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W J Welch

Publications and source records attributed to W J Welch.

At least 73 records · Page 4Linked to original sources
PubMed1995

Validation of miniature ultrasonic transit-time flow probes for measurement of renal blood flow in rats.

This study validates the accuracy of miniature ultrasonic transit-time flow probes for measuring renal blood flow (RBF) in the rat. Probes for 1-mm and 2-mm vessels were calibrated ex vivo using excised arteries at varying flow rates and hematocrit (Hct). Correlation between measured and true flow rates for the 2-mm probe were identical (r = 1.0) at both normal and subnormal Hct values. Correlation for the 1-mm probe was high (r = 0.994) at normal Hct, but varied at both high flow rates and subnormal Hct values. In vivo correlation of RBF measurements using the 1-mm probe with the clearance and extraction of p-aminohippuric acid showed a high correlation (r = 0.84; n = 72, P < 0.0001) over a wide range of flow rates (0.5-21 ml/min) and Hct (36-74%). Zero flow levels remained steady, averaging -0.2 +/- 0.2 ml/min during occlusion in the living animal and -0.1 +/- 0.3 ml/min after exsanguination. This study shows that the ultrasonic transit-time flowmeter (1-mm and 2-mm probes) is a reasonably accurate and reliable method with which to measure RBF in the anesthetized, acute-instrumented rat.

Animals↗
PubMed1995

Renal vasodilation with L-arginine. Effects of dietary salt.

Infusion of L-arginine, the substrate for nitric oxide synthase, causes renal vasodilation. Since dietary salt restriction blunts the renal vasoconstrictor response to inhibition of nitric oxide synthase, we investigated the hypothesis that dietary salt intake determines the renal vascular response to L-arginine. Bolus intravenous doses of L-arginine given to anesthetized Sprague-Dawley rats caused dose-dependent increases in renal blood flow and decreases in renal vascular resistance, whereas D-arginine was not effective. The response to L-arginine was prevented by pretreatment with NG-nitro-L-arginine methyl ester. Compared with rats adapted to a high salt diet, those adapted to a low salt diet were more sensitive to the reductions in blood pressure and renal vascular resistance (threshold dose of L-arginine for renal vascular resistance: low salt, 2.9 +/- 0.9 mumol . kg-1 versus high salt, 20.0 +/- 6.2; P < .025), but the maximal changes in renal vascular resistance were similar (low salt, -43 +/- 5% versus high salt, -34 +/- 5%; P = NS). Bolus doses of L-glycine also caused dose-dependent renal vasodilation, but the renal vasodilator responses were not affected by salt intake. Preinfusion of L-arginine augmented the renal vasoconstrictor response to NG-nitro-L-arginine methyl ester in low salt but not high salt rats; after L-arginine dietary salt no longer significantly affected the renal vasoconstrictor response to NG-nitro-L-arginine methyl ester. In conclusion, renal vasodilation is more sensitive to L-arginine during salt restriction. This effect is specific for L-arginine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Clinical implications of the stress response.

A field of research that began with a curious observation in Drosophila has resulted in a new understanding of how cells respond to sudden and adverse changes in their environment. In addition through the study of the structure/function of the stress proteins, especially those which function as molecular chaperones, new insights into the details by which proteins are synthesized and acquire their final biologically active conformation have been realized. Equally exciting is the progress being made as it relates the potential diagnostic and therapeutic applications of the stress-response proteins. The use of stress proteins as the next generation of vaccines and/or their use as potentially powerful adjuvants, capable of stimulating both T and B cell responses to a particular antigen of interest appear close to becoming a reality. One wonders how many more surprises are in store for us as we continue to explore this evolutionally conserved cellular stress response.

Adaptation, Physiological↗
PubMed1994

Complex environment of nascent polypeptide chains.

Nascent polypeptides enter into high molecular weight complexes with other proteins during chain elongation in vitro and in vivo. The nature of these complexes was investigated using an in vitro translation system programmed with a single mRNA lacking a translational termination codon. Complexes containing nascent polypeptides (molecular mass < 20 kDa), the molecular chaperone hsp 73 and other unidentified proteins can be released from the translationally arrested polysomes by puromycin treatment. The apparent native molecular mass of the nascent chain binding complex was determined to be > 700 kDa by gel-filtration analysis. Complexes between the nascent polypeptide and at least hsp 73 appear to be sensitive to (disrupted by) ATP. The presence of ATP also dramatically alters the sensitivity of the nascent polypeptide chains to digestion by exogenous protease. Collectively, our data indicate that there may be a cytoplasmic machinery, including hsp 70, which comprises a nascent polypeptide chain binding complex.

Adenosine Triphosphate↗
PubMed1994

Intramitochondrial folding and assembly of medium-chain acyl-CoA dehydrogenase (MCAD). Demonstration of impaired transfer of K304E-variant MCAD from its complex with hsp60 to the native tetramer.

We incubated in vitro translated precursor of medium-chain acyl-CoA dehydrogenase (MCAD) with isolated rat liver mitochondria and fractionated the solubilized mitochondria on gel filtration. After a 5-min import into mitochondria, MCAD was recovered exclusively as a high molecular weight (hMr) complex (700,000), while after a 10-min import, it was recovered mainly in the hMr complex and mature tetramer, with a small amount in monomer. Either a further 15-min chase or exposure to ATP caused a marked decrease of MCAD in the hMr complex and an increase in the mature tetramer in comparable amounts, suggesting that the hMr complex was the precursor of tetramer. No monomer was detected in either case. Using specific antibodies, we have shown that the hMr complex represented a complex of MCAD and heat-shock protein 60 (hsp60), and, that upon import into mitochondria, unfolded MCAD first formed a transient complex with mitochondrial heat-shock protein 70 (hsp70mit) and then transferred to hsp60 to complete its folding into an assembly-competent conformation. We also examined the assembly of K304E MCAD, which is a prevalent variant enzyme among patients with MCAD deficiency. The assembly of the K304E into its tetrameric form was severely impaired. The binding of K304E with hsp70mit and its transfer from hsp70mit to hsp60 were normal. However, the hsp60 complex of K304E was much more stable than the wild-type counterpart upon a 15-min chase or exposure to ATP, suggesting that the folding in, or the transfer of K304E subunit to tetramer from, the complex with hsp60 was impaired.

Acyl-CoA Dehydrogenase↗
PubMed1994

Both viral (adenovirus E1B) and cellular (hsp 70, p53) components interact with centrosomes.

Human 293 cells, transformed by and expressing the early region of the adenovirus genome (i.e., E1A and E1B), contain a phase-dense cytoplasmic structure situated in close proximity to the nucleus. Via indirect immunofluorescence studies such structures have been previously shown to contain both the adenovirus E1B (55 kDa) protein as well as the tumor suppressor gene product p53. Here we show that such structures also stain positive for the cytoplasmic hsp 70 proteins. Such phase-dense structures containing hsp 70, p53, and adenovirus E1B are not unique to 293 cells but also are observed in rodent cell lines stabily transfected with the early region of the adenovirus genome. Using an antibody against a centrosomal protein, pericentrin, we show that these cytoplasmic phase-dense structures are in close proximity to the centrosome. Cell fractionation studies revealed such structures to be highly detergent insoluble. However, like the centrosome, the cytoplasmic phase-dense structures could be rendered detergent soluble following treatment of the cells with agents that disrupt the integrity of the cytoskeleton. While the phase-dense structures appear in close proximity to the centrosome in interphase cells, during mitosis the centrosome and the phase-dense bodies separate from one another. Owing to these observations we examined whether hsp70 and p53 might also co-localize with the centrosome in other cell types not expressing the adenovirus E1A/E1B proteins. We show that a portion of both hsp70 and p53 indeed are present within the centrosome in Hela, COS, and 3T3 cells. These observations raise the possibility that components like hsp70 and p53 may participate in the mechanism(s) controlling cell division in mammalian cells.

Adenovirus E1B Proteins↗
PubMed1994

Renal vasoconstriction during inhibition of NO synthase: effects of dietary salt.

Since dietary salt loading enhances nitric oxide (NO) generation in the kidney, we investigated the hypothesis that changes in salt intake have specific effects on vascular resistance in the kidney mediated by the L-arginine-NO pathway. We contrasted changes in renal and hindquarter vascular resistances (RVR and HQVR) in anesthetized rats during intravenous infusions of graded doses of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). Groups (N = 8 to 10) of rats were maintained on a high salt (HS) or low salt (LS) diet for two weeks. Compared to those on LS, rats on HS had a greater increase in mean arterial pressure (delta MAP; +32 +/- 4 vs. +22 +/- 3%; P = 0.05) and RVR (+160 +/- 17 vs. +83 +/- 10%; P < 0.005) and a greater fall in renal blood flow (delta RBF; -47 +/- 3 vs. -32 +/- 4%; P < 0.01); changes in HQVR were similar in the two groups. The enhanced RVR response to L-NAME in HS rats could not be ascribed to the higher renal perfusion pressure (RPP) since it persisted in rats whose RPP was controlled by adjustment of a suprarenal aortic clamp. Changes in RVR with an NO donor (SIN-1) were similar in HS and LS rats. L-NAME reduced plasma renin activity in both HS and LS rats. After inhibition of ACE with captopril, or of angiotensin II type I (AT1) receptor with losartan, the increase in RVR with L-NAME remained greater in HS than LS rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1994

A eukaryotic cytosolic chaperonin is associated with a high molecular weight intermediate in the assembly of hepatitis B virus capsid, a multimeric particle.

We have established a system for assembly of hepatitis B virus capsid, a homomultimer of the viral core polypeptide, using cell-free transcription-linked translation. The mature particles that are produced are indistinguishable from authentic viral capsids by four criteria: velocity sedimentation, buoyant density, protease resistance, and electron microscopic appearance. Production of unassembled core polypeptides can be uncoupled from production of capsid particles by decreasing core mRNA concentration. Addition of excess unlabeled core polypeptides allows the chase of the unassembled polypeptides into mature capsids. Using this cell-free system, we demonstrate that assembly of capsids proceeds by way of a novel high molecular weight intermediate. Upon isolation, the high molecular weight intermediate is productive of mature capsids when energy substrates are manipulated. A 60-kD protein related to the chaperonin t-complex polypeptide 1 (TCP-1) is found in association with core polypeptides in two different assembly intermediates, but is not associated with either the initial unassembled polypeptides or with the final mature capsid product. These findings implicate TCP-1 or a related chaperonin in viral assembly and raise the possibility that eukaryotic cytosolic chaperonins may play a distinctive role in multimer assembly apart from their involvement in assisting monomer folding.

Amino Acid Sequence↗
PubMed1994

Involvement of 70-kD heat-shock proteins in peroxisomal import.

This report describes the involvement of 70-kD heat-shock proteins (hsp70) in the import of proteins into mammalian peroxisomes. Employing a microinjection-based assay (Walton, P. A., S. J. Gould, J. R. Feramisco, and S. Subramani. 1992. Mol. Cell Biol. 12:531-541), we demonstrate that proteins of the hsp70 family were associated with proteins being imported into the peroxisomal matrix. Import of peroxisomal proteins could be inhibited by coinjection of antibodies directed against the constitutive hsp70 proteins (hsp73). In a permeabilized-cell assay (Wendland and Subramani. 1993. J. Cell Biol. 120:675-685), antibodies directed against hsp70 proteins were shown to inhibit peroxisomal protein import. Inhibition could be overcome by the addition of exogenous hsp70 proteins. Purified rat liver peroxisomes were shown to have associated hsp70 proteins. The amount of associated hsp70 was increased under conditions of peroxisomal proliferation. Furthermore, proteinase protection assays indicated that the hsp70 molecules were located on the outside of the peroxisomal membrane. Finally, the process of heat-shocking cells resulted in a considerable delay in the import of peroxisomal proteins. Taken together, these results indicate that heat-shock proteins of the cytoplasmic hsp70 family are involved in the import of peroxisomal proteins.

Animals↗
PubMed1994

Renal vasoconstriction with U-46,619; role of arachidonate metabolites.

Thromboxane A2 (TxA2) or its stable mimetic U-46,619 can increase the generation of arachidonate metabolites. Therefore, these studies were designed to investigate the role of prostaglandins, TxA2, and leukotrienes in the renal vascular response to U-46,619. Anesthetized rats were studied during a basal period and during an intra-aortic infusion of vehicle or U-46,619 (1 micrograms/kg per minute). U-46,619 reduced the GFR and the RBF without changing the mean arterial pressure or the femoral vascular resistance. All of the effects of U-46,619 were blocked by pretreatment with the TxA2/prostaglandin H2 receptor antagonist SQ-29,548. Pretreatment with the cyclo-oxygenase inhibitor indomethacin did not modify the renal vascular response to U-46,619. However, pretreatment with the TxA2 synthesis inhibitor UK-38,485 or with the leukotriene D4/E4 antagonist LY-163,443 markedly blunted the U-46,619-induced increase in renal vascular resistance and the decrease in GFR. These results indicate that the renal vascular response to U-46,619 is receptor mediated and is promoted by TxA2 and leukotriene D4/E4.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1993

ATP-induced protein-Hsp70 complex dissociation requires K+ but not ATP hydrolysis.

The molecular chaperone proteins, particularly Hsp60 and Hsp70, have been implicated in essential cell functions under both normal and stress conditions (reviewed in refs 1-5). Members of the family of heat-shock proteins of M(r) 70K, Hsp70, bind to unfolded proteins and short peptides. Addition of Mg-ATP results in the dissociation of the substrate polypeptides from the chaperone, but as ATP-gamma S (an ATP analogue that is only slowly hydrolysable) cannot substitute for ATP in this reaction, it has been concluded that ATP hydrolysis is necessary to dissociate Hsp70-substrate protein complexes. By independently measuring the rates of ATP hydrolysis and substrate protein dissociation, we show here that Mg-ATP binding but not Mg-ATP hydrolysis is essential for substrate dissociation. We also show that there is an absolute requirement for K+ for the effect of Mg-ATP: only the combination of K+ and Mg-ATP will cause the conformational change in Hsp70 that is necessary for substrate dissociation. Moreover, in the absence of K+, Mg-ATP favours complex formation. We consider these results in terms of a G-protein-like model.

Adenosine Triphosphatases↗
PubMed1993

Heat shock protein hsp70 in patients with axillary lymph node-negative breast cancer: prognostic implications.

BACKGROUND: Cell synthesis of heat shock (stress-response) proteins is increased by a variety of environmental and pathophysiological stressful conditions. The 70-kd heat shock protein (hsp70) is thought to be involved in protein-protein interactions including those of the protein products of the human c-myc oncogene and the p53 (also known as TP53) tumor suppressor gene. PURPOSE: The purpose of this study was to investigate whether elevated hsp70 expression may be an indicator of biological stress experienced by a breast cancer and may, therefore, predict disease outcome. METHODS: Levels of hsp70 were determined by Western blot analysis in primary breast tumors from patients with negative axillary lymph nodes. We performed exploratory data analyses on a set of 162 primary breast cancers and constructed prognostic indexes of hsp70 expression levels. The optimal cutpoint for hsp70 expression was considered to be the value yielding the greatest separation for disease-free survival for the resulting two groups of patients. That cutpoint was then validated in a set of 345 tumors by univariate and multivariate analyses. Data were analyzed for overall survival, disease-free survival, tumor size, and patient age, as well as estrogen receptor and progesterone receptor status, ploidy (DNA content), and percentage of cells in S phase as determined by flow cytometry. RESULTS: Expression of hsp70 emerged as a useful prognostic factor, both in univariate and in multivariate analyses. Patients whose tumors had high expression of hsp70 had significantly shorter disease-free survival (P = .006). The other statistically significant factors were S-phase fraction (P = .008) and tumor size (P = .01). For patients who received adjuvant therapy, hsp70 was the only independent predictor of disease recurrence (P = .05). For those with tumors 1-3 cm in diameter, hsp70 (P = .008) and S-phase fraction (P = .02) were statistically significant predictors of recurrence. CONCLUSIONS: Measurement of hsp70 expression in primary tumors from patients with node-negative breast cancer may be useful in identifying patients at high risk for disease recurrence and thus may affect decisions regarding treatment after surgery. IMPLICATIONS: Future studies should be performed to determine if detection of hsp70 by immunohistochemistry can be used to predict clinical outcome and to better understand the relationships between hsp70 and the effects of various treatment modalities.

Analysis of Variance↗
PubMed1993

Heat shock proteins functioning as molecular chaperones: their roles in normal and stressed cells.

In response to either elevated temperatures or several other metabolic insults, cells from all organisms respond by increasing the expression of so-called heat shock proteins (hsp or stress proteins). In general, the stress response appears to represent a universal cellular defence mechanism. The increased expression and accumulation of the stress proteins provides the cell with an added degree of protection. Studies over the past few years have revealed a role for some of the stress proteins as being intimately involved in protein maturation. Members of the hsp 70 family, distributed throughout various intracellular compartments, interact transiently with other proteins undergoing synthesis, translocation, or higher ordered assembly. Although not yet proven, it has been suggested that members of the hsp 70 family function to slow down or retard the premature folding of proteins in the course of synthesis and translocation. Yet another family of stress proteins, the hsp 60 or GroEL proteins (chaperonins), appear to function as catalysts of protein folding. Here I discuss the role of those stress proteins functioning as molecular chaperones, both within the normal cell and in the cell subjected to metabolic stress.

Animals↗
PubMed1993

Nitric oxide mediates renal vasodilation during erythropoietin-induced polycythemia.

The renal blood flow (RBF) of patients with polycythemia rubra vera is increased despite the high hematocrit (Hct) which elevates the whole blood viscosity. Since blood viscosity determines the shear force on the endothelium which is a major stimulus to nitric oxide (NO) release, we investigated the hypothesis that renal vasodilation during erythropoietin-induced erythrocytosis is mediated by the L-arginine-NO pathway. Groups of Sprague-Dawley rats received thrice weekly injections of erythropoietin (E) for two to five weeks; responses were contrasted with normal rats (N) which received sham injections. The first group was studied after five weeks of erythropoietin injections which led to sharp increases in Hct (E: 72 +/- 3 vs. N: 44 +/- 1%) and mean arterial pressure (MAP: 126 +/- 3 vs. 107 +/- 3 mm Hg). These rats had an elevated basal RBF whether measured by the clearance and renal extraction of PAH or by a transit-time renal blood flow meter. Subsequent groups were studied after two to three weeks of erythropoietin which raised the Hct more modestly to 59 +/- 2%. In this group, the basal MAP was similar in E and N rats. Graded doses of the NO synthase inhibitor, N omega-monomethyl-L-arginine (L-NMA) led to a steeper rise in MAP in E than N; at the highest doses, the MAP had increased by 36 +/- 2 in E and 23 +/- 3 mm Hg in N (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

How cells respond to stress.

During emergencies, cells produce stress proteins that repair damage. Inquiry into how they work offers promise for coping with infection, autoimmune disease and even cancer.

Adenosine Triphosphate↗
PubMed1993

The constitutive and stress inducible forms of hsp 70 exhibit functional similarities and interact with one another in an ATP-dependent fashion.

Mammalian cells constitutively express a cytosolic and nuclear form of heat shock protein (hsp) 70, referred to here as hsp 73. In response to heat shock or other metabolic insults, increased expression of another cytosolic and nuclear form of hsp 70, hsp 72, is observed. The constitutively expressed hsp 73, and stress-inducible hsp 72, are highly related proteins. Still unclear, however, is exactly why most eukaryotic cells, in contrast to prokaryotic cells, express a novel form of hsp 70 (i.e., hsp 72) after experiencing stress. To address this question, we prepared antibodies specific to either hsp 72 or hsp 73 and have compared a number of biological properties of the two proteins, both in vivo and in vitro. Using metabolic pulse-chase labeling and immunoprecipitation analysis, both the hsp 72 and hsp 73 specific antibodies were found to coprecipitate a significant number of newly synthesized proteins. Such interactions appeared transient and sensitive to ATP. Consequently, we suspect that both hsp 72 and hsp 73 function as molecular chaperones, interacting transiently with nascent polypeptides. During the course of these studies, we routinely observed that antibodies specific to hsp 73 resulted in the coprecipitation of hsp 72. Similarly, antibodies specific to hsp 72 were capable of coprecipitating hsp 73. Using a number of different approaches, we show that the constitutively expressed, pre-existing hsp 73 rapidly forms a stable complex with the newly synthesized stress inducible hsp 72. As is demonstrated by double-label indirect immunofluorescence, both proteins exhibit a coincident locale within the cell. Moreover, injection of antibodies specific to hsp 73 into living cells effectively blocks the ability of both hsp 73 and hsp 72 to redistribute from the cytoplasm into the nucleus and nucleolus after heat shock. These results are discussed as they relate to the possible structure and function of the constitutive (hsp 73) and highly stress inducible (hsp 72) forms of hsp 70, both within the normal cell as well as in the cell experiencing stress.

Adenosine Triphosphate↗
PubMed1992

Nitric oxide synthase in macula densa regulates glomerular capillary pressure.

Tubular-fluid reabsorption by specialized cells of the nephron at the junction of the ascending limb of the loop of Henle and the distal convoluted tubule, termed the macula densa, releases compounds causing vasoconstriction of the adjacent afferent arteriole. Activation of this tubuloglomerular feedback response reduces glomerular capillary pressure of the nephron and, hence, the glomerular filtration rate. The tubuloglomerular feedback response functions in a negative-feedback mode to relate glomerular capillary pressure to tubular-fluid delivery and reabsorption. This system has been implicated in renal autoregulation, renin release, and longterm body fluid and blood-pressure homeostasis. Here we report that arginine-derived nitric oxide, generated in the macula densa, is an additional intercellular signaling molecule that is released during tubular-fluid reabsorption and counters the vasoconstriction of the afferent arteriole. Antibody to rat cerebellar constitutive nitric oxide synthase stained rat macula densa cells specifically. Microperfusion of the macula densa segment of single nephrons with N omega-methyl-L-arginine (an inhibitor of nitric oxide synthase) or with pyocyanin (a lipid-soluble inhibitor of endothelium-derived relaxation factor) showed that generation of nitric oxide can vasodilate the afferent arteriole and increase glomerular capillary pressure; this effect was blocked by drugs that prevent tubular-fluid reabsorption. We conclude that nitric oxide synthase in macula densa cells is activated by tubular-fluid reabsorption and mediates a vasodilating component to the tubuloglomerular feedback response. These findings imply a role for arginine-derived nitric oxide in body fluid-volume and blood-pressure homeostasis, in addition to its established roles in modulation of vascular tone by the endothelium and in neurotransmission.

Amino Acid Oxidoreductases↗