Molecular properties of a stratum corneum model lipid system: large unilamellar vesicles.

Stratum corneum lipids are relatively complex, and there is little detailed understanding of their chemical and physical properties at the molecular level. Large unilamellar vesicles (LUVs) with lipid compositions similar to those of stratum corneum were prepared at pH 9 with commercially available lipids. This system was used as a model system for molecular studies of stratum corneum lipids. LUVs were chosen as the model system as they are comparatively more stable and can be characterized more quantitatively in terms of lipid concentration, surface area, and volume than model systems such as lipid mixture suspensions, lipid films, and small unilamellar vesicles. Results from freeze-fracture and cryo electron microscopy studies of our LUVs showed spherical vesicles. Quasi-elastic light scattering measurements revealed a narrow size distribution, centering around 119 nm. At room temperature, the LUVs were stable for several weeks at pH 9 and for more than 15 h but less than 24 h at pH 6. Differential scanning calorimetry measurements indicated broad endothermic transitions centered near 60-65 degrees C, closely matching the transition temperature reported for stratum corneum lipid extracts. Spin probes, 5-doxylstearic acid and 12-doxylstearic acid, were used for electron paramagnetic resonance (EPR) studies of the molecular dynamics of the lipids. EPR results indicated more restricted motion near the polar headgroup region than near the center of the alkyl chain region. Motional profiles of the spin labels near the polar headgroup and within the alkyl chain region in the LUVs were obtained as a function of temperature, ranging from 25 to 90 degrees C. We also found that the partitioning between the lipid and aqueous phases for each spin probe was temperature dependent and was generally correlated with phase transitions observed by differential scanning calorimetry and with alkyl chain mobility observed by EPR. Thus, this LUV system is well suited for additional molecular studies under different experimental conditions.     

Mutations in the X-linked E1 alpha subunit of pyruvate dehydrogenase: exon skipping, insertion of duplicate sequence, and missense mutations leading to the deficiency of the pyruvate dehydrogenase complex.

Human pyruvate dehydrogenase (PDH)-complex deficiency is an inborn error of metabolism that is extremely heterogeneous in its presentation and clinical course. In a study of 14 patients (7 females and 7 males), we have found a mutation in the coding region of the E1 alpha gene in all 14 patients. Two female patients had the same 7-bp deletion at nt 927; another female patient had a 3-bp deletion at nt 931. Another female patient was found to have a deletion of exon 6 in her cDNA. Two other female patients were found to have insertions, one of 13 bp at nt 981 and one of 46 bp at nucleotide 1078. Two male patients were found to have a 4-bp insertion at nucleotide 1163. The remaining six patients all had missense mutations. A male patient and a female patient both had an A1133G mutation. The other missense mutations were C214T, C615A, and C787G (two patients). Five of these mutations are novel mutations, five have been previously reported in other patients, and two were published observations in other patients in an E1 alpha-mutation summary. In the four cases where parent DNA was available, only one mother was found to be a carrier of the same mutation as her child.     

Bacterial organisms suitable for filamentous cell immobilization

Summary Several bacterial strains commonly used in biotechnology have been studied for induced filamentous growth at a wide range of sublethal 18-crown-6 concentrations. Only the rod shape bacteriaEscheriehia coli, Salmonella typhimurium andBacillus sp exhibit filamentous morphology, irrespective of whether the organism is gram stain positive or negative.     

Effect of temperature on the biaxial mechanics of excised lung parenchyma of the dog.

The influence of temperature on the mechanical properties of excised saline-filled lung parenchyma of the dog was studied at low lung volume. The motivation of this study was to determine whether lung tissue material without the influence of surface tension undergoes a phase transition in the 20-40 degrees C range, as does synthetic elastin studied by Urry in 1984-1986. Dynamic biaxial and uniaxial tensile tests were done, and strain vs. Lagrangian stress curves were recorded during slow cooling and heating between 40 and 10 degrees C. To emphasize the effects of elastin, strains (defined as stretch ratio minus one) were kept below 30%. A slight decrease in compliance occurred with cooling over the entire temperature range. This effect may be attributed to collagen. It was accompanied by a gradual increase in length as the tissue cooled, an effect that may be attributed to elastin. This process was partially reversible with reheating. However, this effect is in contrast with the sudden drastic change in mechanical properties of synthetic elastin described by Urry. Hysteresis, creep, and stress relaxation were small at these low strains. Possible causes of these effects are discussed.     

Interleukin 2-induced tyrosine phosphorylation. Interleukin 2 receptor beta is tyrosine phosphorylated

Interaction of interleukin 2 (IL2) with its high affinity membrane receptor complex (IL2R) is sufficient to induce proliferation of T lymphocytes. However, the biochemical mechanisms by which IL2 induces this process remain unresolved. The IL2R complex consists of at least two distinct polypeptides that bind IL2, a 75-kDa intermediate affinity subunit (IL2R beta) and a 55-kDa low affinity subunit (IL2R alpha). As indicated by Western blotting with anti-phosphotyrosine-specific antibodies and confirmed by phosphoamino acid analysis, we now demonstrate that interaction of the T cell growth factor interleukin 2 (IL2) with its high affinity receptor on IL2-sensitive human peripheral blood lymphoblasts induces tyrosine phosphorylation of proteins of 92, 80, 78, 70-75, and 57 kDa. IL2 induced tyrosine phosphorylation in YT 2C2 cells which express only the 75-kDa intermediate affinity IL2 binding molecule (IL2R beta) but not in cells which either express only the 55-kDa low affinity IL2 receptor molecule (IL2R alpha) or no IL2-binding sites. Therefore, IL2R beta, in the absence of IL2R alpha, appears sufficient to transduce the transmembrane signal leading to tyrosine phosphorylation. Two different antibodies reactive with phosphotyrosine specifically immunoprecipitated IL2R beta cross-linked to radiolabeled IL2. These findings suggest that IL2R beta is a substrate for the tyrosine kinase which is activated by IL2 binding to its receptor. Thus, like several other growth factor receptors, activation of the IL2R results in an increase in tyrosine phosphorylation with the receptor itself serving as one substrate.     

Concanavalin A Receptors Associated with Rat Brain Synaptic Junctions Are High Mannose-Type Oligosaccharides

Abstract: Glycoproteins were isolated from a rat brain synaptic junction fraction by affinity chromatography on Concanavalin A-agarose. The isolated glycoproteins were digested with pronase and radiolabeled with ¹²⁵I-Bolton Hunter reagent, and ¹²⁵I-Concanavalin A-binding glycopeptides were isolated by chromatography on Concanavalin A-agarose. Treatment of the ¹²⁵I-Concanavalin A-binding glycopeptides with either α-mannosidase or endo-β- N -acetylglucosaminidase-C₁₁ abolished their interaction with Concanavalin A. The pronase digest was reacted with endo-β-N-acetylglucosaminidase-C₁₁ and released oligosaccharides were reduced with NaB³H₄. Following affinity chromatography on Concanavalin A-agarose, Concanavalin A-binding [³H]oligosaccharides were chromatographed on Biogel P₄. Two major oligosaccharides corresponding to standard carbohydrates containing eight and five mannose residues were identified. Treatment of these oligosaccharides with α-mannosidase converted them to smaller saccharides having a mobility on Biogel P₄ columns equal to the standard disaccharide mannose-β-1-4- N '-acetylglucosamine. These results demonstrate that the Concanavalin A receptor activity associated with CNS synaptic junctions resides in asparaginelinked oligosaccharides of the high-mannose type.     

Effect of glucose on protein phosphorylation in rat pancreatic islets

Isolated rat pancreatic islets, incubated in the presence of extracellular ³²Pi to steady state ³²P incorporation into cellular phosphopeptides, were exposed to glucose for 10 min. Glucose (16.7 mM) significantly stimulated the phosphorylation of six phosphoproteins with molecular weights of 15,000, 35,000, 49,000, 64,000, 93,000 and 138,000. Mannoheptulose (16.7 mM) markedly inhibited glucose-stimulated phosphorylation of these six phosphoproteins. This protein phosphorylation might be important in mediating glucose-stimulated insulin release.     

Spin-label studies of the lipid and protein components of erythrocyte membranes. A comparison of electron paramagnetic resonance and saturation transfer electron paramagnetic resonance methods.

We have used both a protein spin label and a lipid spin probe to study some of the slow motions of proteins and of lipids, respectively, in intact erythrocyte membranes. Three electron paramagnetic resonance (EPR) methods, conventional (V1) EPR, second harmonic out-of-phase absorption saturation transfer (ST) EPR (V'2), and first harmonic out-of-phase dispersion ST EPR (U'1) were used to compare the experimental methods and spectral sensitivities with different kinds of molecular motions in human erythrocyte membranes under different experimental conditions. The results show that the V'2 display is relatively more sensitive to the protein motion, while the U'1 display appears more sensitive to the lipid motions, and the V'2 display is substantially more convenient to obtain than the U'1 display.     

Efflux of beta-galactosidase products from Escherichia coli.

Several different strains of Escherichia coli were grown on a variety of carbon sources under various growth conditions. Lactose was added (usually at mid-log phase), and the concentrations of the products of beta-galactosidase action on this sugar (galactose, glucose, and allolactose) were determined at various times thereafter in the total culture and in the medium. It was found that with each strain, with all carbon sources, and under all of the conditions studied, a very large proportion of the products were found in the medium. Control studies were carried out which showed that these results were not artifacts of the method of separating the cells from the medium. The results also did not arise from the secretion of beta-galactosidase into the medium, from the diffusion of substrates and products into and out of the cells due to leaks in the membrane, or from faults in the method of sugar analysis. In addition, the results showed that there were very high levels of products inside the cells under the conditions used and that the efflux of the products was rapid. The efflux might be energetically advantageous to the cell as well as being a means of storing excess products until needed.