Selective isolation of individual cell surface proteins from tissue culture cells by a cleavable biotin label

A method was developed to isolate cell surface proteins by a simple two-step procedure. Hepatocyte cell surface proteins were labeled by a cleavable biotin derivative in a covalent pulse reaction. Under the described conditions, NHS-SS-biotin proved to be an impermeant, cell surface-specific label which does not affect the impermeant, cell surface-specific label which does not affect the viability of rat hepatocytes. Biotinylated cell surface proteins could be selectively separated under non-denaturing conditions from non-biotinylated proteins and biotin-containing carboxylases by avidin affinity chromatography and sulfhydryl-mediated elution. Subsequent to alkylation of the eluted protein, individual cell surface proteins could be isolated by immunoprecipitation as shown for a selected Mr 120,000 glycoprotein gp120 of the hepatocyte plasma membrane. Using this technique, a transit time of gp120 from the endoplasmic reticulum to the cell surface of 2 h was determined. The results show that the combination of labeling with a cleavable biotin derivative, non-denaturing avidin affinity chromatography and immunoprecipitation is a useful method to isolate and study individual cell surface proteins.     

A Model for Ulnar Dysmelia

The treatment of pregnant rats with the carbonic anhydrase inhibitor, acetazolamide, produced gross limb malformations primarily affecting the forepaw, but also producing variable ulnar dysmelia. Analysis of the cytoarchitecture of the ulnar dysmelic limbs showed the presence of cartilaginous and fibrocartilaginous connections between the ulnar and radial chondroepiphyses, with variable deformation of the radial chondroepiphysis by the tethering effect (although the growth plate, per se, did not appear affected at the stage of development studied). The extremely variable experimental appearances duplicated most of the variations seen in the human disease analogue, and suggest this drug-induced embryopathy may be useful as a model for the study of postaxial forelimb deformities in the postnatal phase in order to adequately assess the structural changes of disparate growth between radius and ulna due to the presence of the cellular continuity between the two distal chondroepiphyses.     

Analysis of the SIM1 Contribution to Polygenic Obesity in the French Population

SIM1 (single‐minded 1) haploinsufficiency is responsible for obesity in both humans and mice, but the contribution of frequent DNA variation to polygenic obesity is unknown. Sequencing of all exons, exon/intron boundaries, 870 base pairs (bp) of the putative promoter, and 1,095 bp of the 3′UTR of SIM1 gene in 143 obese children and 24 control adults identified 13 common variants. After analysis of the linkage disequilibrium (LD) structure, association study of eight variants was performed in 1,275 obese children and severely obese adults, in 1,395 control subjects, and in 578 obesity‐selected pedigrees. A nominal evidence of association was found for the nonsynonymous variant P352T C/A (rs3734354) (P = 0.01, OR = 0.81 (0.70–0.95)), the +2,004 TGA ?/insT SNP (rs35180395) (P = 0.02, OR = 1.21 (1.02–1.43)), the +2,215A/G TGA SNP (rs9386126) (P = 0.002, OR = 0.81 (0.71–0.93)), and pooled childhood/adult obesity. Even though transmission disequilibrium test (TDT) further supported the association of P352T and +2,004 ?/inst T with obesity, none of these nominal associations remained significant after a multiple testing Bonferroni correction. Therefore, our study excludes a major contribution of SIM1 common variants in exons, 5′ and 3′ UTR regions in polygenic obesity susceptibility in French Europeans.     

Decreased intramolecular turnover of L-fucose in membrane glycoproteins of rat liver during liver regeneration

In plasma membrane glycoproteins of rat liver L-fucose undergoes a rapid intramolecular turnover in that fucose residues are removed from the glycoproteins (Tauber, R., Park, C.S. & Reutter, W. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4026-4029). The present paper demonstrates that the intramolecular turnover of L-fucose is markedly decreased during liver regeneration. Turnover half-lives of L-fucose were measured in regenerating liver by pulse-chase experiments in five plasma membrane glycoproteins (Mr 60,000 (gp60), 80,000 (gp80), 120,000 (gp120), 140,000 (gp140), and 160,000 (gp160). The glycoproteins were isolated from plasma membranes by concanavalin A-Sepharose affinity chromatography and semipreparative NaDodSO4 polyacrylamide gel electrophoresis. L-Fucose turned over in the five glycoproteins with heterogeneous half-lives ranging from 22 h (gp160) to 49 h (gp120). The protein moieties of the glycoproteins were degraded with half-lives ranging from 56 h (gp80) to 107 h (gp140). Relative to the half-life of the protein backbone the half-live of L-fucose was increased in the five membrane glycoproteins by 70% (gp60), 150% (gp80), 182% (gp120), 60% (gp140) and 16% (gp160) during liver regeneration when compared to normal liver. The data show that L-fucose turns over in different membrane glycoproteins with individual rates, and that loss of L-fucose from plasma membrane glycoproteins is reduced in rapidly proliferating liver after partial hepatectomy.     

A simple and novel volumetric method to metre low gas flows from laboratory-scale bioreactors and its application on laboratory sludge digesters

A novel, cheap and easy to use method has been developed to measure low gas yields in bioreactors, based on the principle of bubble counting via digital imaging techniques and pattern recognition. No external hardware control is required for the measurements, and the device can be turned into a multichannel tool without further detector accessories. The method proved to perform outstandingly according to the testing and calibrating measurements against standard gas flow and revealed a short periodicity in the gas yields of two parallel laboratory-scale mesophilic biogas reactors providing well analysable data about them. For exact characterization of the oscillation, the data were converted into Morell wavelet spectra, which showed that every feeding period had a characteristic and similarly shaped wavelet profile, thereby this rhythm must have had an immanent source in the community metabolism, and it was not an artefact.