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S Lin

Publications and source records attributed to S Lin.

At least 559 records · Page 31Linked to original sources
PubMed1987

Specific interaction of vinculin with alpha-actinin.

Vinculin and alpha-actinin are cytoskeletal proteins present at focal contacts of the ventral surface of cultured fibroblasts. We labelled alpha-actinin with an acceptor fluorophore and vinculin with a donor. A mixture of vinculin and alpha-actinin showed a 28% quench, due to energy transfer, suggesting an interaction. Quench of vinculin was dependent on the concentration of alpha-actinin; Scatchard analysis gives a dissociation constant in the microM range. Quench was inhibited by excess unlabelled alpha-actinin, and by reaction of the acceptor protein with p-chloromercuribenzoate. We found that vinculin had a slightly greater elution volume in a gel filtration column equilibrated with alpha-actinin, indicating a higher effective Stokes radius due to the interaction of the two proteins.

Actinin↗
PubMed1987

Biochemical studies on the effect of Clostridium difficile toxin B on actin in vivo and in vitro.

We describe a simplified procedure for purification of Clostridium difficile toxin B. In this procedure, cytotoxicity is associated with a single protein band with a molecular mass of 230 kilodaltons. We used direct fluorescent staining of actin filaments to study the effect of this toxin on cultured cells. Morphologic changes were preceded by a decrease in the number and length of stress fibers followed by their disappearance with condensation of cellular actin around the nucleus. We then showed that cells treated with either cytochalasin B or toxin B had a significant increase in the monomeric actin pool as quantitated by DNase I inhibition. In contrast to the cytochalasins, toxin B had no direct effect on the rate or extent of actin polymerization or network formation in vitro. Cytoplasmic extracts of toxin B-treated cells had a significantly lower level of modulating activity on actin assembly and interactions in vitro compared with extracts of untreated cells. These results suggest that the action of toxin B on cells is due to direct or indirect effects on cellular proteins involved in controlling the state of actin assembly in the cells.

Actin Cytoskeleton↗
PubMed1986

Purification and initial characterization of a protein from skeletal muscle that caps the barbed ends of actin filaments.

We describe herein the purification of a protein from skeletal muscle that binds to ("caps") the morphologically defined barbed end of actin filaments. This actin-capping protein appeared to be a heterodimer with chemically and immunologically distinct subunits of Mr = 36,000 (alpha) and 32,000 (beta), Rs = 37 A, s20,w = 4.0 S, and a calculated native molecular weight of approximately 61,000. The protein was obtained in milligram quantities at greater than 95% purity from acetone powder of chicken skeletal muscle by extraction in 0.6 M KI, precipitation with ammonium sulfate, sequential chromatographic steps on DEAE-cellulose, hydroxylapatite, and Sephacryl S-200, followed by preparative rate zonal sucrose density gradient centrifugation. In immunoblots of myofibrillar proteins, affinity-purified antibodies selectively recognized protein bands of the same molecular weight as the subunits of the capping protein to which they were made, indicating that the isolated capping protein is a native myofibrillar protein, and not a proteolytic digestion product of a larger muscle protein. A specific interaction of the capping protein with the barbed end of actin filaments was indicated by its ability to inhibit actin filament assembly nucleated by spectrin-band 4.1-actin complex in 0.4 mM Mg2+, accelerate actin filament formation and increase the critical concentration of actin in 2-5 mM Mg2+, 75-100 mM KCl, and inhibit the addition of actin monomers to the barbed end of heavy meromyosin-decorated actin filaments as determined by electron microscopy. All of these effects occurred at nanomolar concentrations of capping protein and micromolar concentrations of actin, suggesting a high affinity interaction.

Actins↗
PubMed1986

Transformation-dependent increases in endogenous cytochalasin-like activity in chicken embryo fibroblasts infected by Rous sarcoma virus.

Transformation of chicken embryo fibroblasts by infection with Rous sarcoma virus has been shown to cause disruption of actin filament organization as seen with fluorescence staining techniques. This study is an attempt to use quantitative biochemical techniques to compare actin-related parameters in normal and transformed cells. Normal cells and cells infected with a temperature-sensitive mutant virus (NY68) and grown at the restrictive temperature of 41.5 degrees C have normal bundles of actin filaments, or F-actin; these cells also have about the same number of high-affinity cytochalasin binding sites at the ends of F-actin (approximately 5 pmol of sites per mg of cellular protein; Kd, 20 nM). In contrast, infected cells grown at the permissive temperature of 37 degrees C have a more diffuse pattern of actin filaments, and the number of cytochalasin binding sites in these transformed cells was below the level of detection. DNase I inhibition assays showed that the percent of unpolymerized actin, or G-actin, in cell extracts was not significantly different between normal and transformed cells (approximately 50%). In assays of cell extracts for endogenous cytochalasin-like activity on actin filaments (i.e., retardation of filament assembly at the fast-growing end, inhibition of cytochalasin binding to actin "nuclei," and decrease of low-shear viscosity of solutions of actin filaments), infected cells at 37 degrees C showed a higher level of activity per mg of protein than did uninfected cells or infected cells at 41.5 degrees C. These results suggest that the increase in endogenous cytochalasin-like activity in transformed cells may relate to the decrease in measurable cytochalasin binding sites and the abnormal distribution of actin filaments previously seen by fluorescence staining techniques.

Actins↗
PubMed1986

Acute hepatotoxicity induced by hepatotoxins in Suncus murinus.

A comparative study was conducted to contrast the hepatotoxicity of several chemicals in the musk shrew (Suncus murinus) versus other common laboratory species (mouse or rat), and the following results were obtained from serum enzymes (SGOT and SGPT) and histopathological findings of liver specimens. The sensitivity of Suncus liver to CCl4 was different from that of mouse liver. The sensitivity of Suncus liver to beta-D-galactosamine was weaker than that of rat liver. The sensitivity of Suncus liver to ethanol was stronger than that of mouse liver. After a single oral administration of ethanol (99.5% v/v, 0.1 ml/50 g body weight), the gallbladder of Suncus became enlarged and dark blue in color. A striking fatty degeneration was seen 24 h after a single ip administration of amethopterin at 50 mg/kg in Suncus liver.

Alanine Transaminase↗
PubMed1986

A re-examination of the interaction of vinculin with actin.

Vinculin prepared by published procedures (i.e., Feramisco, J. R., and K. Burridge, 1980, J. Biol. Chem., 255:1194-1199) contains contaminants that have been shown by Evans et al. (Evans, R. R., R. M. Robson, and M. H. Stromer, 1984, J. Biol. Chem., 259:3916-3924) to reduce the low-shear viscosity of F-actin solutions. In this study we separated contaminants from conventional vinculin preparations by hydroxylapatite chromatography. We found that although the contaminants represented a small fraction (less than or equal to 5%) of the total protein in the conventional vinculin preparations, they were responsible for practically all of the filament capping and bundling activities previously attributed to vinculin. In addition, we examined the size of the molecule(s) responsible for the observed capping activity and found that its apparent molecular weight under denaturing conditions in sodium dodecyl sulfate (SDS) polyacrylamide gels fell within a broad range of 23,000-33,000. These results contrast with the observation that under nondenaturing conditions, the activity migrated in gel filtration columns at a position that corresponded to the Stoke's radius of a much bigger molecule. Since the migration of the activity in these chromatographic experiments is independent of the presence of vinculin, it is unlikely that the active protein associates with vinculin with high affinity under the conditions examined.

Actin Cytoskeleton↗
PubMed1986

Studies on proteins that co-purify with smooth muscle vinculin: identification of immunologically related species in focal adhesions of nonmuscle and Z-lines of muscle cells.

Membrane extracts from chicken smooth muscle contain, along with filamin, vinculin and alpha actinin, a group of polypeptides that have the ability to interact with the "barbed end" of actin filaments. These low molecular mass polypeptides were designated as HA1 (Wilkins, J.A., and S. Lin, 1986, J. Cell Biol., 102:1085-1092). In this study, polyclonal antibodies raised against the HA1 preparation were used to study the cellular localization and tissue distribution of these polypeptides. Immunofluorescence experiments revealed a primary localization of staining at the ends of stress fibers on the ventral surface of cultured chicken embryo fibroblasts, i.e., those areas known as the focal adhesions. Specific staining was also seen at the Z-lines of both skeletal muscle myofibrils and cultured embryonic heart cells. Immunoblotting analyses of proteins from different tissues prepared to avoid proteolytic degradation showed a much different pattern than that of HA1 itself. Immunoreactive polypeptides with reduced molecular masses of 200,000 and 150,000 D were found in smooth muscle and fibroblasts while 200 and 60 kD polypeptides were found in cardiac muscle tissue. The antibodies recognized 60- and 31-kD polypeptides on immunoblots of chicken breast muscle. The results from this study strongly suggest that the polypeptides in HA1 arose from proteolysis of high molecular mass molecules. The studies also raise the possibility that immunologically related proteins in muscle and nonmuscle cells may be involved in linking actin filaments to Z-lines and membranes, respectively.

Actins↗
PubMed1986

Resuscitation with Fluosol-DA 20%--tolerance to sepsis.

Any compromise of the reticuloendothelial system (RES) or host defense in general would militate against the potential benefit of fluorocarbon emulsions as oxygen-carrying resuscitation fluids. The relationship between lethal hemorrhagic shock resuscitation with Fluosol-DA 20% and subsequent host depression was examined in a rat model. Host tolerance to a standard intra-abdominal polymicrobial septic challenge was assessed 5 days after hemorrhagic shock. Resuscitation with fluorocarbon was more effective than no resuscitation and is equal to Ringer's lactate. The addition of O2 did not enhance survival. In response to a septic challenge, survival of animals resuscitated with Ringer's lactate was similar to the control group, and fluorocarbon-treated animals had a significantly lower survival. It appears in this study that supplemental oxygen is not needed and that fluorocarbon emulsions act as a volume expander only. The enhanced mortality to a septic challenge may indicate a compromise in the host defense system induced by fluorocarbons, and clinical trials should proceed with caution accordingly.

Animals↗
PubMed1986

An alternative to DRGs. A clinically meaningful and cost-reducing approach.

A statistical methodology based on the Cox proportional hazards model (a survival time analysis method), an alternative to the approach underlying DRGs, is presented. The method is used to obtain an estimate of the length-of-stay (LOS) distribution of a patient incorporating either patient-specific or hospital variables. A percentile of the distribution chosen to minimize prediction error serves as the assigned LOS. Absolute deviation is used as the loss function both to determine the choice of a predicted LOS and to examine how well the scheme works. Multiple assignment schemes may also be developed from this approach. The results of the method, tested on a national probability sample of 4,608 psychiatric patients treated in psychiatric units of general hospitals, suggest that with respect to average absolute deviation, the proposed methodology may provide a scheme that is superior to the present DRG scheme. For the sample, the average percent improvement obtained using the median of the estimated LOS distribution as the predicted LOS over the sample mean of the DRG group is 19%. A two assignment strategy results in average improvements over DRGs of 43%.

Cost Control↗
PubMed1986

Actin filament organization of the Dunning R3327 rat prostatic adenocarcinoma system: correlation with metastatic potential.

Recently, Volk, Geiger, and Raz (Cancer Res., 44: 811-824, 1984) addressed the question of whether variations in actin organization in clones of the murine K-1735 melanoma tumor correlated with their metastatic capability. Using immunofluorescence techniques, they found that clones which had a more ordered actin network were less metastatic, whereas clones having a diffuse actin staining pattern were more metastatic. Similarly, we have found that in the Dunning rat R3327 prostatic adenocarcinoma tumor system, the non-metastatic (less than 0.1%) H-prostatic tumor cell line has a prominent network of actin filament bundles, whereas the highly metastatic (greater than 90%) MatLyLu cell line has a diffuse actin staining pattern. In the low-metastatic (less than 10%) AT1 cell line an intermediate actin organization between H and MatLyLu was observed. Analysis of cell extracts from H- and MatLyLu-cells revealed differences in the level of activity of cellular proteins which affect actin filament assembly and structure in a manner similar to that of the cytochalasins, fungal metabolites which bind with high affinity to the fast-growing end of actin filaments. Extracts of MatLyLu were significantly more effective than those of H-cells in decreasing the extent of actin filament network formation and in inhibiting the rate of filament assembly by blocking monomer addition onto the fast-growing end. Measurements of spin-lattice nuclear magnetic resonance water proton relaxation times (T1) were made in surgically removed tumor tissue from four sublines (H, AT1, MatLyLu, and MatLu) of the Dunning R3327 tumor system. The highly metastatic cell lines had significantly longer water proton T1 relaxation times than did the lines with low metastatic potential. These differences in T1 may reflect the observed alterations in organization of actin filaments within these various sublines of the Dunning R3327 prostatic adenocarcinoma tumor system.

Actin Cytoskeleton↗
PubMed1985

Pathophysiology of ammonia intoxication.

Ammonia intoxication affects postsynaptic inhibition and disturbs inhibitory neuronal interactions. This study investigated whether or not the effect of ammonia on postsynaptic inhibition was associated with a change of the EEG, i.e., a change in the function of the central nervous system such as in an encephalopathy. We showed that the effect of ammonia on postsynaptic inhibition was associated with a marked change of the EEG, and that this change was not due to an effect of ammonia on the brain stem reticular activating system. In addition, it was shown that in the central nervous system a NH+4 concentration of about 1 mumol/g affected postsynaptic inhibition. Because ammonia simultaneously affected postsynaptic inhibition and the EEG at a NH+4 tissue concentration comparable to that observed in encephalopathy, it is proposed that a dysfunction of postsynaptic inhibition caused the encephalopathy due to ammonia intoxication by simultaneously disturbing inhibitory neuronal interactions in many regions of the central nervous system.

Acetates↗
PubMed1985

Ammonia intoxication: effects on cerebral cortex and spinal cord.

The effect of an acute systemic ammonia intoxication on the metabolic states of the cerebral cortex and the spinal cord of the same animal was studied in the cat. The intravenous infusion of ammonium acetate (2 and 4 mmol/kg body weight/30 min) increased the gross levels of tissue NH4+, glutamine, glutamine/glutamate ratio, lactate, and the lactate/pyruvate ratio in the cerebral cortex and the spinal cord. Pyruvate increased, but significantly only in the spinal cord; aspartate decreased, but significantly only in the cerebral cortex. The infusion of ammonium acetate did not significantly change the levels of phosphocreatine, ATP, ADP, AMP, total adenine nucleotides, adenylate energy charge, glucose, glutamate, alpha-ketoglutarate, and malate in either tissue. The changes of NH4+, glutamine, and lactate levels as well as glutamine/glutamate and lactate/pyruvate ratios in the spinal cord correlated significantly with the corresponding changes of these metabolites in the cerebral cortex. Thus, cerebral cortex and spinal cord show certain specific and comparable metabolic changes in response to a systemic ammonia intoxication. The effect of ammonia intoxication on the increases of glutamine and lactate levels is discussed.

Acetates↗