Dexamethasone increases neutral sphingomyelinase activity and sphingosine levels in 3T3-L1 fibroblasts

The activity of neutral sphingomyelinase (EC 3.1.4.12) in a plasma membrane enriched fraction was found to be increased in dexamethasone treated cells. The elevation of sphingomyelinase activity was blocked by cycloheximide indicating that protein synthesis was required for the steroid action. Ceramidase (EC3.5.1.23) activity was unaffected by the dexamethasone treatment. Levels of sphingosine in 3T3-L1 Cells were also increased after treatment with 10(-7) M dexamethasone for 2 and 4 hours.     

Studies on the stimulation of dolichol-mediated glycosylation by dexamethasone in HeLa cells.

The effect of the addition of 1 microM-dexamethasone on the temporal sequence of the glycosylation process has been studied in HeLa S3G cells. In the presence of delipidized serum, dexamethasone caused an increase in 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and an accelerated synthesis of dolichols. These events were followed by an increase in the transfer of mannose from GDP-mannose to mannolipid. An increase in the rate of synthesis of lipid-linked oligosaccharides and a stimulation of glycosylation were observed in cells grown in the presence of delipidized serum in the culture medium. The data are consistent with the view that cellular syntheses of lipids and glycoproteins are co-ordinately controlled.     

Primary culture of normal rat adrenocortical cells. I. Culture conditions for optimal growth and function

Rat adrenocortical cells retained their differentiated characteristics over 2 wk in culture without a specific requirement for additives other than inorganic salts, amino acids, vitamins, and fetal bovine serum. The cells were maintained free from fibroblast overgrowth by substitution of D-value in place of L-valine in the medium. Corticotropin (ACTH) inhibited the growth of adrenocortical cells in this medium and the effect was reversible. The adrenocortical cells had a limited capacity for growth as reflected by total cell counts and [3H]thymidine uptake with cells from young animals demonstrated a greater potential for DNA synthesis than cells obtained from mature animals. A very sensitive assay for ACTH using a small number of cells in primary culture also is described.     

Coordinate repression of cholesterol biosynthesis and cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase by glucocorticoids in HeLa cells.

The stability constants for the calcium and magnesium complexes of rhodanese are >10⁵m^?1 at both high and low substrate concentrations. The stoichiometry of alkaline earth metal ion binding totals close to 1 per 18,500 molecular weight. The usual assay reagents contain sufficient amounts of these metal ions to maintain added enzyme in its metal-complexed form. When reaction mixtures are treated with oxalate to remove calcium ions, inhibition of rhodanese activity is virtually complete under circumstances such that the contribution of magnesium ion is low.Zinc and a number of transition metal ions are inhibitors of rhodanese activity. Studies of the concentration dependence of these effects with zinc, copper, and nickel showed that: 1) Some cyanide complexes of these metals are competitive with the donor substrate, thiosulfate ion. The binding of the copper and zinc complexes is mutually competitive. 2) Another cyanide species of copper appears to combine with the free enzyme to form a functionally active complex. 3) The zinc cyanide species with a net positive charge is an inhibitor competitive with the acceptor substrate, cyanide ion.All of these observations are consistent with a model in which metal ions serve as the electrophilic site of rhodanese.     

Comparison of the steroidogenic and melanotropic activities of corticotropin, alpha-melanotropin and analogs with their lipolytic activities in rat and rabbit adipocytes.

The ability of alpha-melanotropin and a series of synthetic peptides related to adrenocorticotropin (ACTH) to stimulate steroidogenesis in isolated rat adrenal cells, melanin dispersion in frog melanophores and lipolysis in rat and rabbit fat cells have been studied. It was found that the steroidogenic activity closely paralleled the lipolytic activity of these peptides in rat fat cells, whereas the melanocyte stimulating activity paralleled the lipolytic activity in rabbit fat cells. These results indicate that the structural requirements for stimulating steroidogenesis in isolated rat adrenal cells and lipolysis in isolated rat fat cells are quite similar. The structural features required for eliciting lipolysis in rabbit fat cells appear to be very similar to those necessary for stimulating frog melanophores. The possibility that regulation of lipid metabolism in the rabbit may be a new function acquired by melanotropin is discussed.     

Cytochrome oxidase and ascorbic acid in the normal and regenerating tail of the scincid lizard, Mabuya carinata. A histophysiological study.

The concentration of ascorbic acid (AA) and the histochemical distribution of this vitamin together with cytochrome oxidase have been investigated in the normal and regenerating tail of the Scincid lizard, Mabuya carinata. An interesting aspect of this investigation is the observation of a total lack of cytochrome oxidase in both the normal and regenerating tail of the lizard, except for the differentiating phase. On the other hand, AA has been found to be present in the normal and regenerating tail with above normal levels during wound healing (twofold) and differentiation (fivefold). In the light of the poor cytochrome oxidase activity, the higher content of AA noted during regeneration has been construed to play a possible role in the respiratory mechanics of the regenerating lizard tail. Further, the importance of AA in cellular metabolism and the wound healing and differentiative processes have also been discussed.     

The structural and functional interrelationships of muscarinic acetylcholine receptor subtypes

Molecular cloning of the genes encoding the muscarinic acetylcholine receptors has shown that receptor subtypes classified on the basis of pharmacological properties are related polypeptides encoded by distinct genes. These studies have also revealed the existence of novel muscarinic receptor subtypes. Functional analysis of each of the subtypes expressed in mammalian cells indicates that the different subtypes activate distinct biochemical pathways, a finding that explains the tissue-specific physiological response elicited by the neurotransmitter, acetylcholine.     

Functionally distinct G proteins selectively couple different receptors to PI hydrolysis in the same cell

The number of G proteins identified by molecular cloning exceeds the number of known G protein functions. Here we show that a cell can possess multiple G proteins that carry out a similar function, the activation of phospholipase C, but couple selectively to different receptors, which are endogenous to the cell or introduced by DNA transfection. These G proteins (termed Gp) can be distinguished by their sensitivity to pertussis toxin. The assignment of a given Gp pathway to specific receptors is confirmed by the additivity relationships of the PI hydrolysis response mediated by the different receptors. Significantly different amounts of PI hydrolysis are activated through each Gp pathway, suggesting that Gp proteins also differ in their coupling to phospholipase C. These results indicate that distinct Gp pathways in a given cell exist to couple different receptors to PI hydrolysis selectively, and may specify the nature of the cellular response to different receptors by determining the magnitude of PI hydrolysis.     

Target discrimination by surface-immobilized molecular beacons designed to detect Francisella tularensis

A molecular beacon (MB) array was designed based on unique regions of the 16S rRNA of the bacterium Francisella tularensis. Nucleic acid molecular beacons undergo a spontaneous fluorogenic conformational change when they hybridize to specific complementary targets. The array was printed on aldehyde glass or hydrogel slides and evaluated for functioning in presence of complementary oligonucleotide sequences, single-nucleotide mismatch sequences and multiple nucleotide mismatch sequences. Discriminating true target from mismatched targets was found to be dependent on type, number, and location of mismatches within the beacon (i.e. located in the stem or loop regions). Optimal conditions for molecular beacon deposition, and target hybridization were determined for oligonucleotide target mismatch discrimination. The beacon array was stable upon recharging by exposure to an alkaline solution, and repeatedly used. In addition, performance of the beacon array biosensor was compared with molecular beacons in homogeneous solution.     

Heat shock factor 2 is required for maintaining proteostasis against febrile-range thermal stress and polyglutamine aggregation

Heat shock response is characterized by the induction of heat shock proteins (HSPs), which facilitate protein folding, and non-HSP proteins with diverse functions, including protein degradation, and is regulated by heat shock factors (HSFs). HSF1 is a master regulator of HSP expression during heat shock in mammals, as is HSF3 in avians. HSF2 plays roles in development of the brain and reproductive organs. However, the fundamental roles of HSF2 in vertebrate cells have not been identified. Here we find that vertebrate HSF2 is activated during heat shock in the physiological range. HSF2 deficiency reduces threshold for chicken HSF3 or mouse HSF1 activation, resulting in increased HSP expression during mild heat shock. HSF2-null cells are more sensitive to sustained mild heat shock than wild-type cells, associated with the accumulation of ubiquitylated misfolded proteins. Furthermore, loss of HSF2 function increases the accumulation of aggregated polyglutamine protein and shortens the lifespan of R6/2 Huntington's disease mice, partly through αB-crystallin expression. These results identify HSF2 as a major regulator of proteostasis capacity against febrile-range thermal stress and suggest that HSF2 could be a promising therapeutic target for protein-misfolding diseases.