Biomedical subjects

D L Clemens

Publications and source records attributed to D L Clemens.

At least 37 records · Page 2Linked to original sources

PubMed1995

Characterization of recombinant human liver thermolabile phenol sulfotransferase with minoxidil as the substrate.

Minoxidil, a potent antihypertensive agent and hair growth stimulator, is metabolized by phenol sulfotransferase to its activated form, minoxidil sulfate. The thermostable form of phenol sulfotransferase was reported to be the enzyme that catalyzed the reaction. Our previous findings with partially purified human platelet preparations indicated that the thermolabile form of phenol sulfotransferase also catalyzed the sulfation of minoxidil. To confirm and to characterize precisely the activity of thermolabile phenol sulfotransferase toward minoxidil, we investigated the ability of the enzyme expressed from a human liver cDNA clone to sulfate minoxidil during testing of thermal stability and of inhibition by 2,6-dichloro-4-nitrophenol and NaCl. The cDNA encoded thermolabile phenol sulfotransferase activity assayed with minoxidil behaved in the same fashion as the activity measured with dopamine, a finding that confirmed that this enzyme activity sulfated minoxidil. Thus, thermolabile phenol sulfotransferase must be taken into account with the thermostable enzyme when estimating the human tissue sulfotransferase contribution to minoxidil sulfation.

Animals↗

PubMed1995

Characterization of the Mycobacterium tuberculosis phagosome and evidence that phagosomal maturation is inhibited.

We have used the cryosection immunogold technique to study the composition of the Mycobacterium tuberculosis phagosome. We have used quantitative immunogold staining to determine the distribution of several known markers of the endosomal-lysosomal pathway in human monocytes after ingestion of either M. tuberculosis, Legionella pneumophila, or polystyrene beads. Compared with the other phagocytic particles studied, the M. tuberculosis phagosome exhibits delayed clearance of major histocompatibility complex (MHC) class I molecules, relatively intense staining for MHC class II molecules and the endosomal marker transferrin receptor, and relatively weak staining for the lysosomal membrane glycoproteins, CD63, LAMP-1, and LAMP-2 and the lysosomal acid protease, cathepsin D. In contrast to M. tuberculosis, the L. pneumophila phagosome rapidly clears MHC class I molecules and excludes all endosomal-lysosomal markers studied. In contrast to both live M. tuberculosis and L. pneumophila phagosomes, phagosomes containing either polystyrene beads or heat-killed M. tuberculosis stain intensely for lysosomal membrane glycoproteins and cathepsin D. These findings suggest that (a) M. tuberculosis retards the maturation of its phagosome along the endosomal-lysosomal pathway and resides in a compartment with endosomal, as opposed to lysosomal, characteristics; and (b) the intraphagosomal pathway, i.e., the pathway followed by several intracellular parasites that inhibit phagosome-lysosome fusion, is heterogeneous.

Antigens, CD↗

PubMed1995

Purification, characterization, and genetic analysis of Mycobacterium tuberculosis urease, a potentially critical determinant of host-pathogen interaction.

Mycobacterium tuberculosis urease (urea amidohydrolase [EC 3.5.1.5]) was purified and shown to contain three subunits: two small subunits, each approximately 11,000 Da, and a large subunit of 62,000 Da. The N-terminal sequences of the three subunits were homologous to those of the A, B, and C subunits, respectively, of other bacterial ureases. M. tuberculosis urease was specific for urea, with a Km of 0.3 mM, and did not hydrolyze thiourea, hydroxyurea, arginine, or asparagine. The enzyme was active over a broad pH range (optimal activity at pH 7.2) and was remarkably stable against heating to 60 degrees C and resistant to denaturation with urea. The enzyme was not inhibited by 1 mM EDTA but was inhibited by N-ethylmaleimide, hydroxyurea, acetohydroxamate, and phenylphosphorodiamidate. Urease activity was readily detectable in M. tuberculosis growing in nitrogen-rich broth, but expression increased 10-fold upon nitrogen deprivation, which is consistent with a role for the enzyme in nitrogen acquisition by the bacterium. The gene cluster encoding urease was shown to have organizational similarities to urease gene clusters of other bacteria. The nucleotide sequence of the M. tuberculosis urease gene cluster revealed open reading frames corresponding to the urease A, B, and C subunits, as well as to the urease accessory molecules F and G.

Amino Acid Sequence↗

PubMed1994

Glutamine synthetase of Mycobacterium tuberculosis: extracellular release and characterization of its enzymatic activity.

We have investigated the activity and extracellular release of glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] of Mycobacterium tuberculosis. The purified, homogeneous M. tuberculosis glutamine synthetase appears to consist of 12 most likely identical subunits of M(r) 58,000, arranged in two superimpose hexagons. In the catalysis of L-glutamine, the enzyme has an apparent Km for L-glutamate of approximately 3 mM at the pH optimum of 7.5. M. tuberculosis releases a large proportion (approximately 30%) of its total measurable enzyme activity into the culture medium, a feature that is highly specific for pathogenic mycobacteria. Immunogold electron microscopy revealed that M. tuberculosis also releases the enzyme into its phagosome in infected human monocytes. Two potentially important roles for glutamine synthase in the pathogenesis of M. tuberculosis infection are (i) the synthesis of L-glutamine, a major component of the cell wall of pathogenic but not nonpathogenic mycobacteria, and (ii) the modulation of the ammonia level in the M. tuberculosis phagosome, which may in turn influence phagosomal pH and phagosomelysosome fusion.

Amino Acid Sequence↗

PubMed1994

Long-term ethanol feeding selectively impairs the attachment of rat perivenous hepatocytes to extracellular matrix substrates.

BACKGROUND/AIMS: We have previously shown that long-term ethanol consumption by rats results in a profound decrease in hepatocyte attachment to various extracellular matrix substrates. The present study investigated whether differences in attachment exist between cells isolated from either the periportal or perivenous regions of the liver. METHODS: Rats received long-term ethanol, and hepatocytes were selectively isolated by the digitonin-collagenase perfusion method. The ability of periportal and perivenous cells isolated from ethanol-fed and pair-fed control rats to attach to plates coated with either laminin, fibronectin, or type I collagen was then assayed. RESULTS: With all substrates, the attachment of perivenous hepatocytes isolated from ethanol-fed animals was significantly impaired. Time-course studies showed that although the rate of attachment of perivenous cells from ethanol-fed animals was only slightly reduced, a dramatic decrease in absolute attachment was observed. Furthermore, the perivenous cells isolated from ethanol-fed animals detached more readily from the substrate-coated plates than the corresponding periportal cells or either periportal or perivenous cells from pair-fed controls. CONCLUSIONS: Long-term ethanol consumption impairs hepatocyte-extracellular matrix interactions more severely in the perivenous region of the liver. This finding could be relevant to the pathological changes observed in alcoholic liver injury.

Animals↗

PubMed1994

Effects of ethanol feeding on the interaction of rat hepatocytes with laminin peptides.

Laminin, a complex glycoprotein of the extracellular matrix, contains a number of biologically active sites. These sites are involved in cell growth, attachment, differentiation, and gene expression. Our previous studies have shown that chronic ethanol consumption by rats impairs hepatocyte attachment to various components of the extracellular matrix including laminin. In this study, three synthetic peptides (PA22-2, YIGSR, and RGD) that correspond to three distinct functional sites on the laminin molecule were used to investigate the effect of ethanol consumption on their cognate receptors. Initially, varying concentrations of each peptide were incubated with isolated hepatocytes from ethanol-fed and pair-fed control rats. These hepatocytes were then assayed for the ability to attach to laminin. The results indicated that all three peptides effectively inhibited laminin-mediated cell adhesion: the degree of inhibition appeared similar between pair-fed controls and ethanol-fed animals. Of the three peptides, PA22-2 showed the most dramatic inhibition of attachment. Therefore, we investigated the ability of hepatocytes to attach directly to PA22-2 itself. Attachment of hepatocytes from ethanol-fed animals to PA22-2 was impaired by 30% after 4 days and 90% by 14 days. Conversely, no significant difference in attachment to the entire laminin molecule was observed in ethanol-fed animals at these early time points. These results indicated that the ethanol-induced impairment of hepatocyte attachment to laminin may be caused by the decreased interaction of hepatocytes with specific functional sites on the laminin molecule and that specific receptors on the hepatocyte may be affected differently.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcoholism↗

PubMed1993

Hypoexpression of major histocompatibility complex molecules on Legionella pneumophila phagosomes and phagolysosomes.

Legionella pneumophila is a facultative intracellular pathogen that parasitizes host mononuclear phagocytes. Cell-mediated immunity is pivotal to host defense against L. pneumophila, and the infected host cell may play a central role in processing and presenting parasite antigens to lymphocytes mediating cell-mediated immune response. However, in the case of L. pneumophila and intracellular parasites in general, little is known about the intracellular trafficking of parasite antigens, the influence of parasite infection on major histocompatibility complex (MHC) expression, or the relationship of MHC molecules to sites of parasite replication. To learn more about this, we have used flow cytometry to study the expression of HLA-DR by monocytes infected with L. pneumophila and cryosection immunogold electron microscopy to study the distribution of MHC class I and II molecules on L. pneumophila phagosomes. Flow cytometry analysis demonstrated that L. pneumophila infection has little effect on the overall expression of HLA-DR by monocytes. Cryosection immunogold studies revealed abundant staining for MHC class I and II molecules on the plasma membrane of infected monocytes but little or no staining on the membranes of mature L. pneumophila phagosomes. Cryosection immunogold studies of an avirulent mutant of L. pneumophila that, unlike the wild type, does not inhibit phagosome-lysosome fusion and subsequently survives but does not multiply in a phagolysosome yielded similar results. We have previously found that MHC class I and II molecules are excluded from nascent phagosomes during coiling and conventional phagocytosis. The present work demonstrates that MHC molecules do not accumulate appreciably in the L. pneumophila phagosome as it matures and at a point in the life cycle of the organism in which it is replicating and producing immunoprotective T-cell antigens. This suggests that L. pneumophila does not reside in a typical endosomal compartment in the host cell and that L. pneumophila antigens may encounter MHC molecules at extraphagosomal sites within the host cell.

Animals↗

PubMed1992

Membrane sorting during phagocytosis: selective exclusion of major histocompatibility complex molecules but not complement receptor CR3 during conventional and coiling phagocytosis.

We have used immunocytochemical techniques and enzyme cytochemistry to examine the distribution of plasma membrane proteins during coiling phagocytosis of Legionella pneumophila and conventional phagocytosis of Escherichia coli. Whereas class I and class II major histocompatibility complex (MHC) molecules are relatively excluded from nascent phagosomes during conventional and coiling phagocytosis, the CR3 complement receptor persists in nascent phagosomes. The staining pattern for alkaline phosphatase activity resembles that of MHC molecules, with a marked exclusion of phosphatase activity from L. pneumophila coils and nascent phagosomes. The staining pattern for 5'-nucleotidase activity, on the other hand, resembles that of CR3 with intense staining in the inner layers of L. pneumophila coils. These results demonstrate that the cell has the ability to exclude selectively certain membrane proteins from the nascent phagosome during phagocytosis, thereby producing a phagosomal membrane markedly different from the plasma membrane from which it is derived.

5'-Nucleotidase↗

PubMed1992

Expression of Aleutian mink disease parvovirus capsid proteins by a recombinant vaccinia virus: self-assembly of capsid proteins into particles.

A portion of a cDNA clone containing coding sequences for both structural proteins (VP1 and VP2) of Aleutian mink disease parvovirus (ADV) was inserted into recombinant vaccinia viruses, VV:ADSP. Immunohistochemical staining of VV:ADSP-infected cells revealed that the ADV antigen was readily detected and localized in the nuclei of infected cells. Analysis of VV:ADSP-infected cell lystates indicated that both VP1 and VP2 were produced and comigrated with authentic VP1 and VP2 from ADV-infected Crandell feline kidney cells. These results suggested, therefore, that both VP1 and VP2 were synthesized from a single cloned transcript. CsCl density gradient centrifugation of partially purified VV:ADSP-infected cell lysates indicated that the majority of the antigen was located in a fraction with a density near 1.33 g/ml, indicative of empty ADV particles. Subsequent electron microscopic examination revealed the presence of 27-nm icosahedral virion-like structures at the same density, suggesting that the proteins self-assembled into empty virions. Furthermore, sera from eight of eight mice inoculated with VV:ADSP contained ADV-specific antibodies and two of these eight serum samples had neutralizing activity, indicating that the particles produced in VV:ADSP-infected cells were immunogenic. Finally, when lysates from VV:ADSP-infected cells were compared with standard ADV antigens in counterimmunoelectrophoresis assays, a similar pattern of specific reactivity was observed for sera from normal and infected mink.

Aleutian Mink Disease↗

PubMed1992

Localization of components of the renin-angiotensin system within the kidney and sustained release of angiotensins from isolated and perfused kidney.

We found colocalization of renin, angiotensin (Ang) I and Ang II in juxtaglomerular (JG) cells of the kidney. Coexistence of Ang II in renin granules was demonstrated by electron microscopic immunogold labeling of these components. Coexistence of both Ang I and Ang II in the high density renin storage granules were also demonstrated by gradient centrifugation of renal homogenate. These findings supported the synthesis of Ang I and Ang II in juxtaglomerular cells. Isolated and cultured JG cells showed the synthesis of Ang I, Ang II and renin. Ang I and Ang II were secreted from isolated and perfused rat kidneys at steady rates over 2 hr. Their secretion rates were proportional to that of renin. The rate of Ang II secretion from the kidney was higher than that from the vascular bed. Ang II was also found in renal lymph. These findings indicate that a large amount of Ang II is generated in JG cells by the intracellular action of renin and may play a significant role in the regulation of renal function.

Angiotensins↗

PubMed1989

Regulated expression of the feline panleukopenia virus P38 promoter on extrachromosomal FPV/EBV chimeric plasmids.

Feline panleukopenia virus/Epstein-Barr virus (FPV/EBV) chimeric expression plasmids were constructed to study regulation of the structural protein gene of the parvovirus, FPV, in a homologous cell culture system. Detection and quantitation of activity from the native FPV promoter, P38, was facilitated by fusing the Escherichia coli lacZ gene with the FPV structural protein gene. Feline cell lines which stably maintained these plasmids extrachromosomally were established. Constitutive beta-galactosidase activity was low but increased up to 40-fold after infection with FPV. Expression of beta-galactosidase was only detected when the FPV/lacZ gene was oriented in the same transcriptional direction as the Epstein-Barr virus gene coding for EBNA-1. When a small open reading frame upstream of the FPV/lacZ initiation codon was deleted, beta-galactosidase expression increased another 4.7- to 26-fold. These changes in beta-galactosidase activity indicate that expression of the FPV structural protein gene is regulated both transcriptionally and posttranscriptionally.

Animals↗

PubMed1986

Generation of angiotensinogen by cultured neuroblastoma and glioma cells.

Cultured neuroblastoma cells and neuroblastoma-glioma cells have been shown to contain renin activity, angiotensin-converting enzyme activity, and angiotensins. It has been assumed that these cells also produce angiotensinogen as the substrate of an intracellular renin-angiotensin system. However, measurements of angiotensinogen have not been reported in the neuroblastoma or neuroblastoma-glioma cells, and the possibility that the cells generate angiotensins from fetal bovine angiotensinogen has not been eliminated. In this work angiotensinogen was shown to accumulate in the serum-free medium of thoroughly washed neuroblastoma cells (mouse Neuro-2A and rat B103) and glioma cells (rat C6). Separate experiments demonstrated that mouse Neuro-2A cells continue to produce angiotensinogen even after two passages in a defined serum-free culture medium. Further evidence that the angiotensinogen was not a contaminant from fetal bovine serum was obtained by the use of a monoclonal antibody raised against angiotensinogen of rat plasma. The angiotensinogen of Neuro-2A and C6-glioma cells is bound by the monoclonal antibody, whereas fetal bovine angiotensinogen is not bound. These results are consistent with the hypothesis that angiotensinogen is produced locally in the brain and in neuroblastoma cells as a substrate for an intracellular renin-angiotensin system.

Angiotensinogen↗

PubMed1986

Subcellular localization of angiotensin-converting enzyme in cultured neuroblastoma cells.

Angiotensin-converting enzyme (ACE) activity of Neuro-2A mouse neuroblastoma cells was found predominantly in particulate fractions. Density gradient centrifugation of the particulate fractions showed ACE activity in light fractions of the gradient, a result suggesting a plasma membrane localization. This was confirmed using the aqueous two-phase polymer system of plasma membrane isolation. The rapid and energy-independent hydrolysis of exogenous substrate by ACE of intact cells and the sensitivity of the enzyme of intact cells to proteases indicate further that the active site of ACE is oriented extracellularly.

Animals↗

PubMed1984

Generation of angiotensins in cultured pheochromocytoma cells.

Cultured rat pheochromocytoma cells, PC-12, were found to contain renin-like activity. The activity which was inhibited by monospecific antibodies to rat renal renin showed a pH optimum between 6 and 7, indicating that it was a specific renin. The cells also contained angiotensin I and angiotensin I converting enzyme. Angiotensin II/III were also detected in lysate. These findings indicated an intrinsic pathway of angiotensin formation in pheochromocytoma. The formation of angiotensins in pheochromocytoma cells suggests modulating functions of angiotensin on neurotransmitter release from adrenal medulla.

Adrenal Medulla↗

PubMed1982

Brain renin.

Although the brain contains cathepsins at high concentrations which exhibit a non-specific renin-like activity at acidic pH, the presence of specific renin in the brain has been demonstrated by characterizing its specific properties. Renin was separated from cathepsin by affinity chromatography on casein-Sepharose. Brain renin showed neutral pH optima for the reaction to generate angiotensin I. The presence of inactive prorenin was also found. The isoelectric points of brain renin were significantly lower differences from that of renal or plasma renin. Immunohistochemical studies demonstrated a wide-spread localization of renin in many different regions. Angiotensin II, the final product of the prohormone-to-hormone conversion reaction mediated by renin and angiotensin converting enzyme, was found to exist in the same cell as renin by immunohistochemical studies of brain sections and with cloned and cultured neuroblastoma cells. This is the first demonstration of the mechanism of peptide hormone formation in neuronal cells. Similar intracellular formation was demonstrated in gonadotrophs of adenohypophysis. Coexistence of renin and angiotensin II was demonstrated in some cells. Electrophysiological studies have shown that angiotensin II functions to disinhibit the inhibition of neuronal response to electrical stimuli in the hippocampus.

Angiotensin II↗

PubMed1982

Molecular characterization of inactive renin: complete purification of prorenin in hog kidney and isolation of inactive renin from neuroblastoma cells: evidence for 2 different types of inactive renin.

To determine the molecular properties of inactive renin and its relationship to active renin, inactive renin in hog kidney was purified by devising affinity chromatography. Electrophoretically homogeneous inactive renin was prepared by 3 million-fold purification. It consists of a single polypeptide chain and undergoes reduction in molecular weight from 50,000 to 38,000 upon activation by proteases but not by dissociative treatment. This type of inactive renin is considered as a zymogen. However, a stable complex of renin and its inhibitor with a molecular weight of 110,000 was found in cultured neuroblastoma cells indicating the presence of a second type of inactive renin.

Animals↗

PubMed1981

Renin, angiotensins, and angiotensin-converting enzyme in neuroblastoma cells: evidence for intracellular formation of angiotensins.

The mechanism of formation of various peptide hormones in neuronal cells in the brain is not clear. The question of whether brain angiotensin II is formed by an extracellular mechanism as in the peripheral system or by an intracellular mechanism can be answered by using cloned cells in culture. We have screened several neuroblastoma cell lines of rat and mouse origin and found at least three cell lines that contain renin (EC 3.4.99.19), angiotensin-converting enzyme (dipeptidyl carboxypeptidase; peptidyldipeptide hydrolase, EC 3.4.15.1), and angiotensins I and II. This finding was interpreted to indicate that in these cells angiotensin formation takes place by an intracellular mechanism, in contrast to the extracellular mechanism well known to occur in plasma. This study also demonstrates the existence of viable and cloned cell lines that produce renin.

Angiotensin I↗

PubMed1980

Immunohistochemical localization of renin in mouse brain.

Immunohistochemical studies of mouse brain by the peroxidase-antiperoxidase method using monospecific antimouse renin antibodies has revealed the intracellular localization of renin in stellate and small ovoid cells. Renin-containing ovoid cells were observed in the spinal cord, medulla oblongata, pons, granular layers of the cerebellum, deep cerebellar region, and the lamina terminalis; whereas immunostainable stellate cells were found in the cerebral cortex, hippocampus, dentate gyrus and septum. Intracellular localization of renin rather than intravascular localization supports an endogenous origin of this enzyme in the brain. Wide distribution in different types of cells suggests different types of regulatory mechanisms. Double immunostaining with antigalactocerebroside and antirenin antibodies indicates the presence of renin in oligodendrocytes.

Animals↗