Gene transfection of the HuLy-m2 (Leu-9) antigen into mouse L cells

Human DNA was transfected into mouse L cells and tk⁺ HuLy-m2⁺ (= CD7⁺) transfectants isolated after growth in hypoxanthine, aminopterin, thymidine medium and repeated cloning. After several cycles of transfection, > 90% of HuLy-m2⁺ L cells could be detected, by rosetting and by cytofluorography, which showed the transfectants to have a density of CD7 two to five times that found on peripheral blood lymphocytes. Despite this, the 37 kd CD7⁺ dimer could only be identified with difficulty using cell-surface radioiodination and sodium dodecyl sulfate-polyacrylamide gel electrophoresis techniques. An antiserum was produced (C3H anti-HuLy-m2⁺ L cells) which, after absorption, was shown to react with HuLy-m2 antigens present on human thymocytes and lymphocytes and on CD7⁺ transfected L cells.Abbreviations BSA bovine serum albumin - DME Dulbecco's modified Eagle's medium - EDTA ethylenediamine-tetraacetate - HAT hypoxanthine, aminopterin, thymidine - HSV herpes simplex virus - PBL peripheral blood lymphocyte - PBS phosphate-buffered saline - RFC rosette-forming cell - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - tk thymidine kinase     

Characterization of the chicken oocyte receptor for low and very low density lipoproteins

The chicken oocyte receptor for low and very low density lipoproteins has been identified and characterized. Receptor activity present in octyl-beta-D-glucoside extracts of oocyte membranes was measured by a solid phase filtration assay, and the receptor was visualized by ligand blotting. The protein had an apparent Mr of 95,000 in sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions and exhibited high affinity for apolipoprotein B-containing lipoproteins, but not for high density lipoproteins or lipoproteins in which lysine residues had been reductively methylated. Binding of lipoproteins was sensitive to EDTA, suramin, and treatment with Pronase. In these aspects, the avian oocyte system was analogous to the mammalian low density lipoprotein receptor in somatic cells. Furthermore, a structural relationship between the mammalian and avian receptors was revealed by immunoblotting: polyclonal antibodies directed against the purified bovine low density lipoprotein receptor reacted selectively with the 95-kDa chicken receptor present in crude oocyte membrane extracts.     

Neuropeptide gene expression and neural activity: Assessing a working hypothesis in nucleus caudalis and dorsal horn neurons expressing preproenkephalin and preprodynorphin

1. The working hypothesis that neuropeptide gene expression in a neuron is an indicator of that neuron's physiological activity is discussed. 2. Representative examples from the literature are presented to support the hypothesis. 3. Further, we discuss the regulation of expression of two opioid peptides, preproenkephalin and preprodynorphin, in laminae I and II of the spinal cord and in nucleus caudalis of the trigeminal nuclear complex, where they may play a role in pain modulation. 4. The expression of the opioid peptide genes can be induced by both painful and nonnoxious stimuli in neurons in time-dependent and sensory-specific fashions.     

The thalamocortical contribution to epilepsy

The experimental literature has dealt intensively with the cortical contribution to epilepsy. Possibly because of the direction of technological advance, much less attention has been paid to the role of other structures. A model which emphasizes the role of some of those non-cortical structures, specifically that of thalamocortical modulation of cortical excitability, is developed. Some aspects of the petit mal seizure, a seizure type considered by some investigators to involve thalamocortical mechanisms, are predicted by the model. Although the thalamocortical mechanisms under study are not the only mechanism underlying seizures, a full understanding of the phenomenology of epilepsy needs to take into account the role of subcortical modification of cortical activities in addition to other mechanisms. Gloor has described two types of epileptogenesis: type I characteristic of non-convulsive seizure and type II characteristic of convulsions. There is disagreement as to whether or not the two mechanisms represent qualitatively different phenomena. Utilizing the thalamocortical model, it can be shown that the two types of epileptogenesis are qualitatively different. Furthermore, the thalamocortical model leads to a possible explanation of clinically different profiles of antipileptic efficacy of medications.     

Novel heme-binding component in the serum of the channel catfish (Ictalurus punctatus)

The serum of the channel catfish (Ictalurus punctatus) was examined for heme- and hemoglobin-binding proteins. Electrophoretic mobility retardation assays failed to detect a hemoglobin-binding material similar to mammalian haptoglobin; however, a heme-binding component (not previously described) was identified in catfish seru. The heme-binding component was purified by gel filtration chromatography; electrophoretic analyses suggested it to be composed of two polypeptide subunits of molecular masses about 115 and 98 kDa. This composition is inconsistent with hemopexin, the known heme-binding serum protein of mammals. Although it was not fully saturated with heme, the catfish component contained detectable heme in normal sera. When complexed by the binding material, heme was used as an iron source by isolates of the bacterial Gram-negative genusAeromonas; the capacity of other bacteria to use the complex was not tested. The physiological function of the catfish heme-binding serum protein is presently not clear.     

Optimization of growth conditions for the production of proteolytically-sensitive proteins in the periplasmic space of Escherichia coli

Summary The expression of many secreted recombinant proteins in Gram-negative bacteria is limited by degradation in the periplasmic space. We have previously shown that the production of protein A--lactamase, a secreted fusion protein highly sensitive to proteolysis in Escherichia coli, can be increased in mutant strains deficient in up to three cell-envelope-associated proteolytic activities. In this work we investigated the effect of fermentation conditions on suppressing any residual proteolytic activity in various protease-deficient strains. Optimal production of the fusion protein was observed in cells grown under mildly acidic conditions (5.5pH6.0) and at low temperatures. These conditios were shown to specifically decrease the rate of proteolysis. In addition, a further increase in production was observed in cultures supplemented with 0.5 to 0.75 mM zinc chloride. This may relate to the inhibition of a cell envelope protease by Zn²⁺ ions. Offsprint requests to: G. Georgiou