Altered localization of antigen recognized by proteinuriainducing monoclonal antibody in experimental nephrosis

Change in the localization of the antigen recognized by the proteinuria-inducing monoclonal antibody (MA) 5-1-6 in experimental nephrosis was studied by indirect and biotin-avidin immunofluorescence, and immunoperoxidase at light and electron microscopical levels. The proteinuric state was induced by the administration of the aminonucleoside of puromycin (PAN) or adriamycin. The antigen decreased in quantity and/or its distribution changed with an increase in the amount of protein excreted in both experimental models. Recovery from the alterations observed during the development and proteinuria appeared to occur when PAN-induced proteinuria subsided. This antigenic molecule may thus be essential for maintaining the normal permselectivity of glomerular capillary walls.     

Isolation and regional localization of a large collection (2,000) of single-copy DNA fragments on human chromosome 3 for mapping and cloning tumor suppressor genes

Summary A collection of 2,000 lambda phage-carrying human single-copy inserts (> 700 bp) were isolated from two chromosome-3 flow-sorted libraries. The single-copy DNA fragments were first sorted into 3p and 3q locations and about 700 3p fragments were regionally mapped using a deletion mapping panel comprised of two humanhamster and two-human-mouse cell hybrids, each containing a chromosome 3 with different deletions in the short arm. The hybrids were extensively mapped with a set of standard 3p markers physically localized or ordered by linkage. The deletion mapping panel divided the short arm into five distinct subregions (A-E). The 3p fragments were distributed on 3p regions as follows: region A, 26%; B, 31%; C, 4%; D, 4% and E, 35%. We screened 300 single-copy DNA fragments from the distal part of 3p (regions A and B) with ten restriction endonucleases for their ability to detect restriction fragment length polymorphisms (RFLPs). Of these fragments 110 (36%) were found to detect useful RFLPs: 35% detected polymorphisms with frequency of heterozygosity of 40% or higher, and 25% with frequency of 30% or higher. All polymorphisms originated from single loci and most of them were of the base pair substitution type. These RFLP markers make it possible to construct a fine linkage map that will span the distal part of chromosome 3p and encompasses the von Hippel-Lindau disease locus. The large number of single-copy fragments (2,000) spaced every 100–150 kb on chromosome 3 will make a significant contribution to mapping and sequencing the entire chromosome 3. The 300 conserved chromosome 3 probes will increase the existing knowledge of man-mouse homologies.     

Biosynthesis of lysosomal cathepsins B and H in cultured rat hepatocytes

The biosynthesis of lysosomal cysteine proteases, cathepsins B and H, was investigated by using pulse-chase experiments in vivo in primary cultures of rat hepatocytes. Cathepsins B and H were isolated from either total cell extracts or culture medium labeled with [35S]methionine by immunoprecipitation and analyzed for their molecular forms. Within 60 min of chase, cellular proforms of cathepsins B of 39 kDa and H of 41 kDa were converted to single-chain form cathepsins B of 29 kDa and H of 28 kDa, respectively, and persisted as these forms even after 12-h chase periods. The proforms of cathepsins B and H derived from pulse-labeling experiments showed complete susceptibility to endoglycosidase H treatment, indicating that these proenzymes bear high-mannose-type oligosaccharides at the stage of initial events of biosynthesis. In the presence of tunicamycin, unglycosylated proenzymes of cathepsins B of 35 kDa and H of 34 kDa were found to be secreted into the extracellular medium without undergoing proteolytic processing. Furthermore, in the presence of swainsonine, a potent inhibitor of Golgi mannosidase II, considerable amounts of the proenzymes were secreted and accumulated in the medium during chasing periods. These results suggest that the oligosaccharide moiety of these enzymes would be necessary for the intracellular sorting mechanism. In monensin-treated cells, the conversion of intracellular proenzymes to mature enzymes was significantly inhibited and the proenzymes were secreted into the medium. In the presence of chloroquine or ammonium chloride, proteolytic processing of the proenzymes was completely prevented and the enhanced secretion of proenzymes was observed. These results suggest that in the presence of lysosomotropic amines the intracellular sorting of proenzymes might not occur properly during biosynthesis.     

Insulin-degrading enzyme is capable of degrading receptor-bound insulin

In the investigation of the intracellular sites of insulin degradation, it might be important whether receptor-bound insulin could be a substrate for insulin-degrading enzyme (IDE). Insulin receptor and IDE were purified from rat liver using a wheat germ agglutinin column and monoclonal anti-IDE antibody affinity column, respectively. [125I]insulin-receptor complex was incubated with various amounts of IDE at 0 degree C in the presence of disuccinimidyl suberate and analyzed by reduced 7.5% SDS-PAGE and autoradiography. With increasing amounts of IDE, the radioactivity of 135 kd band (insulin receptor alpha-subunit) decreased, whereas that of 110 kd band (IDE) appeared then gradually increased, suggesting that IDE could bind to receptor-bound insulin. During incubation of insulin-receptor complex with IDE at 37 degrees C, about half of the [125I]insulin was dissociated from the complex. However, the time course of [125I]insulin degradation in this incubation was essentially identical to that of free [125I]insulin degradation. Cross-linked, non-dissociable receptor-bound [125I]insulin was also degraded by IDE. Rebinding studies to IM-9 cells showed that the receptor binding activity of dissociated [125I]insulin from insulin-receptor complex incubated with IDE was significantly (p less than 0.001) decreased as compared with that without the enzyme. These results, therefore, show that IDE could recognize and degrade receptor-bound insulin, and suggest that IDE may be involved in insulin metabolism during receptor-mediated endocytosis through the degradation of receptor-bound insulin in early neutral vesicles before their internal pH is acidified.     

A novel actin label: a fluorescent probe at glutamine-41 and its consequences

By peptide isolation and analysis, it has been shown that the dansyl fluorophore of dansylcadaverine [N-(5-aminopentyl)-5-(dimethylamino)naphthalene-1-sulfonamide] transfers to Gln-41 of actin from rabbit skeletal muscle when the reaction is catalyzed by guinea pig liver transglutaminase. As a function of time, the degree of labeling asymptotically approaches 1 mol of dansyl/l mol of actin. About 80-85% of the attached dansyl fluorophore was found at Gln-41. Such labeled G-actin polymerizes to the same extent as control actin, but the polymerization rate is greater and the critical concentration is less than for control actin. Complete polymerization is accompanied by a 1.5-2.0-fold increase in the emission intensity of the attached fluorophore. Labeled F-actin thus obtained activates myosin subfragment 1 (S-1) Mg2+-ATPase activity with the same Kapp, and to the same Vmax, as control actin; moreover, when such labeled F-actin is cross-linked to S-1 by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, the resulting superactivation of Mg2+-ATPase is the same as that attained with control actin. The attributes of this label thus make it an ideal reporter of events in the N-terminal 10-kilodalton region of actin, and a new topological point for proximity mapping.     

Fractionation and Estimation of Particle-Attached and Unattached Bradyrhizobium japonicum Strains in Soils

Rhizobial cells attached or unattached to soil particles were estimated. Nonsterile soils into which antibiotic-resistant mutants of Bradyrhizobium japonicum had been introduced were fractionated by a centrifugation technique into two fractions: A, which contained mainly rhizobial cells attached to soil particles, and F, which contained mainly rhizobial cells unattached to them. Rhizobial counts decreased in both fractions during incubation of the soil at 30°C, with a concomitant decrease in the proportion of the count of fraction F to that of fraction A. Sonication of fraction A of the soil incubated for more than 3 weeks caused an increase in the rhizobial count. The ratio of the count of fraction A estimated by the plant infection method to that estimated by the dilution plate method increased after 5 days of soil incubation. More than 90% of the indigenous rhizobia in an agricultural field existed in fraction A. These results suggest that the majority of rhizobial cells are attached to soil particles.     

Quantitation of the Myelin-Associated Glycoprotein in Human Nervous Tissue from Controls and Multiple Sclerosis Patients

Myelin-associated glycoprotein (MAG) was measured by radioimmunoassay in the human CNS and peripheral nervous system (PNS). The level of MAG, expressed as ng/microgram of total protein, was approximately 20-fold higher in whole homogenates of cerebral white matter (4.7 +/- 0.60) than of peripheral nerve (0.12-0.28). MAG concentrations were only slightly higher in the isolated myelin fractions from these tissues: CNS myelin, 5.6 ng/microgram; PNS myelin, 0.37 ng/microgram. The levels of MAG were measured in nine plaques, periplaque regions, and areas of macroscopically normal-appearing white matter (NAWM) from six separate multiple sclerosis brains and compared with the levels of other myelin proteins in the same samples. MAG and other myelin proteins were reduced to very low levels in plaques. The levels of MAG and basic protein (BP) and the activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in periplaque areas were significantly lower than those in control white matter, and MAG and BP levels were also significantly reduced in NAWM. In a periplaque region and NAWM from the most rapidly progressing case of multiple sclerosis examined, the MAG content was between 30 and 35% of the control level, whereas BP and PLP levels and CNP activity were between 50 and 85% of control values. The reduction of MAG content in periplaque regions from all nine multiple sclerosis plaques examined was significantly greater than the reductions of BP level and CNP activity. In NAWM samples, the mean reduction of MAG content was also greater than the reductions of BP level and CNP activity, but the difference was only statistically significant in comparison to CNP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth and Aspartate Kinase Activity in Wheat Cell Suspension Culture: Effects of Lysine Analogs and Aspartate-Derived Amino Acids

The effects of lysine analogs and aspartate-derived amino acidson the growth of wheat cell suspension culture were studied.S-(2-Aminoethyl)-L-cysteine (AEC), -hydroxylysine (DHL) andtrans-lysene caused complete growth inhibition at 1.0 mM. Thegrowth inhibition of lysine analogs were, in the order of decreasingeffectiveness; AECDHL, trans-lysene>oxalysine, homolysineand lysyne. cis-Lysene and methyllysine were not inhibitoryeven at concentrations of 10 mM. Lysine effectively relievedgrowth inhibition induced by the lysine analogs. Lysine plusthreonine showed concerted inhibition, which was relieved bythe addition of methionine. Activity of aspartate kinase extracted from wheat cell suspensionculture was strongly inhibited by L-lysine; 0.75 to 1 mM oflysine was required for half-maximal inhibition. Threonine andmethionine, individually or in combination with lysine, showedno inhibitory effect on the enzyme activity. S-Adenosylmethionine,when added with lysine in equimolar concentrations, enhancedthe feedback inhibition by lysine, lowering the concentrationof lysine for half-maximal inhibition to 0.13 mM. The aspartatekinase isolated from the cells cultured in the presence of 5mM lysine did not differ in regulatory properties from the enzymefrom the cells cultured without lysine. AEC at 5 mM inhibitedthe enzyme activity by 50%. Other lysine analogs were not inhibitoryto the enzyme activity even at 10 mM. Growth inhibition of wheat suspension culture by aspartate-derivedamino acids and lysine analogs were discussed in relation totheir inhibitory effects on aspartate kinase activity. (Received October 25, 1985; Accepted February 26, 1986)     

Glucosylation of Salicyl Alcohol by Gardenia jasminoides Cell Cultures

Cultured cells of Gardenia jasminoides produced both salicinand isosalicin from exogenously supplied salicyl alcohol. Theglucosylation activity of the cells was highest in the exponentialphase of growth and ca. 70% of the added substrate was convertedto the glucosides within 4 days. The rate of glucosylation wasalso dependent on the medium composition such as auxin and sucroseconcentrations. The ratio of salicin to isosalicin formed fromsalicyl alcohol was influenced by the growth stage of the culturedcells. Salicin was converted to isosalicin when exogenouslyadded to the culture. (Received October 11, 1985; Accepted March 10, 1986)     

Biochemical Characterization of α-Adrenergic Receptors in Human Brain and Changes in Alzheimer-Type Dementia

Abstract Using ligand binding techniques, we studied α-adrenergic receptors in brains obtained at autopsy from seven histologically normal controls and seven patients with histopathologically verified Alzheimer-type dementia (ATD). Binding of the α-adrenergic antagonists [³H]prazosin and [³H]yohimbine to membranes of human brains exhibited characteristics compatible with α₁- and α₂-adrenergic receptors, respectively. Binding of both ligands was saturable and reversible, with dissociation constants of 0.15 nM for [³H]prazosin and 5.5 nM for [³H]yohimbine. [³H]Prazosin binding was highest in the hippocampus and frontal cortex and lowest in the caudate and putamen in the control brains. [³H]Yohimbine binding was highest in the nucleus basalis of Meynert (NbM) and frontal cortex and lowest in the caudate and cerebellar hemisphere in the control brains. Compared with values for the controls, [³H]prazosin binding sites were significantly reduced in number in the hippocampus and cerebellar hemisphere, and [³H]yohimbine binding sites were significantly reduced in number in the NbM in the ATD brains. These results suggest that α₁ and α₂-adrenergic receptors are present in the human brain and that there are significant changes in numbers of both receptors in selected regions in patients with ATD.