Ultrastructural and biochemical identification of con A receptors in the desmosome

Correlated ultrastructural and biochemical methods were used to identify and localize Concanavalin A (Con A) receptors in the desmosomes of bovine epidermis. Specific carbohydrate residues were labeled with ferritin-Con A in thin sections of tissue embedded in a hydrophilic resin. Quantitative mapping of ferritin distribution in labeled desmosomes revealed that Con A receptors are localized in the intercellular zone and concentrated along the desmosomal midline or central dense stratum. Labeling was almost entirely absent when sections were treated with ferritin-Con A in the presence of 0.1 M α-methyl mannoside, a hapten-inhibitor of Con A. “Whole” desmosomes and desmosomal intercellular regions (desmosomal “cores”) were purified from bovine muzzle epidermis. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis reveals a limited number of major desmosomal protein constituents. Certain of these are glycoproteins and are greatly enriched in the core fraction. Almost all the desmosomal glycoproteins are intensely labeled when electrophoretic gels of whole desmosome or core fractions are exposed to fluorescent Concanavalin A.     

Norepinephrine Metabolism in Man Using Deuterium Labeling: Turnover of 4-Hydroxy-3-Methoxymandelic Acid

Abstract: 4-Hydroxy-3-methoxymandelic acid (HMMA; VMA) labeled with three deuterium atoms was used to study the turnover and fate of HMMA following intravenous injection. Five healthy men were given a pulse dose of 5.0 μmol of labeled HMMA. Plasma and urinary levels of both endogenous and labeled HMMA were subsequently followed by gas chromatography-mass spectrometry using selected ion detection. The kinetic parameters were determined both with and without compensation for the pool expansion caused by the injection of labeled HMMA. The urinary recovery of labeled HMMA was 85 × 10% (mean ± SD). No conversion of HMMA t o 4-hydroxy-3-methoxyphenyl glycol (HMPG) occurred. The biological half-life of HMMA was 0.54 ± 0.22 h. The apparent volume of distribution was 0.36 ± 0.11 L/kg. The production rate or body turnover was 1.27 ± 0.51 μmol HMM/h and urinary excretion rate was 0.82 ± 0.22 μmol/h. These results show that HMMA is turning over rapidly in a relatively small volume of distribution and that, unlike HMPG, it is an end metabolite of norepinephrine in man.     

Lipid-polyethylene glycol interactions: I. Induction of fusion between liposomes

Summary Fusion between unilamellar vesicles of both egg phosphatidylcholine and bovine phosphatidylserine was induced by polyethylene glycol. Aggregation and fusion events were monitored by electron microscopy and turbidity measurements. The threshold concentration of polyethylene glycol for aggregation and fusion is found to be independent of lipid concentration. Typically, aggregation of phosphatidylcholine vesicles starts at 2.5% (wt/wt) polyethylene glycol, but fusion is not significant until the polyethylene glycol concentration reaches 35%. Multilamellar vesicles were formed as a result of fusion.Abbreviations PEG Polyethylene glycol - IMP Intramembranous particle - PC Phosphatidylcholine - PS Phosphatidylserine - SUV Small unilamellar vesicles - MLV Multilamellar vesicles - DPPC Dipalmitoyl phosphatidylcholine - DSC Differential scanning calorimetry     

Lipid-polyethylene glycol interactions: II. Formation of defects in bilayers

Summary Polyethylene glycol, a known cell fusogen, is found to induce the formation of structural defects in egg phosphatidylcholine multilamellar vesicles, as shown by freeze-fracture microscopy.³¹P NMR spectra of these vesicles reveal the existence of a nonbilayer (isotropic) phase. The observed disruption in the bilayers is believed to be associated with an intermediate stage of membrane fusion.Abbreviations PEG Polyethylene glycol - IMP Intramembranous particle - PC Phosphatidylcholine - PS Phosphatidylserine - SUV Small unilamellar vesicles - MLV Multilamellar vesicles - DPPC Dipalmitoyl phosphatidylcholine - DSC Differential scanning calorimetry - DMPC Dimyristoylphosphatidylcholine - T _c Phase transition temperature     

Repair of DNA damage after exposure to 4-nitroquinoline-1-oxide in heterokaryons derived from xeroderma pigmentosum cells

Xeroderma pigmentosum (XP) cells are dificient in the repair of damage induced by ultraviolet irradiation. Excision-repair-deficient XP cell strains have been classified into 7 distinct complementation groups, according to results of studies on cell fusion and UV irradiation. XP cells are not only abnormally sensitive to UV, but also to a variety of chemical carcinogens, including 4-nitroquinoline-1-oxide (4NQO). Complementation analysis with XP strains from 4 different complementation groups with respect to the repair of 4NQO-induced DNA damage revealed that the classification of the strains into complementation groups with respect to 4NQO-induced repair coincides with the classification based on the repair of UV damage.     

Regulation of calcium fluxes in rat pancreatic islets: Quinine mimics the dual effect of glucose on calcium movements

The effects of quinine and 9-aminoacridine, two blockers of potassium conductance in islet cells, on ⁴⁵Ca efflux and insulin release from perifused islets were investigated in order to elucidate the mechanisms by which glucose initially reduces ⁴⁵Ca efflux and later stimulates calcium inflow in islet cells. In the absence of glucose, 100 μM quinine stimulated ⁴⁵Ca net uptake, ⁴⁵Ca outflow rate and insulin release. Quinine also dramatically enhanced the cationic and the secretory response to intermediate concentrations of glucose, but had little effect on ⁴⁵Ca net uptake, ⁴⁵Ca fractional outflow rate and insulin release at a high glucose concentration (16.7 mM). The ability of quinine to stimulate ⁴⁵Ca efflux depended on the presence of extracellular calcium, suggesting that it reflects a stimulation of calcium entry in the islet cells. In the absence of extracellular calcium, quinine provoked a sustained decrease in ⁴⁵Ca efflux. Such an inhibitory effect was not additive to that of glucose, and was reduced at low extracellular Na⁺ concentration. At a low concentration (5 μM), quinine, although reducing ⁸⁶Rb efflux from the islets to the same extent as a non-insulinotropic glucose concentration (4.4 mM), failed to inhibit ⁴⁵Ca efflux. In the presence of extracellular calcium, 9-aminoacridine produced an important but transient increase in ⁴⁵Ca outflow rate and insulin release from islets perifused in the absence of glucose. In the absence of extracellular calcium, 9-aminoacridine, however, failed to reduced ⁴⁵Ca efflux from perifused islets. It is concluded that quinine, by reducing K⁺ conductance, reproduces the effect of glucose to activate voltage-sensitive calcium channels and to stimulate the entry of calcium into the B-cell. However, the glucose-induced inhibition of calcium outflow rate, which may also participate in the intracellular accumulation of calcium, does not appear to be mediated by changes in K⁺ conductance.     

Detection of the associated state of membrane proteins by polyacrylamide gradient gel electrophoresis with non-denaturing detergents Application to band 3 protein from erythrocyte membranes

Polyacrylamide gradient gel electrophoresis was carried out in micellar solutions of various detergents which differ in degree of potency to denature proteins. From the application of this method to band 3 protein from erythrocyte membranes, it was suggested that the procedure was useful in studying the molecular state of membrane proteins.The electrophoretic behaviors of human and bovine band 3 protein did not show any species specificity in either a denature state and a state resembling the native state. As well as in nonionic detergent solutions, the dimeric and tetrameric structures of bovine band 3 protein were preserved in sodium deoxycholate solution, in which protein complexes maintained in nonionic detergent solutions are frequently dissociated. Even in dodecyltrimethylammonium bromide solution, which is a denaturant for water-soluble proteins, part of the band 3 protein was still present as the oligomer. The results suggest that the oligomeric form of band 3 protein is the stable structure and that the dimer and tetramer possibly coexist in membranes.     

Carbohydrate binding activity of a lectin-like glycoprotein from stems and leaves of Dolichosbiflorus

A glycoprotein from the stems and leaves of the $\text{Dolichos}$$\text{biflorus}$ plant that cross reacts with antibodies to the seed lectin has been found to bind to affinity columns of blood group A + H substance covalently linked to Sepharose. This binding of the cross reactive material to the affinity resin differs from that of the seed lectin in that it is easily dissociated with 0.15 M NaCl. Affinity electrophoresis using entrapped blood group A + H substance shows that the carbohydrate binding activity of the cross reactive material is weakly inhibited with N-acetyl-D

-galactosamine and N-acetyl-D

-glucosamine. Glucose, mannose and galactose gave no inhibition when tested at concentrations of 50 mM. These data indicate that the specificity of the cross reactive material is somewhat different from the N-acetyl-D

-galactosamine specificity of the seed lectin. The significance of these findings is discussed in relation to the structural similarities of the cross reactive material and the seed lectin.     

A radioimmunoassay of plasma estradiol-17beta with the use of polyethylene glycol to separate free and antibody-bound hormone

A radioimmunoassay for plasma estradiol-17β was developed using polyethylene glycol to separate free from antibody-bound hormone. Specificity for estradiol-17β was achieved by a modified celite microcolunm procedure in which estradiol was.separated from interfering estrogens, including estrone. Using trace ³H-estradiol to monitor procedural losses, the method was shown to be sensitive and accurate. Intra- and inter-assay coefficient of variation of the method was 8.7 and 10.6%, respectively. Polyethylene glycol used for antibody precipitation appears to be a generally applicable method for steroid hormone radioimmunoassays. The simplicity, precision and rapid analysis, coupled with its lack of time dependence and ease in automation, makes this a convenient and practical method.