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C S Giometti

Publications and source records attributed to C S Giometti.

At least 19 recordsLinked to original sources
PubMed1995

Polyacrylamide gel electrophoretic methods in the separation of structural muscle proteins.

Polyacrylamide gel electrophoresis plays a major role in analyzing the function of muscle structural proteins. This review describes one- and two-dimensional gel electrophoretic methods for qualitative and quantitative investigation of the muscle proteins, with special emphasis on determination of protein phosphorylation. The electrophoretic studies established the subunit structures of the muscle proteins, characterized their multiple forms, revealed changes in subunit composition or shifts in isoform distribution of specific proteins during development, upon stimulation or denervation of the muscle. Protein phosphorylation during muscle contraction is preferentially studied by two-dimensional gel electrophoresis. The same method demonstrated protein alterations in human neuromuscular diseases.

Animals↗
PubMed1995

Analysis of proteins from human breast epithelial cells using two-dimensional gel electrophoresis.

The human breast is a highly specialized, complex tissue comprised of a heterogeneous population of cells with varying functions. Interactions between the different cell types, changes in their relative abundance, state of differentiation and function in response to stimuli, as well as the alterations that lead to the aberrant growth associated with malignancy are poorly understood. Two-dimensional gel electrophoresis is being used to compare the proteins found in different breast cells in order to identify the gene products that are common or specific to particular cell types so as to provide markers that will be useful in studies of normal breast cell differentiation and the dedifferentiation or blocked differentiation characteristic of cancer. Protein patterns have been obtained from cells prepared for electrophoresis immediately after isolation from human milk, from cells cultured for fewer than ten passages after isolation from healthy breast tissue removed during reduction mammoplasty, and from cells maintained in long-term tissue culture after isolation from the pleural effusions of patients with breast carcinomas. Differential expression of cytokeratins 8, 18, and 19, shown previously to be predominantly expressed by epithelial cells in the luminal layer of breast tissue, was observed among the cells analyzed. Other non-cytokeratin proteins were also found to be differentially expressed in subsets of both the normal and tumor cells. A composite human breast cell protein pattern was created which includes all the commonly and specifically expressed proteins found in this study. This pattern will be the basis for continuing studies of proteins in the human breast.

Amino Acid Sequence↗
PubMed1994

Identification of a heritable deficiency of the folate-dependent enzyme 10-formyltetrahydrofolate dehydrogenase in mice.

During the analysis of liver protein expression in the offspring of male mice irradiated with fission-spectrum neutrons, one offspring displayed a heritable 50% decrease in the abundance of two proteins. Homozygous mice lacking detectable quantities of these proteins were obtained through breeding. Characterization of this protein deficiency has identified these liver proteins as forms of the enzyme 10-formyltetrahydrofolate dehydrogenase (10-formyl-THF DH; 10-formyltetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.6). NH2-terminal sequence analysis demonstrated that both proteins share identical sequences in the first 25 residues, and this sequence matches (96% identity) that of rat and human 10-formyl-THF DH. In addition, these proteins showed cross-reactivity to polyclonal antiserum raised against purified rat 10-formyl-THF DH. Southern (DNA) blot analysis revealed a restriction fragment length polymorphism consistent with a deletion mutation in the 10-formyl-THF DH structural gene in homozygous mice. Results of Northern (RNA) blot analysis demonstrated the absence of 10-formyl-THF DH mRNA in mice lacking 10-formyl-THF DH protein. Furthermore, liver cytosolic 10-formyl-THF DH enzymatic activity was undetectable in homozygotes. Measurement of hepatic folate pools showed that in homozygotes the total folate pool is decreased and the level of tetrahydrofolate is markedly depleted.

Animals↗
PubMed1994

Altered protein expression detected in the F1 offspring of male mice exposed to fission neutrons.

Liver protein expression in F1 offspring arising from spermatogonia exposed to 60 cGy of fission spectrum neutrons from the JANUS reactor was compared to that in offspring from unexposed spermatogonia by using two-dimensional electrophoresis (2DE). Approximately 100 protein spots in 2DE patterns from 167 control offspring and 530 offspring from irradiated sires were monitored for quantitative decreases of 50%, indicative of mutation events causing the loss of one normal copy of a structural gene. Reproducible abnormalities were found only in 3 patterns, all from the offspring of neutron-irradiated sires. Two of the three patterns were from littermates (brother and sister) and both showed an approximately 70% decrease in the amount of liver protein MSN188. The third pattern was from a male mouse sired by a different male and showed an approximately 50% decrease in the abundance of protein MSN94. The decreased abundance of MSN188 and MSN94 was assumed to be due to mutation events referred to as NEUT1 and NEUT2, respectively. Sibling crosses between the 2 mice showing the NEUT1 trait produced offspring with control, decreased and undetectable levels of MSN188 in a ratio of 0.25:0.5:0.25. Test crosses between the F1 offspring expressing the NEUT2 trait back to C57BL/6JANL mice produced offspring expressing normal or decreased amounts of MSN94 in a ratio of 0.5:0.5. Inbreeding of individuals expressing decreased amounts of MSN94 produced mice expressing control, decreased amounts, or no detectable amount of that protein in a ratio of 0.25:0.5:0.25. These results indicate that the decreased abundance of MSN188 or MSN94 originally detected in the F1 offspring is due to a genetically transmissible event. Unlike the heritable protein changes observed previously in the F1 offspring of sires exposed to N-ethyl-N-nitrosourea in which a protein variant was produced, both the NEUT1 and NEUT2 mutation events appear to prevent the production of any protein product. These 2 mutations may thus represent mutation lesions other than point mutations (e.g., deletions or translocations) detectable as quantitative changes in protein expression in the F1 generation.

Animals↗
PubMed1992

Evidence for regulatory genes on mouse chromosome 7 that affect the quantitative expression of proteins in the fetal and newborn liver.

A series of deletions around the albino locus on mouse chromosome 7 is believed to include one or more regulatory genes that control the activities of a cluster of liver enzymes. To further characterize the functions of this region of the mouse genome, we have used quantitative two-dimensional electrophoresis to analyze the effects of two of these deletions, c3H and c14CoS, on the expression of liver proteins. More than 400 distinct protein gene products were quantitated in livers from fetal and newborn wild-type homozygous (cch/cch), heterozygous (cch/c3H or cch/c14CoS), and deletion homozygous (c3H/c3H or c14CoS/c14CoS) mice. Livers of fetal and newborn c3H heterozygotes and homozygous wild-type littermates produced qualitatively identical protein patterns after two-dimensional electrophoresis. In livers of c3H homozygous fetuses, however, abnormal amounts (either increased or decreased relative to homozygous wild-type and heterozygous littermates) of 29 proteins were found. Twenty-eight of these 29 protein anomalies were also found in livers of newborn c3H homozygotes. Livers of fetal and newborn mice homozygous for the c14CoS deletion, which overlaps the c3H deletion and produces a similar phenotype, expressed normal amounts of these proteins. One of the 29 proteins (MSN807) has an amino-terminal sequence similar to a 23-kDa translationally controlled protein abundant in mouse erythroleukemia and sarcoma-180 cells. These results suggest that normal chromosome 7 contains genes, located within the region of the c3H but not the c14CoS deletion, that regulate the abundance of specific proteins in the liver. These proteins cannot be related to the phenotypic alterations shared by the c3H and c14CoS deletions.

Albinism↗
PubMed1992

Mouse liver protein database: a catalog of proteins detected by two-dimensional gel electrophoresis.

Alterations in the abundance or structure of mouse liver proteins are being studied using two-dimensional gel electrophoresis (2-DE) to build a database of protein changes correlating with exposure to ionizing radiation or toxic chemicals. Thus far, studies have included the analysis of proteins from the offspring of exposed parents or from the exposed individuals themselves. In order to characterize and identify proteins found altered by such exposures, sex- and strain-related differences in protein patterns have been analyzed, and the subcellular locations of a large portion of the mapped proteins have been determined. As part of these studies, data are collected and stored using a variety of computer hardware and software tools that allow the accumulation of information on the origin of samples, gel identification, experiment description, and protein similarities and differences. This accumulation of information constitutes the mouse liver protein database. Relational database software is used to tie the different facets of the database together so that the results of a variety of experiments can be compared and interrelated. The database optimizes the information obtained from 2-DE gel sets by allowing use of the data for many purposes, including monitoring of gel resolution to ensure the collection of high quality data and correlation of protein effects induced by different agents. This first edition of the Argonne National Laboratory mouse liver protein database lays the foundation for future work and communication that should elucidate the significance of observed protein effects as possible markers of exposure to toxic agents.

Animals↗
PubMed1992

Use of principal components analysis for mutation detection with two-dimensional electrophoresis protein separations.

The application of two-dimensional electrophoresis (2-DE) to mutation detection requires the capability to monitor each protein in a 2-DE pattern for significant changes in abundance indicative of a mutation event. Previously, mutation searches were done using a univariate outlier detection method in which each protein spot was considered independently in a classical outlier search. An alternative approach to analysis of 2-DE patterns for quantitative changes is a multivariate procedure which takes advantage of the observation that protein spots in a 2-DE pattern often represent correlated rather than independent measurements. We have compared the efficiency of univariate and multivariate procedures for mutation detection using data from the Argonne National Laboratory 2-DE database of mouse liver proteins. Analyses involving a total of over 1500 gels were performed to compare the performance of a multivariate method based on principal components analysis (PCA) with the univariate method. Up to 279 spots from each pattern were used for PCA. First, a simulation was performed to assess the detection efficiency of PCA for single protein spots decreased in abundance by 50%. Then, the ability to detect actual mutations was tested using eight confirmed mutations. Results show that, compared to a univariate approach to analysis of data from the mouse model system, the multivariate method increases the number of protein spots on each 2-DE pattern that can be monitored for quantitative changes indicative of mutations by compensating for variables that contribute to the background quantitative variability of protein spots.

Animals↗
PubMed1992

Biochemical characterization of a variant form of cytosolic epoxide hydrolase induced by parental exposure to N-ethyl-N-nitrosourea.

1. ENU4 mice express a protein variant originally detected in a CBF1 mouse sired by a C57BL/6 mouse exposed to N-ethyl-N-nitrosourea. It appears to be an isoelectric point variant of cytosolic epoxide hydrolase. Affinity purified cytosolic epoxide hydrolase from ENU4 mice has a pI of approximately 5.1 compared to 5.6 in other mouse strains. 2. Clofibrate induced cytosolic epoxide hydrolase to similar levels in five strains of mice. However, CBF1 and ENU4 mice were more sensitive to the induction of palmitoyl CoA oxidase activity. 3. Except for isoelectric point, the physico- and immunochemical properties of cytosolic epoxide hydrolase from ENU4 mice were similar to those of the other mouse strains. Substrate specificities for five of six substrates tested were also similar.

Animals↗
PubMed1992

Muscle sarcomere lesions and thrombosis after spaceflight and suspension unloading.

Spaceflight (flight) and tail suspension-hindlimb unloading (unloaded) produced significant decreases in fiber cross-sectional areas of the adductor longus (AL), a slow-twitch antigravity muscle. However, the mean wet weight of the flight AL muscles was near normal, whereas that of the suspension unloaded AL muscles was significantly reduced. Interstitial edema within the flight AL, but not in the unloaded AL, appeared to account for this apparent disagreement. In both experimental conditions, the slow-twitch oxidative fibers atrophied more than the fast-twitch oxidative-glycolytic and fast-twitch glycolytic fibers. Immunostaining showed that slow-twitch oxidative fibers expressed fast myosin, producing hybrid fibers containing slow and fast myosin isoforms. Two-dimensional gel electrophoresis of flight AL muscles revealed increased content of fast myosin light chains and decreased amounts of slow myosin light chains and fatty acid-binding protein. In the flight AL, absolute mitochondrial content decreased, but the relatively greater breakdown of myofibrillar proteins maintained mitochondrial concentration near normal in the central intermyofibrillar regions of fibers. Subsarcolemmal mitochondria were preferentially lost and reduced below normal concentration. Elevated fiber immunostaining for ubiquitin conjugates was suggestive of ubiquitin-mediated breakdown of myofibrillar proteins. On return to weight bearing for 8-11 h, the weakened atrophic muscles exhibited eccentric contraction-like lesions (hyperextension of sarcomeres with A-band filaments pulled apart and fragmented), tearing of the supporting connective tissue, and thrombosis of the microcirculation. Segmental necrosis of muscle fibers, denervation of neuromuscular junctions, and extravasation of red blood cells were minimal. Lymphocyte antibody markers did not indicate a significant immune reaction. The flight AL exhibited threefold more eccentric-like lesions than the unloaded AL; the high reentry G forces experienced by the flight animals, but not the unloaded group, possibly accounted for this difference. Muscle atrophy appears to increase the susceptibility to form eccentric contraction-like lesions after reloading; this may reflect weakening of the myofibrils and extracellular matrix. Microcirculation was also compromised by spaceflight, such that there was increased formation of thrombi in the post-capillary venules and capillaries. This blockage led to edema by 8-11 h after resumption of weight bearing by the COSMOS 2044 rats. The present findings indicate that defective microcirculation most likely accounted for the extensive tissue necrosis and microhemorrhages observed for COSMOS 1887 rats killed 2 days after landing.

Animals↗
PubMed1991

A comparative study of the effects of clofibrate, ciprofibrate, WY-14,643, and di-(2-ethylhexyl)-phthalate on liver protein expression in mice.

Chemical, physical, or mechanical insults to a cellular system cause changes in the rates of protein synthesis or degradation. To define patterns of quantitative protein changes and identify the biochemical basis of those changes, we are studying fluctuations in mouse liver protein abundance in response to factors such as stages of maturation, changes in diet, and exposure to chemicals. Two-dimensional gel electrophoresis (2DE) together with computer-assisted data analysis is used to collect quantitative data from groups of 2DE patterns. Our analysis of liver protein expression in mice treated with four different chemicals known to cause peroxisome proliferation serves as an example of the changes in protein expression that can be monitored by using this approach. The database we are building indicates that changes specific to a particular treatment as well as generalized effects can be identified, providing the opportunity to identify patterns of protein expression that are indicators of biological response to different types of cellular insults.

Animals↗
PubMed1991

Association of WR-1065 with CHO AA8 cells, nuclei, and nucleoids.

The radioprotector WR-1065 (N-(2-mercaptoethyl)-1,3-diaminopropane) has been shown to be the active moiety involved in protecting mammalian cells from the cytotoxic and mutagenic effects of ionizing radiation after administration of WR-1065 or the phosphorylated form, WR-2721. Initial experiments demonstrated that, in our hands, WR-1065 protects Chinese hamster AA8 cells from killing by (a) mechanism(s) other than induction of hypoxia. AA8 cells were then incubated in the presence of [14C]WR-1065 to determine whether association of WR-1065 in vivo was random or targeted to the nucleus or the nuclear matrix. The kinetics of incorporation of labeled material showed rapid incorporation for the first 30 min and little, if any, additional incorporation over the next 2.5 h. Examination of nuclei and nucleoids generated from the AA8 cells indicated that approximately 10% of the drug was localized in the nucleus and the drug that remained was not dislodged with repeated washes of the filters. Association kinetics of the drug with nuclei and nucleoids indicated that there was little increase in drug association with time, suggesting that there may be a limited number of strong association sites in the nucleus, but these sites are either with DNA or with matrix proteins. Exposure of the AA8 cells to 6 Gy of 60Co gamma rays did not significantly alter the association of the drug with AA8 cells. Incubating AA8 cells in [14C]WR-1065 for 30 min and then incubating in drug-free medium indicated that nearly all of the drug was lost from cells within the first 5 min of incubation in drug-free medium. The low level of tightly bound matrix-associated label may be important in generating alterations in matrix organization that have been observed previously in this laboratory.

Animals↗
PubMed1990

Characterization of a protein that appears in the nervous system of the moth Manduca sexta coincident with neuronal death.

Two-dimensional gel electrophoresis was used to locate potential neuronal death-related proteins in the moth Manduca sexta. Protein patterns of ganglia of pharate adult moths (taken prior to adult ecdysis) compared with protein patterns of one-day-old adults revealed reproducible changes in protein patterns. An acidic protein of approximately 40,000 Da was present in all samples from adult moths undergoing neuronal death and essentially absent from pharate adult samples.

Aging↗
PubMed1990

The expression of myosin light chains and tropomyosin in human muscle biopsies with histochemical type 1 and type 2 fiber deficiency.

Two-dimensional gel electrophoresis (2DE) was used to compare the protein composition of human muscle biopsies that were shown by histochemical staining to be deficient in either type 1 or type 2 fibers. Distinct quantitative differences were found in the myofibrillar protein composition of muscle from a 43-year-old woman with proximal limb weakness that showed almost total absence of type 1 fibers and muscle from a 2.5-year-old girl with congenital myopathy in which there was severe lack of type 2 fibers. These data indicate that human type 1 and type 2 muscle fibers express distinct isoforms of myosin light chains and alpha-tropomyosin.

Adult↗
PubMed1990

The analysis of recessive lethal mutations in mice by using two-dimensional gel electrophoresis of liver proteins.

We have used two-dimensional gel electrophoresis (2DE) coupled with computer-assisted data analysis to analyze liver-protein expression in mice known to be heterozygous carriers of recessive lethal mutations induced in In(1)1Rk or In(7)13Rk inversion stocks by exposure to either triethylene melamine or ionizing radiation. Carriers of 8 different mutations and corresponding littermate controls (average of 17 individuals in each group) were screened for liver-protein differences. Both qualitative and quantitative protein differences were detected that correlated with unique pedigrees among the mouse stocks analyzed. Such strain-specific differences demonstrated that quantitative differences (either increases or decreases) in protein abundance of greater than 25% can be readily detected by using this 2DE system. Thus the 50% reduction in expression of a protein expected in the event of a structural gene deletion is well within the level of detection. No significant quantitative decreases in protein expression that correlated with the recessive lethal mutations were detected, however.

Animals↗
PubMed1988

A heritable variant of mouse liver ornithine aminotransferase (EC 2.6.1.13) induced by ethylnitrosourea.

A variant of ornithine aminotransferase (OAT, EC 2.6.1.13) has been detected in an offspring of a male mouse treated with ethylnitrosourea. The evidence presented to support the identification of the protein variant (ENU 2) as altered OAT includes (a) a corresponding 50% decrease in the abundance of a protein, located one charge unit basic to the variant, which comigrates on two-dimensional gel patterns with purified mouse liver OAT; (b) the binding of anti-rat-OAT antibody to the variant; (c) the increased abundance of the variant protein in the livers of mice fed a high protein diet (85% casein); and (d) purification of the variant through an OAT purification protocol.

Animals↗
PubMed1988

Comparison of rat and mouse ornithine aminotransferase with respect to molecular properties and regulation of synthesis.

A comparative study of the synthesis patterns and molecular properties of mouse and rat ornithine aminotransferase (OAT) was conducted. The two enzymes were found to be very similar with respect to catalytic properties, two-dimensional electrophoresis patterns of tryptic digests, amino acid compositions, and antibody cross-reactivity. In vitro translation assays for OAT synthesis on free polysomes isolated from livers at different times of day showed similar circadian fluctuations in OAT synthesis for both species. However, hybridization measurements revealed no circadian changes in the levels of hybridizable OAT mRNA in these livers. These results demonstrate that the circadian cycling of OAT synthesis is regulated at the level of translation in both the rat and the mouse.

Amino Acids↗