Actin and associated proteins in gastric epithelial cells

A quantitative assessment of the distribution and state of microfilament-related proteins in the heterocellular fundic gastric epithelium was carried out. Actin content, as determined by the DNAase inhibition assay, ranged from 29 to 42 micrograms/mg of tissue protein, depending upon the tissue source. About 60% of the total actin existed in fresh tissue in the polymeric form (F-actin). The distribution of fluorescent-labelled phallicidin demonstrated that F-actin was concentrated predominantly in the acid-secreting oxyntic cells. The patterns of distribution corresponded to the location of the numerous elongated apical surface microvilli seen within oxyntic cell canaliculi. In the isolated apical membrane, actin represented about 10% of the total protein and was present entirely as F-actin. After mild treatment of membranes with Triton X-100, filaments could be readily visualized by negative staining. More extensive Triton X-100 extraction solubilized intrinsic membrane protein and yielded an insoluble residue highly enriched in actin and containing several additional polypeptides. Homogenization and fractionation of the gastric epithelium in low ionic strength media led to the depolymerization of a significant proportion of the tissue actin which was recovered in the homogenate supernatant. When purified by DNAase affinity chromatography, this gastric actin displayed structural and functional properties similar to muscle actin. Incubation of the homogenate supernatant in KCl-Mg2+ induced the formation of actin-rich gels. The gels contained myosin as well as several other peptides that may be actin-binding proteins.     

Interaction of human plasma high-density lipoprotein HDL2b with discoidal complexes of dimyristoylphosphatidylcholine and apolipoprotein A-I

The interaction of HDL2b, a major subclass (d = 1.063 - 1.100 g/ml) of human plasma high-density lipoproteins, with discoidal complexes composed of dimyristoylphosphatidylcholine (DMPC) and apolipoprotein A-I (weight ratio, DMPC/apolipoprotein A-I (2.1 - 2.5:1); dimensions, 10.0 x 4.4 nm) was investigated. Incubation at 37 degrees C for 4.5 h of HDL2b with discoidal complexes resulted in a transfer of DMPC from the discoidal complexes to the HDL2b, a release of lipid-free apolipoprotein A-I from the discoidal complexes during such transfer, and a dissociation of some apolipoprotein A-I from the HDL2b surface. The number of discoidal complexes degraded during interaction with HDL2b depended on the initial molar ratio of HDL2b to discoidal complexes. Approximately one molecule of HDL2b was required for the degradation of one discoidal complex particle, and the degradation process appeared limited by the capacity of the HDL2b for uptake of DMPC. Degradation of discoidal complexes was also observed when human plasma LDL (d = 1.006-1.063 g/ml) was substituted for HDL2b in the interaction mixture.     

Reduction of Brain Calcium After Consumption of Diets Deficient in Calcium or Vitamin D

Abstract: Rats fed diets deficient in calcium or vitamin D for 4 weeks displayed hypocalcemia, as indicated by a 50% reduction in serum calcium and a sevenfold elevation of serum parathyroid hormone. These treatments also decreased the calcium content of brain tissue. On a regional basis. this effect was greatest in the brain stem (24% decrease) and least in striatum (10% decrease). Subcellular analysis indicated that the depletion of brain calcium was greatest in the soluble and the microsomal fractions. Infusion of calcium solutions reversed the depletion of brain calcium produced by dietary deficiencies. In control rats. parathyroidectomy or infusion of parathyroid hormone did not alter the calcium content of brain tissue, although these treatments affected the levels of calcium in the serum. In general, these treatments had no effect on the magnesium content of serum or brain tissue. However, vitamin D deficiency did increase the magnesium content of the myelin and synaptosomal fractions. This increase was reversed by parathyroidectomy. These observations demonstrate that long-term hypocalcemia produces distinct changes in the localization of calcium and magnesium in brain tissue. Furthermore. these studies suggest that though brain calcium levels are influenced by serum concentrations, serum changes must be of large magnitude and long duration for brain calcium levels to be affected.     

K+-stimulated phosphatase of microsomes from gastric mucosa

The light microsomal fraction was isolated from homogenates of rabbit and bullfrog gastric mucosa. On examination with the electron microscope, the light microsomes appear as tubular membranous structures with morphology and dimensions similar to the elements of the smooth-surfaced endoplasmic reticulum seen in intact oxyntic cells. A K⁺-stimulated, Mg⁺⁺-requiring p-nitrophenylphosphatase has been demonstrated in the gastric microsomes. Neither Na⁺ nor ouabain (10^?6–10^?3 M) altered the K⁺-stimulated phosphatase. SCN^? was not very effective as an inhibitor of the gastric microsomal phosphatase, in contrast to the effect of this anion on the ATPase activity; however, the gastric phosphatase as well as the ATPase are destroyed by phospholipase C, thus showing the lipoprotein nature of these enzymes. Kinetics of the K⁺ activation of the microsomal phosphatase suggest that the K⁺-PNPP complex is the active substrate for the enzymic reaction. Rb⁺, NH₄ ⁺ and Cs⁺ will substitute to some degree for K⁺ as an activator of the microsomal phosphatase. It is pointed out that K⁺ is an essential requirement for HCl secretion in intact gastric mucosa and the replacement of K⁺ with Rb⁺, Cs⁺ and NH₄⁺ is discussed. The K⁺-stimulated phosphatase presented in this paper may play a role in the H⁺ secretion process.     

Transformation of large discoidal complexes of apolipoprotein A-I and phosphatidylcholine by lecithin-cholesterol acyltransferase

Using a cholate-dialysis recombination procedure, complexes of apolipoprotein A-I and synthetic phosphatidylcholine (1-palmitoyl-2-oleoylphosphatidylcholine (POPC) or dioleoylphosphatidylcholine (DOPC] were prepared in mixtures at a relatively high molar ratio of 150:1 phosphatidylcholine/apolipoprotein A-I. Particle size distribution analysis by gradient gel electrophoresis of the recombinant mixtures indicated the presence of a series of discrete complexes that included species migrating at RF values observed for discoidal particles in nascent high-density lipoproteins (HDL) in plasma of lecithin-cholesterol acyltransferase-deficient subjects. One of these complex species, designated complex class 6, formed with either phosphatidylcholine, was isolated by gel filtration and characterized at follows: discoidal shape (mean diameter 20.8 nm (POPC) and 19.0 nm (DOPC]; molar ratio, phosphatidylcholine/apolipoprotein A-I, 155:1 (POPC) and 130:1 (DOPC); and both containing 4 molecules of apolipoprotein A-I per particle. Incubation of class 6 complexes with lecithin-cholesterol acyltransferase (EC 2.3.1.43) and a source of unesterified cholesterol (low-density lipoprotein (LDL] was shown by electron microscopy to result in a progressive transformation of the discoidal particles (0 h) to deformable (2.5 h) and to spherical particles (24 h). The spherical particles (diameter 13.6 nm (POPC) and 12.5 nm (DOPC) exhibit sizes at the upper boundary of the interval defining the human plasma (HDL2b)gge (12.9-9.8 nm). The spherical particles contain a cholesteryl ester core that reaches a limiting molar ratio of approx. 50-55:1 cholesteryl ester/apolipoprotein A-I. The deformable particles assume a rectangular shape under negative staining and, relative to the 24-h spherical product, are enriched in phosphatidylcholine. Chemical crosslinking (by dimethyl suberimidate) of the isolated transformation products shows the 24-h spherical particle to contain predominantly 4 apolipoprotein A-I molecules; products produced after intermediate periods of time appear to contain species with 3 and 4 apolipoproteins per particle. Our in vitro studies indicate a potential pathway in the origins of large, apolipoprotein A-I-containing plasma HDL particles. The deformable species observed during transformation were similar in size and shape to particles observed in interstitial fluid.     

Carnitine and acetylcarnitine in red blood cells

Carnitine and acetylcarnitine were found to be present in human erythrocytes. Their presence was not as a factor of leucocyte contamination. Carnitine is present within the erythrocyte at a level comparable to that of the plasma, whilst acetylcarnitine is more concentrated within the cell. Red blood cell carnitine and acetylcarnitine do not freely exchange with plasma but intra-erythrocyte acetylcarnitine has a significant relationship to the plasma levels.     

Early- and intermediate-stage variants of simian immunodeficiency virus replicate efficiently in cells lacking CCR5

Primate lentiviruses are thought to use the chemokine receptor CCR5 as the major coreceptor for entry into cells. Here we show that some variants of simian immunodeficiency virus (SIV) replicate efficiently in peripheral blood mononuclear cells (PBMCs) lacking a functional CCR5. There were differences in the replication patterns of sequential variants that evolved during SIVMne infection; the late-stage pathogenic variants were unable to replicate in PBMCs lacking CCR5, whereas the early- and intermediate-stage viruses replicated as well in PBMCs lacking CCR5 as they did in cells with wild-type CCR5. The coreceptor specificities of these sequential variants were compared using indicator cell lines expressing known SIV coreceptors. Among the known SIV coreceptors, there were none that were functional for the early and intermediate variants but not the late-stage variants, suggesting that the coreceptor used for replication in PBMCs may be a coreceptor that has not yet been described. Because some variants replicate with high efficiency in peripheral blood cells using this as yet uncharacterized cellular receptor, this coreceptor may be important for viral entry of some target cell populations in the host.