Volume-activated chloride permeability can mediate cell volume regulation in a mathematical model of a tight epithelium

Cell volume regulation during anisotonic challenge is investigated in a mathematical model of a tight epithelium. The epithelium is represented as compliant cellular and paracellular compartments bounded by mucosal and serosal bathing media. Model variables include the concentrations of Na, K, and Cl, hydrostatic pressure, and electrical potential, and the mass conservation equations have been formulated for both steady-state and time-dependent problems. Ionic conductance is represented by the Goldman constant field equation (Civan, M.M., and R.J. Bookman. 1982. Journal of Membrane Biology. 65:63-80). A basolateral cotransporter of Na, K, and Cl with 1:1:2 stoichiometry (Geck, P., and E. Heinz. 1980. Annals of the New York Academy of Sciences. 341:57-62.) and volume-activated basolateral ion permeabilities are incorporated in the model. MacRobbie and Ussing (1961. Acta Physiologica Scandinavica. 53:348-365.) reported that the cells of frog skin exhibit osmotic swelling followed by a volume regulatory decrease (VRD) when the serosal bath is diluted to half the initial osmolality. Similar regulation is achieved in the model epithelium when both a basolateral cotransporter and a volume-activated Cl permeation path are included. The observed transepithelial potential changes could only be simulated by allowing volume activation of the basolateral K permeation path. The fractional VRD, or shrinkage as percent of initial swelling, is examined as a function of the hypotonic challenge. The fractional VRD increases with increasing osmotic challenge, but eventually declines under the most severe circumstances. This analysis demonstrates that the VRD response depends on the presence of adequate intracellular chloride stores and the volume sensitivity of the chloride channel.     

Detection of an unbalanced translocation (4;14) in a mildly retarded father and son by flow cytometry

Summary A child with impaired intelligence, minor dysmorphisms, obesity and genital hypoplasia was found to have an apparently balanced translocation, 46,XY,t(4;14)(q12;q13), following cytogenetic analysis. The same rearrangement was also detected in the child's father, who had similar phenotypic abnormalities to his son. Detailed study of flow karyotypes produced from lymphoblastoid cell lines established that in both patients the translocation was in fact unbalanced with approximately 11 million base pairs of DNA (corresponding to about 6.0% of chromosome 4 or 11.0% of chromosome 14) being lost.     

Cell-specific fatty acylation of proteins in cultured cells of neuronal and glial origin

Distinct sets of cellular proteins were labeled with [³H]myristic and [³H]palmitic acids in primary (rat neurons and astroglia) and continuous (murine N1E-115 neuroblastoma and rat C6 glioma) cell cultures derived from the nervous system. Both soluble and membrane proteins were modified by myristate in a hydroxylamine-stable (amide) linkage, while palmitoylated proteins were esterlinked and almost exclusively membrane bound. Chain elongation of both labeled fatty acids prior to acylation was observed, but no protein amide-liked [³H]myristate originating from [³H]palmitate was detected. Fatty acylation profiles differed considerably among most of the cell lines, except for rat astroglial and glioma cells in which myristoylated proteins appeared to be almost identical based on SDS gel electrophoresis. An unidentified 47 kDa myristoylated protein was labeled to a significantly greater extent in astroglial than in glioma cells; the expression of this protein could be related to transformation or development in cells of glial origin.     

Quantification of cytochrome P450 reductase gene expression in human tissues.

We have isolated and sequenced cDNA clones that code for a variant of human cytochrome P450 reductase. An RNase protection assay was used to quantify the corresponding mRNA in adult and fetal tissues. The results demonstrate that, in the samples analyzed, the cytochrome P450 reductase gene displays very little inter-individual variation in its expression in adult liver and is subject to little developmental or tissue-specific regulation.     

Glucocorticoids do not affect the induction of a novel calcium-dependent nitric oxide synthase in rabbit chondrocytes.

Incubation of rabbit articular chondrocytes with interleukin-1 beta caused time-dependent expression of NO synthase, determined as nitrite, after a lag period of 6h. The synthesis of nitrite was concentration-dependent and was inhibited by cycloheximide and NG-monomethyl-L-arginine, but not by dexamethasone or hydrocortisone. The synthesis of NO in the 100,000g supernatant of activated chondrocytes was inhibited by EGTA, but not by the calmodulin inhibitors W-13 or trifluoperazine. The synthesis of NO was half-maximal at approximately 20nM free Ca2+. Endotoxin also induced the expression of this NO synthase. Thus, rabbit articular chondrocytes express a novel inducible NO synthase which is Ca(2+)-dependent, and whose induction is not prevented by glucocorticoids.     

Transfer RNA genes from the hyperthermophilic Archaeon, Methanopyrus kandleri.

Genes encoding the Leu (GAG), Ser (UGA), Gln (UUG) and Lys (UUU) tRNAs have been cloned and sequenced from the deep sea hyperthermophilic Archaeon, Methanopyrus kandleri. Sequences conforming to the TATA box element established for methanogen promoters are located upstream of the tRNA(Gln) and tRNA(Lys) genes. All four of the tRNA genes appear to encode the 3' terminal CCA residues of the mature tRNA. These methanogen tRNAs are predicted to contain most, but not all, invariant residues and are characterized by a high level of G + C base pairing, consistent with the 98 degrees C optimum growth temperature of M. kandleri.     

Polyunsaturated fatty acid incorporation into plasmalogens in plasma membrane of glioma cells is preceded temporally by acylation in microsomes.

Plasmalogens (1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) are major phospholipids in many tissues and cells, particularly of neural origin. Using cultured C6 glioma cells and subcellular fractions isolated on Percoll gradients we investigated selectivity for esterification of several polyunsaturated fatty acids (PUFA) in the sn-2 position of plasmalogens compared to [1-14C]hexadecanol, representative of de novo synthesis of the ether-linked sn-1 position. In whole cells at a final concentration of 105 microM PUFA, 2-4 nmol plasmalogen/mg protein was labeled in 4 h and 10-14 nmol in 24 h, representing 8-15% and 35-50%, respectively, of initial plasmalogen mass. Incorporation of label from hexadecanol was lower than PUFA incorporation (20:5(n-3) greater than 20:4(n-6) greater than 18:3(n-3) much greater than 18:2(n-6)) suggesting deacylation-reacylation at the sn-2 position. Plasmalogens accounted for 50% of total cell ethanolamine phospholipids and 75% in plasma membrane. Using a novel, improved method for extraction of subcellular fractions containing Percoll, plasma membrane also was enriched in plasmalogen relative to microsomes (107.4 +/- 5.2 vs. 40.0 +/- 2.9 nmol/mg protein). Selectivity for esterification at the sn-2 position of plasmalogens with respect to chain length and unsaturation of the fatty acyl chain was similar in both subcellular fractions and reflected that of whole cells. Labeling of plasma membrane with PUFA and fatty alcohol lagged behind that of microsomes. Chase experiments in cells prelabeled with [1-14C]18:3(n-3) for 2 h showed no significant reduction of label in plasmalogen of any subcellular fraction although accumulation of label in the microsomal fraction was slowed initially. Reduction of plasmalogen label (40-50%) did occur in microsomes and plasma membrane when cells prelabeled for 24 h were switched to chase medium with or without chase fatty acid. Our data suggest that esterification of PUFA to plasmalogen may occur at the endoplasmic reticulum with subsequent translocation to plasma membrane resulting in accumulation of relatively stable pools of plasmalogen that are not readily accessible for deacylation-reacylation exchange with newly appearing PUFA. Alternatively, deacylation-reacylation may occur in a more stable phospholipid pool within the plasma membrane but would involve a slower process than at the endoplasmic reticulum.     

The sequence of the major protein stored in ovine ceroid lipofuscinosis is identical with that of the dicyclohexylcarbodiimide-reactive proteolipid of mitochondrial ATP synthase.

The ceroid lipofuscinoses are a group of neurodegenerative lysosomal storage diseases of children and animals that are recessively inherited. In diseased individuals fluorescent storage bodies accumulate in a wide variety of cells, including neurons. Previous studies of these bodies isolated from tissues of affected sheep confirmed that the storage occurs in lysosomes, and showed that the storage body is mostly made of a single protein with an apparent molecular mass of 3500 Da with an N-terminal amino acid sequence that is the same as residues 1-40 of the c-subunit (or dicyclohexylcarbodi-imide-reactive proteolipid) of mitochondrial ATP synthase. In the present work we have shown by direct analysis that the stored protein is identical in sequence with the entire c-subunit of mitochondrial ATP synthase, a very hydrophobic protein of 75 amino acid residues. As far as can be detected by the Edman degradation, the stored protein appears not to have been subject to any post-translational modification other than the correct removal of the mitochondrial import sequences that have been shown in other experiments to be present at the N-terminal of its two different precursors. No other protein accumulates in the storage bodies to any significant extent. Taken with studies of the cDNAs for the c-subunit in normal and diseased sheep, these results indicate that the material that is stored in lysosomes of diseased animals has probably entered mitochondria and has been subjected to the proteolytic processing that is associated with mitochondrial import. This implies that the defect that leads to the lysosomal accumulation concerns the degradative pathway of the c-subunit of ATP synthase. An alternative, but less likely, hypothesis is that for some unknown reason the precursors of subunit c are being directly mis-targeted to lysosomes, where they become processed to yield a protein identical with the protein that is normally found in the mitochondrial ATP synthase assembly, and which then accumulates.     

Small-scale environmental heterogeneity and the analysis of species distributions along gradients

Abstract. The observed distribution of a species along an environmental gradient is strongly affected by environmental variability within a quadrat. Because a quadrat does not represent a point along an environmental gradient, but rather a range of conditions, it is likely to contain species not typically associated with the mean conditions in the quadrat. Systematic relationships exist between a species' true distribution, the observed distribution as a function of mean quadrat environment, and the frequency distribution of the environment within that quadrat. The observed species habitat breadth increases and the observed maximum abundance decreases as within-quadrat environmental heterogeneity increases. If species distributions or beta diversities are to be compared among species or coenoclines, they should be correctedforintra-quadratheterogeneity.Wederive simple corrections for environmental heterogeneity. The distributions of hardwood forest understory species along a soil acidity gradient in the North Carolina piedmont are presented as an example.     

Lipase regulation of muscle triglyceride hydrolysis

The cellular control of intramuscular triglyceride (TG) metabolism involves two major identified lipases: hormone-sensitive lipase (HSL) and lipoprotein lipase (LPL). Recently, the presence of HSL in muscle has been unequivocally demonstrated. However, although it is thought that HSL is responsible for intramuscular TG lipolysis, direct evidence for this is lacking. There is evidence to suggest that HSL and LPL are simultaneously activated under a variety of conditions. The two muscle lipases appear to be turned on by the same signal and function as a coordinated unit in meeting the energy demands of muscle. At a time when HSL is presumably hydrolyzing endogenous TG, LPL is sent to the capillary beds in search of substrate. TG uptake from circulation is highly related to muscle LPL activity. Exercise training increases LPL activity in plasma and in parenchymal cells in muscle. These results suggest that training may increase the capacity to clear TG from circulation and that LPL might have a role in replenishing muscle TG stores that have been decreased with exercise.