PubMed Health⌕ Search

Biomedical subjects

G A Clawson

Publications and source records attributed to G A Clawson.

At least 37 records · Page 2Linked to original sources
PubMed1992

Nuclear enlargement induced by hepatocarcinogens alters ploidy.

Treatment of rats with low doses of hepatocarcinogens is associated with a number of phenomena, including nuclear enlargement and altered nucleocytoplasmic compartmentation, which potentially reflect initiatory changes. Since a number of investigations have indicated that changes in ploidy may relate to the development of altered foci and/or hepatocellular carcinomas in liver, and since nuclear enlargement may be associated with changes in DNA content, we examined the ploidy of rat hepatocytes following essentially nontoxic low-dose carcinogen treatment. Treatments with thioacetamide, 2-acetylaminofluorene, or dimethylnitrosamine were all associated with a notable shift of nuclei from diploid to tetraploid, in the apparent absence of nuclear replication. These changes were similar in magnitude to that associated with the substantial liver regeneration induced by carbon tetrachloride. If it is argued that cell replication is a necessary prerequisite for the completion of initiation, these data suggest that there may be thresholds for initiation.

2-Acetylaminofluorene↗
PubMed1992

Ca(2+)-regulated serine protease associated with the nuclear scaffold.

The nuclear scaffold (NS) is a proteinaceous network of orthogonally arrayed intermediate filament proteins, termed lamins, which is responsible for nuclear structure. Recent work has demonstrated that a subset of lamins A/C is proteolytically cleaved to produce an ATP-binding protein. This proteolytic cleavage is accomplished by a NS protease activity, which shows a considerable selectivity for lamins A/C and is stringently regulated by Ca2+ in vitro, suggesting that it might also participate in control of NS breakdown in various scenarios. Here, we identify the major NS protease as a novel serine protease with a predominantly chymotryptic-like substrate preference, and we show that even transient perturbations in cytosolic Ca2+ have significant effects on the NS protease activity. This NS protease activity shows extensive similarities to the multicatalytic proteinase complex. In addition to a potential role in control of NS breakdown at mitosis and/or under pathological conditions, this NS protease is also strategically located for other functions, such as inactivation of various oncogenic proteins or maturation-promoting factor.

Amino Acid Sequence↗
PubMed1991

Cooperative ATP binding by cloned lamin C.

Cloned human lamin C was expressed in and purified from bacteria and used in ATP binding assays. Scatchard analysis revealed strong positive cooperative and noncooperative binding, with estimated apparent dissociation constants of 3 X 10(-6) and 2 X 10(-5) M, respectively. The binding is strongly pH dependent. ATP binding by lamins A/C (presumably as intermediate filaments) may provide a substantial storage depot for ATP at the peripheral lamina for use by a number of ATP-requiring nuclear scaffold enzymes.

Adenosine Triphosphate↗
PubMed1991

Demethylation of ribosomal RNA by hepatocyte microsomal preparations.

Previous work has shown that hypomethylation of rRNA is an important control for protein synthesis in rat hepatocytes, and that the net hypomethylation appears to arise from cytoplasmic events. Here we show that demethylation of rRNA is catalyzed by microsomal preparations. The rRNA demethylation is dependent upon NADPH and is almost completely inhibited by carbon monoxide. Demethylation appears to increase following galactosamine intoxication, a hepatotoxin which induces hypomethylation of rRNA and inhibition of protein synthesis.

Animals↗
PubMed1991

Interaction of human immunodeficiency virus type I Rev protein with nuclear scaffold nucleoside triphosphatase activity.

Human immunodeficiency virus type I encodes a regulatory protein, termed Rev, which is associated with the appearance of unspliced and partially spliced viral RNAs in the cytoplasm. Rev is believed to function via interaction with a sequence element in the env region of the viral RNA, termed the Rev-responsive element (RRE). In this study, we use a stably transfected, Rev-producing mouse cell line to show that low, functional levels of Rev are associated with the nuclear scaffold (NS). Immunohistochemical studies localize Rev to the NS. Furthermore, immunoblot analyses demonstrate the presence of Rev in NS preparations isolated from Rev-producing cells and document binding of purified Rev protein to isolated NS or to cloned lamin C in vitro. Results with an in vitro RNA transport assay suggest that Rev is associated with a significant defect in transport of RNAs which lack RRE, whereas transport of RRE-containing transcripts proceeds efficiently. This Rev-induced transport defect appears to be mediated via direct inhibition of NS nucleoside triphosphatase, an enzyme thought to be involved in the nucleocytoplasmic transport process. NS preparations isolated from Rev-producing cells show a significantly lower nucleoside triphosphatase activity than those from control preparations. Addition of Rev protein to isolated NS produces a significant inhibition of NS nucleoside triphosphatase activity, which is specifically reversed by addition of RRE transcripts. These data suggest that a major aspect of Rev function may involve selective modulation of host cell nucleocytoplasmic transport mechanisms via interaction with the NS.

Animals↗
PubMed1991

Alterations in nuclear scaffold constituents during carbon tetrachloride-induced liver regeneration.

Liver regeneration was induced in rats by treatment with CCl4, which results in substantial regenerative activity with a sharp mitotic response 2 days after intoxication. Closely paralleling the mitotic index, we observed fourfold increases in nuclear scaffold nucleoside triphosphatase, an activity thought to participate in nucleocytoplasmic RNA transport and in the 46 kD putative enzyme and its selective photolabeling. Because previous work has indicated that the 46 kD protein may be proteolytically derived from lamins A/C by cleavage at a tyrosine residue at aa376, we investigated the response of lamin A/C transcripts during this regeneration. Surprisingly, Northern blot analyses after CCl4 administration showed low levels of lamin A/C transcripts (which appeared to be predominantly poly[A]-), and we found a decrease in immunoprecipitable lamins A/C from in vitro translation of poly(A)- selected RNA. To circumvent potential problems with such analyses, we used reverse transcription/polymerase chain reaction amplification of lamin A/C transcripts from total cytoplasmic RNA. These assays showed a transient, comparatively minor increase in lamin A/C transcripts 1 day after treatment, but levels rapidly declined from 1 to 3 days and were decreased at 3 to 5 days. However, nuclear scaffold protease activity, which shows a considerable selectivity for lamins A/C and may be involved in derivation of the 46 kD protein, increased in parallel to the mitotic response and increases in nucleoside triphosphatase, as assessed using a nonspecific (Azocoll) protease assay. Assays with a specific tyrosine-containing substrate (Z-Y-Sbenzyl) showed an increase that mirrored that observed with the nonspecific substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

The hepatocyte protein synthesis defect induced by galactosamine involves hypomethylation of ribosomal RNA.

D-galactosamine produces an early defect in protein synthesis, independent of its effects on RNA synthesis. Here we show that the defect in protein synthesis is inherent in purified ribosomal subunits in vitro. Further, galactosamine treatment is associated with an 85% decrease in methylation of ribosomal RNA, involving all sites (2'-0-ribose and base positions), intact ribosomes from galactosamine-treated animals can be methylated to a greater extent than control ribosomes and in vitro methylation restores their functional capacity. Statistical analyses of these data, along with those with a number of other hepatotoxins, reveal a correlation coefficient of r = 0.95 (p less than 0.003) between protein synthetic capacity and ribosomal RNA methylation, and linear regression accounts for more than 90% of the observed variation. In contrast, no relationship was found between nucleolar RNA methylation and protein synthetic capacity. A relationship of borderline statistical significance was found between messenger RNA methylation and protein synthetic capacity, but it does not appear consistent with results obtained after CCl4 intoxication. These results lend strong support to the notion that methylation status of ribosomal RNA is an important control for protein synthesis in quiescent hepatocytes and that net hypomethylation is a common response to such divergent hepatotoxins as CCl4, ethionine and D-galactosamine.

Animals↗
PubMed1990

Regulation of nuclear scaffold protease activity by calcium.

Recent investigations have disclosed protease activity which is intimately associated with the nuclear scaffold. This protease activity shows a marked selectivity for lamins A/C, and from them may produce the 46-kDa NTPase thought to participate in nucleocytoplasmic RNA transport. Preliminary characterizations suggested that the protease activity might require Ca2+. In this report, we present evidence showing that nuclear scaffold protease activity is stringently regulated by Ca2+ within the physiologic range. Further, calmodulin antagonists such as calmidazolium and chlorpromazine produce marked inhibition of the protease activity, suggesting modulation by calmodulin or calmodulin-like factors.

Animals↗
PubMed1990

The Mr 46,000 nuclear scaffold ATP-binding protein: identification of the putative nucleoside triphosphatase by proteolysis and monoclonal antibodies directed against lamins A/C.

Previous work suggested that the major Mr 46,000 ATP-binding protein [a putative nucleoside triphosphatase (NTPase)] found in rat liver nuclear scaffold (NS) may be proteolytically derived from lamins A/C. To definitively establish this identification, we undertook a series of photolabeling, proteolysis, and immunoprecipitation experiments. Mice were immunized with human lamin C expressed in bacteria, and monoclonal antibody-producing hybridomas were obtained. The purified monoclonal antibodies all recognized lamins A and C on immunoblots of NS, as well as Mr 46,000 or 34,000 proteolytic fragments as minor components. The Mr 46,000 photolabeled band was the only major NS component photolabeled with low concentrations of azido-ATP, and it was immunoprecipitated with anti-lamin monoclonal antibodies. To preclude the possibility that the photolabeled Mr 46,000 protein represented a minor component which comigrated with the Mr 46,000 lamin fragment and which specifically associated with lamins A/C during immunoprecipitation, a series of proteolytic digestions were undertaken. Digestion of the photolabeled Mr 46,000 peptide with chymotrypsin and staphylococcal protease V8 produced a limited number of photolabeled fragments, all of which comigrated with major stainable fragments produced from the Mr 46,000 lamin fragment. Cyanogen bromide cleavage of the photolabeled Mr 46,000 polypeptide, followed by polyacrylamide gel electrophoresis or high performance liquid chromatography/amino acid analyses, defined the COOH-terminal cleavage site as the Y residue at amino acid 376 and localized the photolabeled site to the COOH-terminal region (amino acids 372-376). In support of this proposed proteolytic cleavage site, specific assays with tyrosine-containing thiobenzyl ester substrate documented the presence of NS protease activity which cleaves at tyrosine residues; this activity shows a Km of 0.2 mM and a Kcat of approximately 250/s. Parallel experiments with mildly proteolyzed cloned lamin C preparations showed selective photolabeling of an Mr 34,000 fragment, which corresponds to a proteolytic breakdown product of the Mr 46,000 NS polypeptide; this Mr 34,000 photolabeled fragment was also immunoprecipitated with anti-lamin monoclonal antibodies and contained the same photolabeled site as the Mr 46,000 peptide. Cloned lamin C preparations were inactive in NTPase assays but did exhibit substantial ATP binding with an apparent KD = 4 x 10(-5) M ATP. These results indicate that the major Mr 46,000 photoaffinity-labeled protein in NS, which represents the putative NTPase thought to participate in nucleocytoplasmic transport, is derived from lamin A or lamin C by NS proteolytic activity which exposes a cryptic ATP-binding site near the highly conserved end of coil-2.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1989

Proteolytic activity associated with the nuclear scaffold. The effect of self-digestion on lamins.

The inner aspect of the nuclear envelope is supported by a peripheral framework called the nuclear scaffold, which consists of both structural and functional proteins. Its major structural components, lamins A-C, form a highly polymerized and insoluble fibrous matrix during interphase of the cell cycle. Functional constituents of the scaffold include the 46-kDa nucleoside triphosphatase which is thought to participate in nucleocytoplasmic transport of mRNA. This 46-kDa component shares an amino-terminal sequence with lamins A and C, indicating that proteolytic remodeling of the nuclear scaffold may contribute to the generation of nucleoside triphosphatase activity (Clawson, G. A., Lackey, A., and Tökés, Z. A. (1988) Exp. Cell Res. 176, 180-186). We report here that neutral protease activity intimately associated with the nuclear scaffold is also a functional constituent. This activity has a considerable selectivity for lamins as shown by self-digestion of scaffold preparations, and it may participate in the remodeling of the nuclear scaffold after treatment with carcinogens.

Animals↗
PubMed1989

Mechanisms of carbon tetrachloride hepatotoxicity.

CCI4 has long served as a model compound for study of hepatotoxicity. While its simple chemical structure held the allure of a simple mechanism of action, decades of study have disclosed a complex series of responses. Significant early damage following CCI4 administration includes: (1) A number of alterations affecting Ca2+ homeostasis, which conspire to redistribute cellular Ca2+ from endoplasmic reticulum and mitochondria to cytosol, and (2) hypomethylation of ribosomal RNA, which disrupts protein synthesis. The genesis of the injury in vivo appears to encompass early 'metabolism-dependent' effects (which appear to be largely independent of CCI4 concentration at the levels studied) and later 'metabolism-independent' effects, which parallel CCI4 concentration. The inability of injured hepatocytes to respond anabolically to early damage may be a critical feature in CCI4 hepatotoxicity.

Animals↗
PubMed1988

Sodium cholate extraction of rat liver nuclear xenobiotic-metabolizing enzymes.

DNA is the purported target of several carcinogenic and mutagenic agents. Nuclear enzymes which could generate or detoxify reactive metabolites are of major concern. Several such enzymes have been identified within nuclei, but obtaining samples with enriched content or activity is difficult, time-consuming, and uses harsh isolation techniques. Extraction of rat liver nuclear suspensions with cholate-containing buffer results in solubilization of 25-30% of the protein. Linear extraction was obtained for total protein and cytochromes P-450 and b5, NADPH-cytochrome P-450 reductase, NADH-cytochrome b5 reductase, DT-diaphorase, and microsomal-like epoxide hydrolase with specific activities comparable to values reported for isolated nuclear membrane, while the yield was five to ten times greater. Detergent extracts of rat liver nuclei were employed to study the comparative response of microsomal and nuclear enzymes to chemical treatment. While the responses to acute inductive (phenobarbital and 3-methylcholanthrene) and toxic (carbon tetrachloride and dibromochloropropane) treatments were qualitatively similar, an initiation-promotion protocol (diethylnitrosamine with phenobarbital promotion) resulted in divergent responses between the enzymes in the two subcellular fractions. Detergent extracts of nuclei offer an efficient means of recovering xenobiotic-metabolizing enzymes from rat liver nuclei, and have been utilized to demonstrate a differential response of nuclear enzymes during preneoplastic development.

Animals↗
PubMed1988

The 46-kDa nucleoside triphosphatase of rat liver nuclear scaffold represents the N-terminal portion of lamins A/C.

The major nucleoside triphosphatase (NTPase) of rat liver nuclear scaffold (NS) or envelope, which is thought to participate in nucleocytoplasmic transport, has been identified via photoaffinity labeling as a 46-kDa polypeptide. This 46-kDa protein was purified by SDS-polyacrylamide gel electrophoresis and cleaved with trypsin. The resulting peptides were purified by HPLC and five were microsequenced. All five peptides appear to be derived from the N-terminal region of lamins A/C. Subsequent experiments with photolabeled NS showed that the 46-kDa polypeptide was selectively immunoprecipitated by antiserum specific to lamins A/C and by affinity-purified anti-lamin antibodies. Photolabeling of nuclei prepared in the presence of protease inhibitors showed predominant labeling of the 46-kDa polypeptide, suggesting that it is an integral nuclear constituent and not an artifact produced during NS preparation. Use of protease inhibitors throughout purification of NS increased the specificity of photolabeling of the 46-kDa band by significantly reducing photolabeling of smaller molecular weight components, which arise by proteolysis. Anti-lamin antibodies also produced a significant inhibition of NTPase activity in NS. These results suggest that the N-terminal portion of lamins A/C represents the 46-kDa NTPase, which, according to previous reports, may participate in RNA transport.

Affinity Labels↗
PubMed1987

Early hypomethylation of 2'-O-ribose moieties in hepatocyte cytoplasmic ribosomal RNA underlies the protein synthetic defect produced by CCl4.

Carbon tetrachloride (CCl4) treatment of rats produces an early defect in methylation of hepatocyte ribosomal RNA, which occurs concurrently with a defect in the protein synthetic capacity of isolated ribosomes. The CCl4-induced methylation defect is specific for the 2'-O-ribose position, and a corresponding proportional increase in m7G base methylation occurs in vivo. Undermethylated ribosomal subunits (rRNA) from CCl4-treated preparations can be methylated in vitro to a much greater extent than those from control preparations, and in vitro methylation restores their functional capacity. In vitro methylation of treated ribosomal subunits (which restores functional capacity) occurs at 2'-O-ribose positions (largely G residues). In contrast, in vitro methylation of control ribosomal subunits (which does not affect functional activity) represents base methylation as m7G, sites which are apparently methylated in treated preparations in vivo. Methylation/demethylation of 2'-O-ribose sites in rRNA exposed on the surface of cytoplasmic ribosomal subunits may represent an important cellular mechanism for controlling protein synthesis in quiescent hepatocytes, and it appears that CCl4 disrupts protein synthesis by inhibiting this 2'-O-ribose methylation.

Animals↗
PubMed1987

Hepatocarcinogen-induced alterations in nuclear RNA compartmentation.

Prelabeled rat liver nuclei were purified, detergent-rinsed to remove cytoplasmic RNA contaminants and incubated in two in vitro RNA transport assays. Appropriate compartmentation of nuclear RNA sequences was maintained in an assay containing polyvinylpyrrolidone (PVP), which prevents nuclear swelling in aqueous media. Under these conditions, poly(A)+ RNA released in the presence of PVP (and ATP) was significantly more active in directing in vitro protein synthesis than poly(A)+ RNA released in the absence of either additive. Further, the excised fifth intron of alpha 1-acid glycoprotein remained nucleus-restricted, while release of poly(A)+ processing catabolites occurred in the absence of PVP. The appropriate compartmentation of nuclear sequences was lost following treatment of rats with the hepatocarcinogen thioacetamide, paralleling the in vivo situation. This assay should prove useful for subsequent studies on nucleocytoplasmic RNA transport in vitro, in particular for investigations relating to the altered RNA transport associated with the initiation of carcinogenesis.

Animals↗
PubMed1987

Cloning of rat "prion-related protein" cDNA.

Rat prion-related protein (PrP) cDNA has been cloned and sequenced. Comparison of this cDNA with those from human, hamster, and mouse indicates extremely high conservation (about 95%). The deduced partial rat PrP possesses: (a) a highly conserved region composed of repetitive sequences in what is presumably an extracellular domain, (b) a hydrophobic transmembrane domain, (c) a highly charged region which should stop membrane transfer, (d) a substantial cytoplasmic domain (which contains all of the nonconservative substitutions and a high proportion of conservative substitutions), and (e) a hydrophobic C-terminus. Dot and Northern blot analyses suggest a limited expression of PrP in rat tissues and indicate that PrP expression is decreased in the brain during the acute phase response systemically. Our results lend support to the notion that PrP is a highly conserved, normal cellular membrane protein of essential (although unknown) biologic function, which may be deposited in fibrillar amyloid form as a result of abnormal processing.

Amino Acid Sequence↗
PubMed1986

Human prion protein cDNA: molecular cloning, chromosomal mapping, and biological implications.

A human complementary DNA whose protein product is considered to be the major component of scrapie-associated fibrils in Creutzfeldt-Jakob disease, kuru, and Gerstmann-Straussler syndrome has been identified and characterized. The extensive homology of this gene sequence to the hamster PrP 27- to 30-kilodalton prion protein complementary DNA clone, and its existence as a single copy in the human genome, leads to the conclusion that this is the human prion gene. This human prion gene has been mapped to human chromosome 20, negating a direct link between the prion protein and Down's syndrome or the amyloid of Alzheimer's disease.

Amino Acid Sequence↗