

Blood Flow Velocity↗

PubMed1996

Numerical simulation of oil-droplet cleavage by surfactant.

We present numerical computations of the deformation of an oil-droplet under the influence of a surface tension gradient generated by the surfactant released at the poles (the Greenspan experiment). We find this deformation to be very small under the pure surface tension gradient. To explain the large deformation of oil droplets observed in Greenspan's experiments, we propose the existence of a phoretic force generated by the concentration gradient of the surfactant. We show that this hypothesis successfully explains the available experimental data and we propose some further tests.

Cell Division↗

PubMed1996

Efficient transcription from the rice tungro bacilliform virus promoter requires elements downstream of the transcription start site.

Elements downstream of the transcription start site enhance the activity of the rice tungro bacilliform virus (RTBV) promoter in protoplasts derived from cultured rice cells. This enhancer region was located to the first 90 nucleotides of the RTBV leader sequence. Within this region, at least two components which act together to enhance expression from the RTBV promoter could be identified. One is a position- and orientation-independent DNA element within a CT-rich region, and the other is a position-dependent element. Either element was found to be capable of acting independently on a heterologous promoter. The enhancer activity of the DNA element correlates with specific binding of nuclear proteins. Nuclear proteins also recognize an RNA transcript covering the first 90 nucleotides of the RTBV leader.

Badnavirus↗

PubMed1996

Perturbations in the spi1p GTPase cycle of Schizosaccharomyces pombe through its GTPase-activating protein and guanine nucleotide exchange factor components result in similar phenotypic consequences.

spi1p of Schizosaccharomyces pombe is a structural homolog of the mammalian GTPase Ran. The distribution between the GTP- and GDP-bound forms of the protein is regulated by evolutionarily conserved gene products, rna1p and pim1p, functioning as GTPase-activating protein (GAP) and guanine nucleotide exchange factor (GEF), respectively. Antibodies to spi1p, pim1p, and rna1p were generated and used to demonstrate that pim1p is exclusively nuclear, while rna1p is cytoplasmic. A loss of pim1p GEF activity or an increase in the rna1p GAP activity correlates with a change in the localization of the GTPase from predominantly nuclear to uniformly distributed, suggesting that the two forms are topologically segregated and that the nucleotide-bound state of spi1p may dictate its intracellular localization. We demonstrate that the phenotype of cells overproducing the GAP resembles the previously reported phenotype of mutants with alterations in the GEF: the cells are arrested in the cell cycle as septated, binucleated cells with highly condensed chromatin, fragmented nuclear envelopes, and abnormally wide septa. Consistent with the expectation that either an increased dosage of the GAP or a mutation in the GEF would lead to an increase of the spi1p-GDP/spi1p-GTP ratio relative to that of wild-type cells, overexpression of the GAP together with a mutation in the GEF is synthetically lethal. The similar phenotypic consequences of altering the functioning of the nuclear GEF or the cytoplasmic GAP suggest that there is a single pool of the spi1p GTPase that shuttles between the nucleus and the cytoplasm. Phenotypically, rna1 null mutants, in which spi1p-GTP would be expected to accumulate, resemble pim1(ts) and rna1p-overproducing cells, in which spi1p-GDP would be expected to accumulate. Taken together, these results support the hypothesis that the balance between the GDP- and GTP-bound forms of spi1p mediates the host of nuclear processes that are adversely affected when the functioning of different components of this system is perturbed in various organisms.

Animals↗

PubMed1996

Immunolocalization of the secretory isoform of Na-K-Cl cotransporter in rat renal intercalated cells.

Two bumetanide-sensitive ion cotransporters that carry Na+, K+, and Cl- in a coupled fashion have been identified. One type, the "absorptive" isoform, carries these ions across the apical plasma membrane of the thick ascending limb of Henle's loop. Another isoform, the "secretory" cotransporter, has been identified in a number of epithelial tissues by physiological means, but its sites of expression in the kidney have not been fully characterized. Complementary DNA believed to code for the secretory isoform (called "BSC2" or "NKCC1") have recently been cloned. This study used a specific affinity-purified antipeptide antibody to this protein for immunolocalization in the rat kidney. Immunoblot studies using this antibody show abundant immunoreactivity against bands of 140-190 and 120 kd in the parotid gland, colon, and stomach, sites where the secretory form of the cotransporter has been identified by physiological techniques. This distribution supports the hypothesis that this isoform represents the secretory form of the cotransporter. Studies in the kidney revealed that the same bands are associated with membrane fractions chiefly in the outer medulla. Immunolocalizations show that immunoreactivity is selectively and intensely localized to the basolateral plasma membrane of a subfraction of outer medullary collecting duct cells. An independently produced monoclonal antibody (T4) specific for Na-K-Cl cotransporter displays the same localization. Dual localizations of cotransporter antibody with respect to antibody specific for principal cells (aquaporin-2) and intercalated cells (band 3 and H(+)-ATPase) show that cotransporter immunoreactivity is localized to alpha-intercalated cells of the outer medullary collecting duct in the rat. This distinctive localization suggests that the secretory form of the cotransporter may play a role in renal NH4+ and/or acid secretion by this cell type.

Amino Acid Sequence↗

PubMed1996

Temporal patterns of aflatoxin-albumin adducts in hepatitis B surface antigen-positive and antigen-negative residents of Daxin, Qidong County, People's Republic of China.

Molecular epidemiological studies of populations at high risk for liver cancer have shown that hepatitis B virus (HBV) and aflatoxin B1 (AFB1) exposure are two major risk factors for this disease. These etiological agents, combined with nutritional deficiencies, are important for the initiation and promotion of liver cancer in various parts of the world. In Qidong, People's Republic of China, liver cancer accounts for 10% of all adult deaths, and both HBV and AFB1 exposures are common. To study temporal and possible chemical-viral interactions in people, serum samples were collected during a longitudinal study designed to measure aflatoxin molecular biomarkers in residents of Daxin Township, Qidong City, People's Republic of China. In this study, the temporal modulation of aflatoxin adduct formation with albumin over multiple lifetimes of serum albumin was examined in both HBV-positive and HBV-negative people in two periods: September-December 1993 (wave 1) and June-September 1994 (wave 2). During the 12-week monitoring period of wave 1, 120 individuals (balanced by gender and HBV status) provided a total of 792 blood samples. AFB1-albumin adducts were detected in all but one of the serum samples. The range of binding detected by RIA in the Daxin population was 0.17-4.39 pmol AFB11/mg albumin with an overall mean +/- SD of 1.51 +/- 0.21 pmol AFB11/mg albumin. The mean +/- SD for weeks 0, 2, 4, 6, 8, 10 and 12 of wave 1 were 1.21 +/- 0.41, 1.58 +/- 0.70, 1.36 +/- 0.52, 1.71 +/- 0.44, 1.18 +/- 0.60, 2.00 +/- 0.59, and 1.68 +/- 0.34 pmol AFB1/mg albumin, respectively. During wave 2, 103 individuals from wave 1 provided a total of 396 blood samples collected monthly over wave 2, with mean +/- SD aflatoxin-albumin adduct levels of 1.19 +/- 0.37, 0.85 +/- 0.45, 0.89 +/- 0.28, and 0.61 +/- 0.15 pmol AFB1/mg albumin. Using linear regression models, the mean aflatoxin-albumin adduct levels increased (P < 0.05) during the 12 weeks of wave 1 and decreased (P < 0.05) over the 4 months of wave 2. Neither HBV surface antigen status nor gender modified either the baseline mean or the temporal trend. High-performance liquid chromatography confirmation was done on a subset of serum samples, and the results show an excellent association between the immunoassay data and high-performance liquid chromatography. Taken together, these data demonstrate that AFB1-albumin is a sensitive and specific biomarker for assessing exposure to this carcinogen in the population in Qidong.

Adult↗

PubMed1996

Intrapancreatic cholecystokinin mediates vagally stimulated exocrine secretion from the rat pancreas.

Although cholecystokinin is localized within neuronal fibres of the pancreas, a physiological role for intrapancreatic cholecystokinin has not been identified. The strategy of this study was to elicit pure vagal stimulation electrically, and to use specific receptor antagonists to identify the mediators of exocrine pancreatic secretion. We conclude that vagal stimulation of the rat pancreas involves ganglionic neurotransmission and release of acetylcholine and cholecystokinin from intrapancreatic, postganglionic fibres. To our knowledge, this is the first study to demonstrate a physiological role for intrapancreatic cholecystokinin.

Animals↗

PubMed1996

[The postnatal changes of follicle-stimulating hormone receptor mRNA level in rat testis].

It was found in this study that the follicle-stimulating hormone receptor (FSHR) mRNA levels varied between days 2 and 60. The FSHR mRNA rised slightly in the neonatal rat testis and then declined. In the prepuberty the content of FSHR mRNA remained at a Constant level. During the puberty the FSHR mRNA increased to its highest level on days 25 and finally it rised again after days 40.

Animals↗