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Biomedical subjects

K Schmid

Publications and source records attributed to K Schmid.

At least 217 records · Page 12Linked to original sources
PubMed1977

Enrichment of the murine natural killer (NK) and mitogen induced cellular cytotoxicity (MICC) cells using preparative free-flow high voltage electrophoresis.

A procedure using preparative free-flow high voltage electrophoresis is described for the fractionation of murine spleen and bone marrow cells so as to obtain cell subpopulations that are either enriched in or depleted of "natural killer" (NK) cells and "mitogen-induced cellular cytotoxicity" (MICC) effector cells. A nearly three fold enrichment in the NK and MICC activities of spleen cells was achieved. The enrichment in these cells could be further increased if the phagocytic cells were removed prior to electrophoresis. When bone marrow cells were fractionated a two and a half fold increase of NK activity, and a one and a half fold enrichment of MICC activity was achieved. In both cases, other fractions were nearly devoid of NK and MICC activity. The cell recovery after electrophoresis averages 70% of the cells applied, and at least 90% of these cells were viable. MICC and NK effector cells could not be separated to a useful extent electrophoretically but were found to be separable using Sephadex C-10 gel filtration columns. The MICC but not the NK cells were retained on these columns.

Animals↗
PubMed1977

Association of a circulating immunosuppressive polypeptide with operative and accidental trauma.

The serum from 109 traumatized patients was examined for immunosuppressive activity which might explain diminished host immune responsiveness following operative or accidental injury. Twenty-eight fo 31 (90%) severely tralmatized patients, 25 of 60 (42%) moderately traumatized patients, and 0 of 18 minimally traumatized patients developed serum which suppressed the response of normal human lymphocytes to phytohemagglutinin. The degree and duration of serum immunosuppressive activity paralleled the severity of the clinical course but did not correlate with serum cortisol or barbiturate levels. Suppressive sera were not cytotoxic. The immunosuppressive factor(s) was contained in a low molecular weight (less than 10,000 daltons) peptide fraction and was present in 5--10 times the amount recoverable from normal serum. By size and activity the trauma serum factor resembled immunoregulatory alpha globulin, a naturally-occurring serum inhibitor of T-lymphocyte reactions. Thus, depressed immunoreactivity following trauma may be due in part to high concentrations of an endogenous immunosuppressive polypeptide.

Adolescent↗
PubMed1977

Binding of dipyridamole to human platelets and to alpha1 acid glycoprotein and its significance for the inhibition of adenosine uptake.

The interactions of dipyridamole with alpha(1) acid glycoprotein of plasma and with human platelets are related to inhibition of adenosine uptake by platelets. Binding studies by equilibrium gel filtration suggested that 1 mol of dipyridamole binds per mol of alpha(1) acid glycoprotein with a dissociation constant of 1.6 muM. Platelets contain two populations of binding sites, one with high and another with lower affinity for the drug. The binding of dipyridamole to the high-affinity sites follows a Michaelis-Menten binding pattern with a dissociation constant of 0.04 muM. Approximately 2 x 10(4) dipyridamole molecules are bound at the high-affinity sites of each platelet. The lower affinity sites bind the drug with a dissociation constant of 4 muM. In the presence of alpha(1) acid glycoprotein of plasma, the binding of dipyridamole to human platelets is inhibited. Correspondingly, the dipyridamole inhibition of adenosine uptake by platelets is reduced 1,000-fold by purified alpha(1) acid glycoprotein. The binding of dipyridamole to human platelets was found to be essential for its inhibition of adenosine uptake by platelets. Dipyridamole decreases the incorporation of [(14)C]adenosine radioactivity in platelet nucleotides and reduces the [(14)C]-ATP to [(14)C]ADP ratio. Purified alpha(1) acid glycoprotein reverses these effects of dipyridamole on adenosine metabolism of platelets in a concentration-dependent manner. An equilibrium of dipyridamole binding to alpha(1) acid glycoprotein and to platelets is proposed.

Adenine Nucleotides↗
PubMed1977

Suppression of tumor-specific cell-mediated cytotoxicity by immunoregulatory alpha-globulin and by immunoregulatory alpha-globulin-like peptides from cancer patients.

The suppression of tumor-specific cell-mediated cytotoxicity by human immunoregulatory alpha-globulin (IRA), by a peptide fraction derived from IRA, and by IRA-like peptides from the serum of cancer patients was studied in a syngeneic murine tumor-host system. Splenic lymphocytes from tumor-immunized mice were cytotoxic specific tumor cells in vitro as measured by the [125]-iododeoxyuridine release microcytotoxicity assay. However, this effect was significantly depressed if 1.25 to 5 mg of IRA per ml were added to the cultures. Pooled lyophilized normal human serum protein was inactive. IRA peptide and IRA-like peptide fractions from cancer patients were also highly suppressive of cell-mediated cytotoxicity at much lower concentrations (0.05 to 0.5 mg/ml). Control human serum peptide, which failed to inhibit the induction of hemolytic plaque-forming cells in sheep erythrocyte-injected mice, had no effect on cell-mediated cytotoxicity. IRA and IRA-like peptide fractions were not cytotoxic to the effector lymphocytes or to the target cells at the concentrations used.

Alpha-Globulins↗
PubMed1976

Raffinose metabolism in Escherichia coli K12. Purification and properties of a new alpha-galactosidase specified by a transmissible plasmid.

The utilization by Escherichia coli K12 of raffinose as sole carbon source depends on a new raffinose transport system, an invertase and an alpha-galactosidase specified by the Raf-plasmid D1021. The alpha-galactosidase was purified to homogeneity from a mutant strain with constitutive synthesis of the enzyme. alpha-Galactosidase hydrolyzes p-nitrophenyl-alpha-D-galactoside (Km 0.14 mM), methyl-alpha-D-galactoside (Km 30mM), melibiose (Km 3.2 mM) and raffinose (Km 60 mM). The enzymatic activity is strongly inhibited by Ag+, p-chloromercuriphenyl sulfonic acid and, to a lesser extent, by iodoacetamide. Isoelectric focusing indicates the existence of one form of alpha-galactosidase with an isoelectric point of 5.1. The purified enzyme has an sw,20 value of 11.7 +/- 0.3S and a molecular weight of 329000 +/- 4000; this value is not reduced at high dilutions. When examined by dodecylsulphate gel electrophoresis, purified alpha-galactosidase yields a single subunit band of molecular weight 82000 suggesting that the intact enzyme consists of four subunits. Amino acid analysis indicates the presence of approximately 712 amino acid residues per quarter molecule including 8 half-cystine residues. No carbohydrate moiety has been detected. High resolution electron micrographs and Markham rotation of alpha-galactosidase show enzyme molecules of approximately 11 x 11 nm containing four globular subunits in a tetragonal arrangement. The plasmid-coded alpha-galactosidase differs from the homologous E. coli enzyme by substrate affinities, cofactor requirements, stability and toluene resistance. It can, therefore, be used as a marker enzyme suitable for the detection in vivo of Raf-plasmids.

Amino Acids↗
PubMed1976

Topography of human plasma alpha1-acid glycoprotein.

Human plasma alpha1-acid glycoprotein, whose linear amino acid sequence has recently been elucidated (Schmid et al. (1973), Biochemistry 12, 2711), was further investigated with regard to its topography. Nitration of this protein and subsequent elucidation of the structures of the peptides containing modified tyrosine indicated that residues 27, 37, 78, 115, 127, and 157 are free, 50 and 91 are in an intermediate state, and 65, 74, 110, and 142 are buried. CD measurements between pH 10 and 12 demonstrated that the buried tyrosines are strongly hydrogen bonded and are probably responsible to a considerable extent for the stability of this protein. Of the three tryptophans of this protein, residue 122 proved to be partially reactive with Koshland reagent while the other two (25 and 160) were found to be unreactive. The state of the two disulfide bonds, established by differential reduction and alkylation with specific reagents, was shown to be of an intermediate type. Using carboxymethylation with bromoacetate at pH 7.0 for 8 days, the three histidines (97, 100, and 171) and methionine 111 could be shown to be in intermediate states. All lysines were treated with trinitrobenzenesulfonate and thus were assumed to be free. Of the 40 carboxylic groups, which were amidated with glycine methyl ester, 32 including the 14 sialyl residues were found to be free, six in an intermediate and the remaining two in a buried state. The present study describes the states of almost half of the amino acid residues of alpha1-acid glycoprotein, a knowledge important for the construction of a preliminary three-dimensional model of this conjugated protein.

Amino Acid Sequence↗
PubMed1976

The interaction of collagen with alpha1-acid glycoprotein.

The influence of alpha1-acid glycoprotein on the formation of fibrous long spacing fibers of collagen has been investigated. It was observed that addition of the glycoprotein to dialyzed collagen solutions caused a significant decrease in the intensity of the circular dichroic spectrum of collagen. This phenomenon, which displays an optimum with respect to glycoprotein, is consistent with previous observations of fibrous long spacing fiber formation. Changes in viscosity of collagen initially dissolved in acetic acid were monitored during dialysis. It was found that a significant increase in viscosity must occur during dialysis of collagen before fibrous long spacing formation could take place. This increase in viscosity can be related directly to removal of acetic acid from the collagen solution. Removal of all sialyl residues from the alpha1-acid glycoprotein with neuraminidase prevents fibrous long spacing formation while removal of up to 35% of the sialyl residues has no effect on the interaction of glycoprotein with collagen. Amino acid composition and radioactivity studies suggest that 45-55% of the insoluble fibrous long spacing fibers is glycoprotein. In contrast to native collagen fibers, reduced fibrous long spacing fibers do not contain histidinohydroxymerodesmosine or hydroxylysinonorleucine. Instead, they contain significant quantities of allysine aldol and epsilon-hydroxynorleucine.

Amino Acids↗
PubMed1976

A single column packing for the gas-liquid chromatographic separation of various biologically important compounds.

The use of a single, commercially available column packing, TabsorbR, is described for the g.l.c. separation of a large number of different compounds. The resolution of the homologous members of the following series of compounds was achieved: (1) saturated fatty acids (C1-C18), (2) normal aliphatic saturated dicarboxylic acids (C2-C14), (3) normal aliphatic saturated alcohols (C1-C24), (4) normal aliphatic saturated amines (C1-C12), (5) the common amino acids except arginine, histidine and cysteine, (6) aliphatic hydrocarbons (C10-C20) and (7) monosaccharides. It should be noted that twenty-two monosaccharides including three hexosamines and two anhydrohexoses, could be resolved as alditol acetates in a single run. In addition, galacturonic, glucuronic and iduronic acids could be separated from one another as their 1,4-lactones. The resolution achieved in these series of compounds was found to be consistent and highly reproducible. It is of further interest that certain isomers of the higher fatty acids and hydrocarbons with one double bond could also be separated from the normal and saturated compounds, respectively. The applicability of "Tabsorb" for the g.l.c. separation, although noted above to be considerably broad, is by far not yet exhausted. These procedures which form the basis for the quantitative determinations of the various compounds studied as demonstrated by analysis of glycopeptides for neutral hexoses and proteins for the amino acids, can readily be adapted to preparative methods. From the biochemical point of view "Tabsorb" is an extremely versatile column packing in that it can be used for the identification of many of the common building blocks of natural products.

Alcohols↗
PubMed1976

The glycosaminoglycans of the human artery and their changes in atherosclerosis.

The changes in levels of glycosaminoglycans (GAGs) of the intima and media of the human artery in atherosclerosis were determined by a recently introduced two-dimensional electrophoresis technique that permits direct measurments of each of these macromolecules. To identify the arterial GAGs, they were fractionated by chromatography on a DEAE-Sephadex A-25 column, and the resulting three fractions (hyaluronic acid [HA], heparan sulfate [HS], and the partially separated chondroitin sulfates B [CSB] and C [CSC]) were analyzed for their electrophoretic mobilities by this electrophoretic method, for their digestability by highly specific hydrolases (leech hyaluronidase, heparinase, and chondroitinases ABC and AC) and for their iduronic acid content. From these studies we concluded that normal and atherosclerotic human aortas contain CSB, CSC, HA, and HS. Further, we demonstrated that CSB is a hybrid consisting of approximately 40% CSA and 60% CSB and that CSC appears to be a polymer consisting essentially of glucuronic acid and N-acetylgalactosamine-6-sulfate. Classical CSA as well as chondroitin (CH) were not present in detectable amounts. In the relatively normal intima, the mean concentrations of the GAGs were found to be 4.7, 20.9, 1.3, and 5.1 mg/g of dry, defatted, decalcified tissue for CSB, CSC, HA, and HS, respectively. With the progression of atherosclerosis, there was a pronounced decrease in the total GAG content (from 32 to 18 mg) associated with a decrease in the CSC and HS levels but without a change in the HA concentrations. Of particular interest, however, was the increase in the CSB level. In the media whose total GAG content averaged approximately 20 mg, no significant changes in these GAG levels were noted with the progression of the disease except for that of CSC. These findings may be important in explaining the increased lipoprotein and collagen deposition in the diseased aorta.

Aorta↗
PubMed1975

Isolation of an immunosuppressive peptide fraction from the serum of cancer patients.

An immunosuppressive peptide fraction was isolated by means of gel filtration, membrane partition, and ion-exchange chromatography from the sera of patients hospitalized for cancer. The resulting peptide fraction, which was heterogeneous as judged by high-voltage electrophoresis, was found to suppress both phytohemagglutinin-induced proliferation of lymphocytes in vitro and the in vivo induction of splenic plaque-forming cells in mice. The specific activity of the peptide fraction, which was isolated from the sera of cancer patients, was significantly increased over that of the unfractionated starting material. Moreover, in control experiments, when the sera of normals or non-cancer-bearing hospitalized individuals were subjected to the same chromatographic techniques, no active peptide fraction could be obtained.

Blood Protein Electrophoresis↗