Glutamine synthetase (GS) expression is reduced in senile dementia of the Alzheimer type

Glutamine synthetase (GS), a metabolic marker of the mature astrocyte, was investigated in the temporal neocortex of postmortem brain samples of 8 cases, either not demented or affected by senile dementia of the Alzheimer type. A negative correlation between the GS protein level and the density of both classical A4 deposits and senile plaques was evidenced. Such a correlation for GS underlies a dysfunction of the astroglial metabolism and particularly of the glutamate and ammonia neutralization. Since GS is sensitive to oxidative lesioning, the changes in GS level that were observed, occurring at the posttranslational stage, might reflect oxidative damage and have severe consequences on the pathological cascade of events.     

Interaction of human alpha 1-antichymotrypsin with chymotrypsin

Human alpha 1-antichymotrypsin reacts with bovine chymotrypsin to form an equimolar complex and this reaction is accompanied by the formation of a free, modified form of the inhibitor. Time-course studies, performed on mixtures containing an excess of native inhibitor and kept at 0 degree C or at 25 degrees C, show that the equimolar complex dissociates spontaneously; this dissociation results in the release of inactive modified alpha 1-antichymotrypsin and of some active enzyme, which is able to recycle with active inhibitor in excess. When all the native inhibitor is used up, the released active enzyme degrades the remaining intact complex into intermediate forms. At the endpoint of the reaction only inactive modified inhibitor and some active chymotrypsin remain. Immunochemical data indicate that, in the complex, a steric hindrance of the antigenic determinants of the inhibitor prevents the formation of the precipitate with specific antiserum. Inactive modified inhibitor, which has dissociated from the complex, has retained antigenic determinants of the native alpha 1-antichymotrypsin.     

Effect of lymphatic cannula outflow height on lung microvascular permeability estimations

Estimates of the pulmonary microvascular membrane reflection coefficient (sigma) and permeability-surface area product (PS) are frequently made with the assumption that a percent change in transmicrovascular fluid flux (Jv) will be represented by an equal percent change in the lymph flow rate (QL) from a single cannulated lung lymph vessel. To test this, we measured QL in seven anesthetized dogs with the outflow end of the lymph cannula set at several heights (H) above and below the lung hilus. The left atrial pressure was then elevated to increase Jv, and QL was again measured at several H's. The percent increase in QL at elevated left atrial pressure depended on H. We used the QL data and lymph and plasma protein concentrations to estimate sigma and PS with a modified form of the Kedem and Katchalsky equations. The calculated values varied considerably with H. Our results indicate that changes in Jv are not represented by equal changes in QL. Therefore, techniques for estimating permeability that depend upon QL as an estimate of Jv may lead to erroneous estimates of sigma and PS.     

Characterization of gangliosides from bovine erythrocyte membranes

Two glucosamine-containing gangliosides, sialosylhexaglycosylceramides, were isolated from bovine erythrocyte membranes. Both gangliosides were hydrolyzed by neuraminidase isolated from Clostridium perfringens to become neutral hexaglycosylceramides. Based on the results of sequential enzymatic hydrolysis and gas chromatography-mass spectrometric analyses of the methylated sugars, the structures of these two gangliosides were shown to be NeuAcalpha2 leads to 3Galbeta1 leads to 4GlcNAcbeta1 leads to 3Galbeta1 leads to 4GlcNAcbeta1 leads to 3Galbeta1 leads to 4Glc-ceramide and NeuGcalpha2 leads to 3Galbeta1 leads to 4GlcNAcbeta1 leads to 3Galbeta1 leads to 4GlcNAcbeta1 leads to 3Galbeta1 leads to 4Glc-ceramide, respectively. In addition, N-acetyl- and N-glycolylneuraminosyllacto-N-neotetraosylceramides, and N-acetyl- and N-glycolylneuraminosyllactosylceramides were also found in bovine erythrocytes. The predominant fatty acids in these two gangliosides were C 22:0 and C 24:0. C-18 sphingosine was the major base detected.     

Structure determination of the glycans of human-serum alpha 1-antichymotrypsin using 1H-NMR spectroscopy and deglycosylation by N-glycanase

alpha 1-Antichymotrypsin purified from normal human serum was separated by affinity chromatography into th ree microheterogeneous forms on a concanavalin-A-Sepharose column: a pass-through (peak 1), a retarded (peak 2) and a bound form (peaks 3 + 4). For each form the asparagine-linked carbohydrate chains were liberated as oligosaccharides by hydrazinolysis, submitted to reduction with NaBH4 after re-N-acetylation and further separated by affinity chromatography on a concanavalin-A-Sepharose column. The complete primary structure of the glycans was determined by high-resolution 1H-NMR spectroscopy. The results indicated the presence of disialyl diantennary and of trisialyl triantennary type glycanic structures, the latter being accompanied by traces of disialylated triantennary oligosaccharide. The N-glycanase was used for the deglycosylation of the unfractionated alpha 1-antichymotrypsin; the successive removal of the N-linked complex-type oligosaccharide side chains of alpha 1-antichymotrypsin was studied in the presence of detergents. From these experiments it is concluded that alpha 1-antichymotrypsin carries four oligosaccharide side chains. Moreover our results show that the peak 1 contains four triantennary glycans, the peak 2 three triantennary and one diantennary glycans while the bound peaks 3 + 4 possess, on average, about one triantennary and three diantennary glycans per molecule. Since we showed that the peak 4 contains mostly diantennary glycans, it can be deduced that in peak 3 there are molecules carrying two triantennary and two diantennary glycans and others carrying one triantennary and three diantennary glycans.     

In vitro susceptibility of Erwinia amylovora (Burrill) Winslow et al. to geraniol and citronellol

The in vitro antimicrobial activity of geraniol and citronellol towards seven strains of Erwinia amylovora , the causal agent of 'fire blight'of Rosaceous plants, was assessed in tube cultures. All of the strains tested at 1 × 10⁵ cfu/ml were inhibited for 24 h by geraniol in the range 600–1500 mg/1, whereas its minimum bactericidal concentration was 800–1700 mg/1. Citronellol was less effective, being bactericidal for only two of seven strains. RIF-NY, isolated from apple orchards, was relatively resistant to geraniol; 1700 mg/1 of the chemical only reduced the growth of an inoculum of 1 × 10⁷ cfu/ml. In general, such terpenoids commenced exerting a bactericidal effect 6 h after addition to the suspensions, even if geraniol added at 1700 mg/1 to 1 × 10³ cfu/ml of five strains, commenced its bactericidal activity earlier than 6 h.     

Identification of the protein responsible for hepatic system N amino acid transport activity.

In the liver, glutamine utilization may be limited by the rate of transport across the plasma membrane by the System N carrier. System N-mediated transport activity has been solubilized from rat liver plasma membrane, partially purified, and then reconstituted into proteoliposomes. To identify the System N carrier protein, monoclonal antibodies were generated against the protein fraction enriched for System N activity. Two antibodies , 3E1-2 and 1E7-3, inhibited System N activity in hepatocytes. These antibodies also immunoprecipitated System N activity from a mixture of solubilized proteins and were specific for antigen recognition in that neither immunoprecipitated System A activity. The antibody recognized a single protein of molecular size 100 kDa by immunoblot analysis. Recognition of this protein by the antibody increased in parallel with the enrichment of System N activity in solubilized membrane fractions. These data suggest that a 100-kDa plasma membrane protein mediates System N transport activity in rat hepatocytes.     

Enhancement of detection for partially methylated alditol acetates by chemical ionization mass spectrometry

Treatment of washed, intact platelets with Bolton-Hunter reagent is a satisfactory method for ¹²⁵I-labeling of many platelet proteins. Analysis by two dimensional polyacrylamide gel electrophoresis and autoradiography shows that the major platelet cytoskeletal proteins and at least four surface-exposed proteins are labeled. The method allows the identification of these labeled proteins in amounts that are below the limits of detection by Coomassie blue staining. Two granule proteins, thrombospondin and fibrinogen, are slightly labeled. Conditions of labeling do not appear to affect platelet structure or function, as assessed by phase-contrast microscopy, ⁵¹CrO₄^2? release, and aggregation in response to thrombin or fibrinogen/adenosine-5′-diphosphate.     

Oligosaccharides accumulated in the kidney of a goat with β-mannosidosis: Mass spectrometry of intact permethylated derivatives

Two oligosaccharides accumulate in the kidney of a goat with β-mannosidosis. These oligosaccharides were isolated and purified from kidney extracts by Bio-Gel P2 gel permeation column chromatography. Their structures were characterized as Manβ1 → 4GlcNAc and Manβ1 → 4G1cNAcβ1 → 4G1cNAc by mass spectrometry of the permethylated intact oligosaccharide alcohols and permethylated native oligosaccharides. Carbohydrate composition analysis, methylation linkage studies, and enzymatic hydrolysis were also performed. Stored in 1 g of kidney were 1.6 μmol of disaccharide and 7.6 μmol of trisaccharide, which was three times that found in the brain of this affected animal (M. Z. Jones and R. A. Laine, 1981, J. Biol. Chem., 256, 5181–5184). In both the brain and kidney of the affected goat, oligosaccharide accumulation was evidently represented by membrane-bound, electron-lucent vacuoles in numerous cell types. While lesions in the brain were associated with profound neurological deficits, functional impairment of the kidney was not apparent. Similar oligosaccharides excreted in urine may be derived from those stored in the kidney. The mass spectrometric methods utilized in this investigation will facilitate comparison of oligosaccharide composition in different tissues and biological samples in β-mannosidosis and other disorders of glycoprotein catabolism.     

Structural and functional significance of association of a low molecular weight chalone from JB-1 ascites tumours with high molecular weight factors

Abstract. The cell-specific inhibitory (chalone) activity of JB-1 ascites tumour cell proliferation has been purified using five different procedures. By combining (1) molecular weight estimations based on ultrafiltration and gel chromatography, and (2) partitioning in organic solvent and ion exchangers, it is concluded that the active factor associates, in a complex manner, with various other components involving both hydrophobic and ionic forces. The active factor appears to be a slightly acidic, hydrophobic peptide (molecular weight 500–1000 D). When assessing the activity in vivo , it appears to be highly dependent on associated serum factors. Thus, the chalone studied appears to interact both structurally and functionally with various associated factors which affect its physicochemical behaviour and biological activity.