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J Moss

Publications and source records attributed to J Moss.

At least 289 records · Page 16Linked to original sources
PubMed1992

Differential interaction of ADP-ribosylation factors 1, 3, and 5 with rat brain Golgi membranes.

Six mammalian ADP-ribosylation factors (ARFs) identified by cDNA cloning were expressed as recombinant proteins (rARFs) that stimulated cholera toxin ADP-ribosyltransferase activity. Microsequencing of soluble ARFs I and II (sARFs I and II), purified from bovine brain, established that they are ARFs 1 and 3, respectively. Rabbit antibodies (IgG) against sARF II reacted similarly with ARFs 1, 2, and 3 (class I) on Western blots. ARFs 1 and 3 were distinguished by their electrophoretic mobilities. Antiserum against rARF 5 cross-reacted partially with rARF 4 but not detectably with rARF 6 and minimally with class I ARFs. Guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma S]) increased recovery of ARF activity and immunoreactivity in organelle fractions separated by density gradient centrifugation, after incubation of rat brain homogenate with ATP and a regenerating system. ARF 1 accumulated in microsomes plus Golgi and Golgi fractions, whereas ARF 5 seemed to localize more specifically in Golgi; the smaller increment in ARF 3 was distributed more evenly among fractions. On incubation of Golgi with a crude ARF fraction, GTP[gamma S], and an ATP-regenerating system, association of ARF activity with Golgi increased with increasing ATP concentration paralleled by increases in immunoreactive ARFs 1 and 5 and, to a lesser degree, ARF 3. Golgi incubated with GTP[gamma S] and purified ARF 1 or 3 bound more ARF 1 than ARF 3. Based on immunoreactivity and assay of ARF activity, individual ARFs 1, 3, and 5 appeared to behave independently and selectively in their GTP-dependent association with Golgi in vitro.

ADP-Ribosylation Factors↗
PubMed1992

Effects of phospholipid and GTP on recombinant ADP-ribosylation factors (ARFs). Molecular basis for differences in requirements for activity of mammalian ARFs.

ADP-ribosylation factors (ARFs) are highly conserved approximately 20-kDa guanine nucleotide-binding proteins that were first identified based on their ability to stimulate the cholera toxin-catalyzed ADP-ribosylation of Gs alpha and thus activate adenylyl cyclase. Proteins with ARF activity have been characterized from different mammalian tissues and exhibited different requirements for activity, stability, and phospholipid. Based on molecular cloning and mRNA distribution, at least six mammalian ARFs, which fall into three classes, have been identified. To test whether individual ARFs might have different requirements for optimal activity, as judged by their ability to enhance cholera toxin ADP-ribosyltransferase activity, four ARFs from classes I, II, and III were produced as recombinant proteins in Escherichia coli and characterized. Recombinant bovine ARF 2 (rARF 2) and human ARF 3 (rARF 3) (class I), human ARF 5 (rARF 5, class II), and human ARF 6 (rARF 6, class III) differed in the effects of phospholipid and detergent on their ability to enhance cholera toxin activity; rARFs 2, 3, and 5 required dimyristoylphosphatidylcholine (DMPC) and cholate, whereas rARF 6 did not require phospholipid/detergent for activity. Further characterization of two of the more divergent ARFs (ARFs 2 and 6) showed that both exhibited guanosine 5'-O-(3-thio)triphosphate binding which was enhanced by DMPC/cholate. In the transferase assay, rARF 2 required approximately 4 microM GTP for half-maximal stimulation of toxin activity, whereas rARF 6 required 0.05 microM GTP. rARF 6 exhibited a delay in activation of toxin not detected with rARF 2 that may be related to a requirement for guanine nucleotide exchange and/or GTP binding. These findings are consistent with the conclusion that the highly conserved members of the ARF family have different requirements for optimal activity.

ADP-Ribosylation Factor 6↗
PubMed1992

Colocalization of nitric oxide synthase and NADPH-diaphorase in the myenteric plexus of the rat gut.

The pattern of distribution and colocalization of nitric oxide-synthase (NOS) and NADPH-diaphorase in the myenteric plexus of whole-mount preparations of the antrum, duodenum, ileum, caecum, proximal colon and distal colon of the rat were investigated using immunohistochemical and histochemical staining techniques. Almost all the myenteric neurons that were NOS-positive in all regions of the gut examined were also stained for NADPH-diaphorase. However, in the stomach, duodenum and ileum, only a few of the NOS-positive nerve fibres in the tertiary and secondary plexuses and circular muscle layer were also stained for NADPH-diaphorase, whereas in the caecum and distal colon almost all the NOS-positive nerve fibres were also stained for NADPH-diaphorase. The results in the present study are consistent with the view that nitric oxide (NO) has a mediating role in gastrointestinal neurotransmission.

Amino Acid Oxidoreductases↗
PubMed1992

Nitric oxide synthase immunoreactivity and NADPH-diaphorase activity in a subpopulation of intrinsic neurones of the guinea-pig heart.

This is the first report of the presence of nitric oxide synthase (NOS) immunoreactivity and NADPH-diaphorase (NADPH-d) activity in a subpopulation of the intrinsic neurones that innervate the heart. A cytochemical technique to detect NADPH-d and antisera raised against purified rat cerebellar NOS were employed to examine the expression of these enzymes by cells in a dissociated cell culture preparation from newborn guinea-pig atria and interatrial septum. Comparison of the results obtained by these two techniques and double-labelling experiments indicate that a subpopulation of intracardiac neurones contain both NADPH-d and NOS. These results indicate that some intracardiac neurones are capable of synthesizing nitric oxide. This raises the possibility that nitric oxide plays a role in the neural control of the heart.

Amino Acid Oxidoreductases↗
PubMed1992

Pertussis toxin-catalyzed ADP-ribosylation of G(o) alpha with mutations at the carboxyl terminus.

The guanine nucleotide-binding protein G(o alpha) has been implicated in the regulation of Ca2+ channels in neural tissues. Covalent modification of G(o alpha) by pertussis toxin-catalyzed ADP-ribosylation of a cysteine (position 351) four amino acids from the carboxyl terminus decouples G(o alpha) from receptor. To define the structural requirements for ADP-ribosylation, preparations of recombinant G(o alpha) with mutations within the five amino acids at the carboxyl terminus were evaluated for their ability to serve as pertussis toxin substrates. As expected, the mutant in which cysteine 351 was replaced by glycine (C351G) was not a toxin substrate. Other inactive mutants were G352D and L353 delta/Y354 delta. Mutations that had no significant effect on toxin-catalyzed ADP-ribosylation included G350D, G350R, Y354 delta, and L353V/Y354 delta. Less active mutants were L353G/Y354 delta, L353A/Y354 delta, and L353G. ADP-ribosylation of the active mutants, like that of wild-type G(o alpha), was enhanced by the beta gamma subunits of bovine transducin. It appears that three of the four terminal amino acids critically influence pertussis toxin-catalyzed ADP-ribosylation of G(o alpha).

Adenosine Diphosphate Ribose↗
PubMed1992

Molecular and immunological characterization of ADP-ribosylarginine hydrolases.

Mono-ADP-ribosylation is a reversible modification of proteins with NAD:arginine ADP-ribosyltransferases and ADP-ribosylarginine hydrolases catalyzing the forward and reverse reactions, respectively. Hydrolase activities were present in a variety of animal species, with the highest specific activities found in rat and mouse brain, spleen, and testis. Rat and mouse hydrolases were dithiothreitol- and Mg(2+)-dependent, whereas the bovine and guinea pig enzymes were dithiothreitol-independent. A rat brain hydrolase was purified approximately 20,000-fold and represented the major approximately 39-kDa protein on denaturing gels. Immunoaffinity-purified rabbit polyclonal antibodies reacted with 39-kDa proteins from turkey erythrocytes and rat, mouse, and calf brains. A rat brain cDNA library was screened using oligonucleotide and polymerase chain reaction-generated cDNA probes. Inserts from two overlapping clones yielded a composite sequence that included a 1086-base pair open reading frame, which contained amino acid sequences found in the purified hydrolase. A hydrolase fusion protein, synthesized in Escherichia coli, reacted with anti-39-kDa polyclonal antibodies and exhibited Mg(2+)- and dithiothreitol-dependent hydrolase activity. A coding region cDNA hybridized readily to a 1.7-kilobase band in rat and mouse poly(A)+ RNA, but poorly to bovine, chicken, rabbit, and human poly(A)+ RNA. The immunological and molecular biological data are consistent with partial conservation of hydrolase structure across animal species.

Amino Acid Sequence↗
PubMed1992

Guanine nucleotide-binding proteins in the intestinal parasite Giardia lamblia. Isolation of a gene encoding an approximately 20-kDa ADP-ribosylation factor.

Giardia lamblia is a protozoan intestinal parasite that has characteristics of both eukaryotes and prokaryotes. To determine whether genes for guanine nucleotide-binding proteins are present in Giardia, genomic DNA and cDNA libraries were screened by polymerase chain reaction and by hybridization with mixed oligonucleotide probes complementary to sequences encoding conserved GTP-binding domains. A gene with a high degree of sequence identity with mammalian ADP-ribosylation factors (ARFs), believed to be important in vesicular transport, was identified. The Giardia ARF gene had a 573-base open reading frame encoding 191 amino acids which are 63-70% identical with known mammalian and yeast ARFs. Sequence conservation among ARFs was greatest in putative GTP-binding domains. A single ARF mRNA species of approximately 750 bases was found in two different Giardia isolates. Primer extension and RNA sequencing of the Giardia ARF transcript revealed a short (6-base) 5'-untranslated region similar in size to those found in other Giardia transcripts. Giardia extracts contained ARF activity, as shown by stimulation of cholera toxin-catalyzed ADP-ribosylation and a Giardia ARF expressed in Escherichia coli as a fusion protein likewise exhibited biochemical activity. Its presence in Giardia is consistent with the view that ARF emerged before the divergence of this protozoan from other eukaryotes (approximately 1.5 billion years ago), and that an ARF-like protein may have been the ancestor of several other classes of signal-transducing guanine nucleotide-binding proteins, including the alpha subunits of the heterotrimeric G proteins.

ADP-Ribosylation Factors↗
PubMed1992

Characterization of the human gene encoding ADP-ribosylation factor 1, a guanine nucleotide-binding activator of cholera toxin.

Mammalian ADP-ribosylation factors (ARFs), approximately 20-kDa guanine nucleotide-binding proteins that stimulate cholera toxin ADP-ribosyltransferase activity, were grouped into three classes based on deduced amino acid sequence. Human ARF 1, a class I ARF, is identical with its bovine counterpart, has a distinctive pattern of tissue and developmental expression, and is encoded by a approximately 1.9-kilobase mRNA. ARF 1 cDNAs were isolated from a human fibroblast cDNA library; one arose via an alternative polyadenylation signal (AA-TACA) 84 nucleotides 5' to the polyadenylation signal (AATAAA) used in the 1815-base pair cDNA. The polyadenylation signals, their respective locations, and the surrounding nucleotide sequences are conserved in human and rat. The human ARF 1 gene, with four introns, spans approximately 16.5 kilobases. Exon 1 (46 base pairs) contains only untranslated sequence. Translation initiates in exon 2, which encodes the sequence GXXXXGK involved in phosphate binding (GTP hydrolysis). The sequence DVGG is encoded in exon 3, and NKQD, which is involved in the interaction with the guanine ring, is interrupted following the codon for Q by intron 4. The carboxyl-terminal 53 amino acids and greater than 1110 base pairs of 3'-untranslated region are encoded in exon 5. Primer extension and mung bean and S1 nuclease mapping indicated multiple transcription initiation sites and were consistent with Northern analyses. The 5'-flanking region has a high GC content but no TATA or CAAT box, as found in housekeeping genes. In addition, the two human class I ARF genes, ARF 1 and ARF 3, have similar exon/intron organizations and use GC-rich promoters.

ADP-Ribosylation Factors↗
PubMed1992

Cyclic AMP-independent effects of cholera toxin on B cell activation. II. Binding of ganglioside GM1 induces B cell activation.

Although the physiologic function of gangliosides is unknown, evidence suggests they play a role in the regulation of cell growth. The binding of ganglioside GM1 by recombinant B subunit of cholera toxin (rCT-B) inhibited mitogen-stimulated B cell proliferation without elevating intracellular cAMP. CT-B paradoxically enhanced the expression of MHC class II (Ia) molecules and minor lymphocyte-stimulating determinants without altering the expression of some other immunologically relevant B cell surface Ag. Increased expression of Ia was not detected until 4 h after stimulation, kinetics similar to those seen when B cells are stimulated with anti-Ig antibody or IL-4, suggesting that the enhancement was not the result of redistribution of existing cell surface markers but rather the result of a new metabolic event. Both the inhibitory and stimulatory effects of CT-B could be blocked by incubation of CT-B with ganglioside GM1. Furthermore, enhancement of the CT-B-mediated effect was seen when additional ganglioside GM1 was incorporated into the B cell membrane. rCT-B with a mutation that interfered with its binding to ganglioside GM1 did not enhance Ia expression. Taken together, these results indicate that the observed effects of CT-B were most likely mediated through the binding of cell surface ganglioside GM1. CT-B-mediated stimulation of Ia expression provides a potential explanation for the previously described ability of CT-B to act as an immunoadjuvant. These results suggest that the binding of ganglioside GM1 has multiple B cell growth-regulating effects.

Animals↗
PubMed1992

Ligation-independent cloning of glutathione S-transferase fusion genes for expression in Escherichia coli.

A plasmid vector has been constructed that allows the ligation-independent cloning of cDNAs in any reading frame and directs their synthesis in Escherichia coli as glutathione S-transferase-linked fusion proteins. The cloning procedure does not require restriction enzyme digestion of the target sequence and does not introduce any additional sequences between the thrombin cleavage site and the foreign protein. Extended single-stranded tails complementary between the vector and insert, generated by the (3'----5') exonuclease activity of T4 DNA polymerase, obviate the need for in vitro ligation prior to bacterial transformation. This cloning procedure is rapid and highly efficient, and has been used successfully to construct a series of fusion proteins to investigate the sequence requirements for efficient thrombin cleavage.

ADP-Ribosylation Factors↗
PubMed1992

GTP but not GDP analogues promote association of ADP-ribosylation factors, 20-kDa protein activators of cholera toxin, with phospholipids and PC-12 cell membranes.

ADP-ribosylation factors (ARFs) are a family of approximately 20-kDa guanine nucleotide-binding proteins initially identified by their ability to enhance cholera toxin ADP-ribosyltransferase activity in the presence of GTP. ARFs have been purified from both membrane and cytosolic fractions. ARF purified from bovine brain cytosol requires phospholipid plus detergent for high affinity guanine nucleotide binding and for optimal enhancement of cholera toxin ADP-ribosyltransferase activity. The phospholipid requirements, combined with a putative role for ARF in vesicular transport, suggested that the soluble protein might interact reversibly with membranes. A polyclonal antibody against purified bovine ARF (sARF II) was used to detect ARF by immunoblot in membrane and soluble fractions from rat pheochromocytoma (PC-12) cell homogenates. ARF was predominantly cytosolic but increased in membranes during incubation of homogenates with nonhydrolyzable GTP analogues guanosine 5'-O-(3-thiotriphosphate), guanylyl-(beta gamma-imido)-diphosphate, and guanylyl-(beta gamma-methylene)-diphosphate, and to a lesser extent, adenosine 5'-O-(3-thiotriphosphate). GTP, GDP, GMP, and ATP were inactive. Cytosolic ARF similarly associated with added phosphatidylserine, phosphatidylinositol, or cardiolipin in GTP gamma S-dependent fashion. ARF binding to phosphatidylserine was reversible and coincident with stimulation of cholera toxin-catalyzed ADP-ribosylation. These observations may reflect a mechanism by which ARF could cycle between soluble and membrane compartments in vivo.

ADP-Ribosylation Factors↗
PubMed1992

The neuromuscular basis of hereditary kyphoscoliosis in the mouse.

We describe a new neuromuscular disorder in the kyphoscoliotic mouse mutant (ky). Mice were killed at ages from birth to 210 days, and tissues were taken for standard light microscopy, histochemistry, nerve ending studies, and electron microscopy. At birth a few myofibers showed phagocytosis ultrastructurally. Between 6 and 25 days there was prominent necrosis and regeneration in soleus, gracilis, paraspinal, and back muscles. At 47 days, these muscles were atrophic and necrosis and regeneration were rare. At 136 days, all muscle groups, including head muscles, showed some degree of myofiber atrophy and gracilis was fibrotic. Prominent intramuscular axonal sprouting was present from 31 days. Peripheral nerves and anterior horn cells were normal. The findings indicate a neuromuscular basis of hereditary kyphoscoliosis in the mouse. The animal may be useful as a model of human muscle disease and scoliosis.

Animals↗
PubMed1992

Diabetic glomerulosclerosis--immunogold ultrastructural studies on the glomerular distribution of type IV collagen and heparan sulphate proteoglycan.

We have undertaken an ultrastructural immunogold investigation of the distribution of type IV collagen and heparan sulphate proteoglycan (HSPG) in glomeruli from the kidneys of one normal control and three patients with diabetes mellitus and proteinuria. The sample included both diffuse and nodular diabetic glomerulosclerosis. In the control and diabetic kidneys, the type IV collagen was present predominantly on the endothelial aspect of the glomerular basement membrane (GBM), and by contrast the HSPG was found mainly on the epithelial side. In the mesangium in both control and diabetic glomeruli, type IV collagen was found predominantly in the central regions, while HSPG was mostly restricted to the region beneath the epithelial cells. Consequently, where there is a marked increase in mesangial matrix with nodule formation in diabetics there is a corresponding increase in the amount of type IV collagen but not of HSPG. Although the three diabetic patients were proteinuric, the HSPG was not decreased in the thickened GBMs.

Adult↗
PubMed1992

Estrogen and progesterone withdrawal increases cerebral vasoreactivity to serotonin in rabbit basilar artery.

An in vitro animal model which examines the effects of sex hormone variations during the menstrual cycle on basilar artery reactivity is presented. Three groups of rabbits were utilized: a chronically depleted control group which received no further hormonal treatment after bilateral surgical oophorectomy (O), simulating menopause, and two groups of intact females, one of which was treated to mimic the estrogen and progesterone surge during the luteal phase (H) and the third group which was acutely estrogen and progesterone depleted after the luteal surges to simulate the immediate premenstrual state (W). We show that both acute and chronic estrogen and progesterone withdrawal significantly increase serotonin sensitivity (ED50) in basilar artery rings. There was no difference between groups for maximum contraction (Tmax) to serotonin, nor optimal resting tension. Furthermore, there was no difference in vasoreactivity and contractility to norepinephrine between groups. In order to distinguish between the effects of chronic and acute treatment we examined acute estrogen and progesterone superfusion in basilar artery rings from intact non-treated female rabbits. Acute superfusion of pre-contracted and non-pre-contracted artery segments resulted in significant dilatation only when supraphysiologic concentrations of estrogen and progesterone were used. We conclude that both acute and chronic female sex hormone withdrawal selectively increases cerebral vasoreactivity to serotonin.

Animals↗
PubMed1992

Conservation of a 23-kDa human transplantation antigen in mammalian species.

A group of transplantation antigens, referred to as tum- antigens, were identified in mouse tumor cells that had been mutagenized to produce variant cells and were recognized by clonal cytolytic T lymphocytes (CTL). Alterations in these variant cells that were recognized by CTL resulted from point mutations in the genes of specific proteins. We have isolated human and bovine cDNA clones that encode the homologs of the mouse tum- antigen P198. This 23.6-kDa protein is highly basic with a predicted pI of 11.55. p23/P198 is highly conserved across mammalian species, with > 94% identity (97% including conservative substitutions) among the human, bovine, and mouse deduced amino acid sequences. The nucleotide sequences of both the coding and 5'- and 3'-untranslated regions from human, bovine, and mouse are also highly conserved with > 88% identity in the coding regions. Hybridization of poly(A)+ RNA from various mammalian sources with cDNA and oligonucleotides specific for the coding region identified two mRNAs of 1.2 and 0.8 kb, whereas probes specific for the 3'-untranslated region between two consensus polyadenylation signals hybridized with the 1.2-kb, but not the 0.8-kb, mRNA. The abundance of the 1.2-kb mRNA relative to that of the 0.8-kb species varied depending upon the cell type. A single predominant transcription initiation site was mapped by primer extension. These studies indicate that this highly basic 23.6-kDa protein is encoded by two major mRNA species that differ only in the length of their 3'-untranslated regions and that the mechanism that gives rise to these two mRNAs, utilization of alternative polyadenylation sites, is conserved across species.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Colocalization of nitric oxide synthase and NADPH-diaphorase in cultured myenteric neurones.

Nitric oxide synthase immunoreactivity and NADPH-diaphorase activity were examined in explant culture preparations of the myenteric plexus from beneath the taenia coli of the guinea-pig caecum. Nitric oxide synthase immunoreactive neurones formed approximately one third of the total neuronal population. NADPH-diaphorase positive neurones, demonstrated histochemically, constituted a similar proportion of the total number of neurones. Immunocytochemistry and NADPH-diaphorase histochemistry performed on the same preparations revealed that all nitric oxide synthase immunoreactive neurones expressed NADPH-diaphorase activity. This histochemical evidence is consistent with the view that nitric oxide may act as a regulatory agent in the guinea-pig caecum.

Amino Acid Oxidoreductases↗