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.     

Factors affecting the elicitation of sesquiterpenoid phytoalexin accumulation by eicosapentaenoic and arachidonic acids in potato

Eicosapentaenoic and arachidonic acids in extracts of Phytophthora infestans mycelium were identified as the most active elicitors of sesquiterpenoid phytoalexin accumulation in potato tuber slices. These fatty acids were found free or esterified in all fractions with elicitor activity including cell wall preparations. Yeast lipase released a major portion of eicosapentaenoic and arachidonic acids from lyophilized mycelium. Concentration response curves comparing the elicitor activity of the polyunsaturated fatty acids to a cell-free sonicate of P. infestans mycelium indicated that the elicitor activity of the sonicated mycelium exceeded that which would be obtained by the amount of eicosapentaenoic and arachidonic acids (free and esterified) present in the mycelium. Upon acid hydrolysis of lyophilized mycelium, elicitor activity was obtained only from the fatty acid fraction. However, the fatty acids accounted for only 21% of the activity of the unhydrolyzed mycelium and the residue did not enhance their activity. Centrifugation of the hydrolysate, obtained from lyophilized mycelium treated with 2n NaOH, 1 molarity NaBH₄ at 100°C, yielded a supernatant fraction with little or no elicitor activity. Addition of this material to the fatty acids restored the activity to that which was present in the unhydrolyzed mycelium. The results indicate that the elicitor activity of the unsaturated fatty acids is enhanced by heat and base-stable factors in the mycelium.     

Studies on the relationship between glycerophosphoglycolipids and lipoteichoic acids. IV. Trigalactosylglycerophospho-acylkojibiosyldiacylglycerol and related compounds from Streptococcus lactis Kiel 42172.

Streptococcus lactis Kiel 42172 contains at least six unusually polar glycerophosphoglycolipids. The predominant one was composed of D-galactose, D-glucose, glycerol, acyl groups and phosphorus in a molar ratio of approx. 3 : 2 : 2 : 3 : 1. By analysis of the breakdown products of HF hydrolysis and Smith-degradation the structure was established to be [Galp (alpha 1 leads to 6)Galp(alpha 1 leads to 3)-sn-glycero(2 comes from 1 alpha Galp)-1-phospho] leads to 6Glcp(alpha 1 leads to 2), acyl leads to Glcp(alpha 1 leads to 3)-acyl2Gro. By HF hydrolysis the other compounds were shown to be in the main also derivatives of GroP leads to 6Glc(alpha 1 leads to 2), acyl leads to 6Glc(alpha 1 leads to 3)acyl2Gro but they released as water-soluble glycosides Gal(alpha 1 leads to 2)Gro, Gal(alpha 1 leads to 3)Gro, Gal(alpha 1 leads to 3)Gro(2 comes from 1 alpha Gal), Gal(alpha 1 leads to 6)Gal(alpha 1 leads to 3)Gro and Gal(alpha 1 leads to 6)Gal-(alpha 1 leads to 6)Gal(alpha 1 leads to 3)Gro(2 comes from 1 alpha Gal), respectively. In the lipid extract Glc(alpha 1 leads to 2), acyl leads to 6Glc(alpha 1 leads to 3)acyl2Gro and GroP leads to 6Glc(alpha 1 leads to 2), acyl leads to 6Glc(alpha 1 leads to 3) acyl2Gro were also observed. This set of compounds is proposed to constitute a biosynthetic series reflecting the individual steps in the synthesis of the lipoteichoic acid of Streptococcus lactis Kiel 42172 which is made up by the same lipid anchor and a non-classical poly(galabiosyl, galactosyl glycerophosphate)-chain (Koch, H.U. and Fischer, W. (1978) Biochemistry 17, 5275--5281).     

Palatal dimensions in 45,X-females

Seventy-two females with 45,X-chromosome complement were examined for palatal dimensions, and the results of the measurements were compared to those of first-degree normal female relatives of the study subjects and population control females. Hard stone casts were prepared for measuring widths and lengths of the maxillary alveolar arch and palatal height between or at the level of the permanent canines, first and second premolars, and first molars with a palatometer and sharp-pointed vernier calipers (0.5 mm). According to the analyses of covariance (history of orthodontic treatment and loss of permanent teeth as cofactors and age as covariate), the differences in palatal dimensions between the groups were statistically significant or highly significant for all dimensions except for palatal height in the posterior segments. The group of the 45,X-females had the highest mean value for palatal height at the level of the canines and systematically the lowest mean values for palatal width in all segments, while no clear difference could be found in the length of the alveolar arch between the 45,X-females and the relatives, the population control group showing the lowest value in the posterior segments. The findings of this study indicate that the narrowed palate rather than the high palate is a frequent but not definite feature in 45,X-females. This may reflect the effect of sex-chromosomes on width but not other dimensions of the palate, resulting in deficiency of transversal growth of the palate possibly through decreased growth of the palatal shelves or through disturbances in the growth of the nasal septum, sutural growth, or--to which the exostosis on the palatal alveolar plates would refer--disturbances in apposition-resorption growth changes of the maxilla.     

A Lead ANRIL Polymorphism Is Associated with Elevated CRP Levels in Periodontitis: A Pilot Case-Control Study

Elevated high sensitive C-reactive protein (hsCRP) is a marker for systemic inflammation and a risk marker for atherosclerotic cardiovascular disease (ACVD), and has also been associated with periodontitis. Inter-individual variation for hsCRP in periodontitis has been shown. ANRIL is the strongest genetic susceptibility locus for both periodontitis and ACVD, and it is speculated that genetic variation in ANRIL may modulate inflammatory processes. Therefore, we explored the possible association between hsCRP plasma levels and a leading ANRIL single nucleotide polymorphism (SNP) in periodontitis patients and controls. 171 healthy subjects with North European descent (115 periodontitis and 56 controls) were included in this case-control study. hsCRP levels were determined and subjects were genotyped for the leading ANRIL SNP rs1333048. In a multivariate analysis, periodontitis, female gender, increasing BMI and homozygosity for the major allele (AA-genotype) of rs1333048 were significantly associated with elevated hsCRP plasma levels (p = 0.012, p = 0.004, p = 0.007 and p = 0.003, respectively). Periodontitis patients with rs1333048 AA-genotype showed higher levels of hsCRP than those carrying the minor C allele (median: 4.5 mg/L vs. 1.6 mg/L, p_adjusted = 0.007). This study is the first to show that, in addition to gender and BMI, also a leading SNP in ANRIL is explanatory for inter-individual variation in hsCRP levels in periodontitis patients of North European descent.