Isolation of Neurospora Crassa a Mating Type Mutants by Repeat Induced Point (Rip) Mutation

In the filamentous fungus, Neurospora crassa, mating type is regulated by a single locus with alternate alleles, termed A and a. The mating type alleles control entry into the sexual cycle, but during vegetative growth they function to elicit heterokaryon incompatibility, such that fusion of A and a hypha results in death of cells along the fusion point. Previous studies have shown that the A allele consists of 5301 bp and has no similarity to the a allele; it is found as a single copy and only within the A genome. The a allele is 3235 bp in length and it, too, is found as a single copy within the a genome. Within the A sequence, a single open reading frame (ORF) of 288 amino acids (mt A-1) is thought to confer fertility and heterokaryon incompatibility. In this study, we have used repeat induced point (RIP) mutation to identify functional regions of the A idiomorph. RIP mutations in mt A-1 resulted in the isolation of sterile, heterokaryon-compatible mutants, while RIP mutations generated in a region outside of mt A-1 resulted in the isolation of mutants capable of mating, but deficient in ascospore formation.     

Specific reduction of delta-opioid receptor binding in transfected NG108-15 cells.

We have recently identified and sequenced the cDNA for an opioid-binding protein with homologies to cell adhesion molecules (OBCAM) (Schofield, P. R., McFarlard, K. C., Hayflick, J. S., Wilcox, J. N., Cho, T. M., Roy, S., Lee, N. M., Loh, H. H., and Seeburg, P. H. (1989) EMBO J. 8, 489-495). Several lines of evidence using antibodies suggest that OBCAM may play a functional role in NG108-15 neuroblastoma x glioma cells, a useful model system that contains a homogeneous population of delta-opioid receptors. A logical extension of this research is to further test this hypothesis. As part of this study, NG108-15 cells were stably transfected with either sense or antisense sequences of a portion of pROM, the rat cDNA for OBCAM. [3H] Diprenorphine binding was greatly reduced in antisense-transfected cells relative to non-transfected cells. Binding to alpha 2-adrenergic, muscarinic, and insulin receptors was unaffected. These results further support the notion that OBCAM or its analogue is part (or a subunit) of an opioid receptor. Furthermore, our observation of an apparently specific reduction in opioid binding in these transfected cells suggests that they may provide a novel genetic approach for studying regulation of the opioid receptor in this defined cell line.     

Activation of MAP kinases by calcium-dependent and calcium-independent pathways. Stimulation by thapsigargin and epidermal growth factor.

In order to determine the effect of calcium mobilization on mitogen-activated protein (MAP) kinase activation, we have treated human foreskin fibroblasts (HSWP cells) and human epidermal carcinoma (A431) cells with thapsigargin. Intracellular free calcium was monitored by single cell image analysis using fura-2 and correlated with MAP kinase stimulation as assessed by immunoprecipitation, kinase renaturation assays and immunoblotting. Thapsigargin stimulated the 44- and 42-kDa MAP kinase isozymes in both cell types with kinetics that were slightly delayed relative to enzyme stimulated by epidermal growth factor. Removal of external calcium did not significantly affect the activation of the MAP kinases by thapsigargin, indicating that intracellular calcium mobilization is sufficient to stimulate the enzymes. However, treatment of cells with EGTA under conditions which deplete both intra- and extracellular calcium inhibited stimulation by thapsigargin but not epidermal growth factor. Stimulation of the MAP kinases by the calcium ionophore ionomycin paralleled the activation observed with thapsigargin in both calcium-containing and calcium-free conditions. These results indicate that there are at least two independent pathways for stimulation of MAP kinase: one that is dependent on intracellular calcium mobilization, and one that is mediated by the tyrosine kinase epidermal growth factor receptor and is calcium-independent.     

Partially folded state of the disulfide-reduced form of human serum albumin as an intermediate for reversible denaturation.

The conformation of the fully disulfide-reduced state of human serum albumin was investigated by tryptophan fluorescence spectrum, CD analyses, and size-exclusion chromatography. Both the reduction of the native disulfide-bonded form under nondenaturing conditions and the refolding of the urea-denatured disulfide-reduced form under reduced conditions yielded almost exactly the same disulfide-reduced state with partially folded unique conformation that was clearly distinguished from either the native or fully denatured state. In addition, the interconversion between the urea-denatured reduced form and the partially folded reduced form was reversible with each other; by reoxidation, the partially folded reduced form was converted to the disulfide-bonded form. The conformation of disulfide-reduced serum albumin was highly variable depending on pH and ionic strength conditions. Thus, we concluded that the disulfide-reduced state with partially folded variable conformation is involved in the reversible interconversion between the denatured reduced form and the native disulfide-bonded form of human serum albumin.     

Methane monooxygenase component B and reductase alter the regioselectivity of the hydroxylase component-catalyzed reactions. A novel role for protein-protein interactions in an oxygenase mechanism.

The soluble methane monooxygenase (MMO) system, consisting of reductase, component B, and hydroxylase (MMOH), catalyzes NADH and O2-dependent monooxygenation of many hydrocarbons. MMOH contains 2 mu-(H or R)oxo-bridged dinuclear iron clusters thought to be the sites of catalysis. Although rapid NADH-coupled turnover requires all three protein components, three less complex systems are also functional: System I, NADH, O2, reductase, and MMOH; System II, H2O2 and oxidized MMOH; System III, MMOH reduced nonenzymatically by 2e- and then exposed to O2 (single turnover). All three systems give the same products, suggesting a common reactive oxygen species. However, the distribution of products observed for most substrates that are hydroxylated in more than one position is different for each system. For several of these substrates, addition of component B to Systems I, II, or III causes the product distributions to shift dramatically. These shifts result in identical product distributions for Systems I and III in which MMOH passes through the 2e- reduced state ([Fe(II).Fe(II)]) during catalysis. In contrast, System II (in which MMOH probably does not become reduced) generally gives a unique product distribution. It is proposed that changes in MMOH structure occurring upon diiron cluster reduction and/or component complex formation cause substrates to be presented differently to the activated oxygen species. Kinetic studies show that component B strongly activates System I and, in most cases, strongly deactivates System II. The effect of component B on product distribution of System I (and III) occurs at less than 5% of the MMOH concentration, while nearly stoichiometric concentrations are required to maximize the rate of System I. This shows that component B has at least two roles in catalysis. EPR monitored titration of reduced MMOH ([Fe(II).Fe(II)]) with component B suggests that the effect of substoichiometric component B on product distribution is due to hysteresis in the MMOH conformational changes.     

Distinct mechanisms of zinc uptake at the apical and basolateral membranes of caco-2 cells.

Zinc uptake mechanisms at the apical and basolateral membrane borders of caco-2 cells were examined. This human-derived cell line possesses many morphological and functional characteristics of absorptive small intestinal cells. By day 14, confluent and well-differentiated monolayers were formed when the cells were grown on porous polycarbonate filters. Labelled zinc was placed on the apical or basal side of the monolayer and its uptake by the cells, as well as its transport across the monolayer, were measured. Zinc uptake by the cells from the apical side was found to be a saturable process (Kt = 41 microM; Vmax = 0.3 nmols/cm2/10 min) with a diffusional term at higher concentrations (1.0 sec/cm). Apical uptake was not affected by metabolic inhibitors or potential zinc ligands. Zinc uptake from the basolateral side was concentration dependent (Kd = 1.3 sec/cm) and was partially inhibited (30%) by ouabain and vanadate, suggesting that the (Na-K)-ATPase on the basolateral membrane is involved in the serosal uptake of zinc by the cell. Transport of zinc across the monolayers from the apical or basolateral compartment was concentration dependent and was not affected by metabolic inhibitors. Zinc transport from the basolateral side was greater than 2-fold greater than apical transport. Hence, separate mechanisms can be distinguished with respect to zinc uptake at the apical and basolateral membranes of caco-2 cells.     

Cooling rate influences cryoprotectant distribution and organ dehydration in freezing wood frogs.

Ice formation in the freeze-tolerant wood frog (Rana sylvatica) induces the production and distribution of the cryoprotectant, glucose. Concomitantly, organs undergo a beneficial dehydration which likely inhibits mechanical injury during freezing. Together, these physiological responses promote freezing survival when frogs are frozen under slow cooling regimes. Rapid cooling, however, is lethal. We tested the hypothesis that the injurious effects of rapid cooling stem from an inadequate distribution of glucose to tissues and an insufficient removal of water from tissues during freezing. Accordingly, we compared glucose and water contents of five organs (liver, heart, skeletal muscle, eye, brain) from wood frogs cooled slowly or rapidly during freezing to -2.5 degrees C. Glucose concentrations in organs from slowly cooled frogs were significantly elevated over unfrozen controls, but no significant increases occurred in rapidly cooled frogs. Organs from slowly cooled frogs contained significantly less water than did those from controls, whereas water contents from rapidly cooled frogs generally were unchanged. Rapid cooling therefore inhibited the production and distribution of cryoprotectant and organ dehydration during freezing. This inhibition may result from an accelerated, premature failure of the cardiovascular system.     

Lithium Enhances Accumulation of [3H]Inositol Radioactivity and Mass of Second Messenger Inositol 1,4,5-Trisphosphate in Monkey Cerebral Cortex Slices

We previously reported that lithium, in the presence of acetylcholine, increased accumulations of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in brain cortex slices from the guinea pig, rabbit, rat, and mouse. In the mouse and rat, the Li(+)-induced increases required supplementation of the medium with inositol. This probably relates to the following facts: (a) Brain cortices of the mouse and rat contain in vivo concentrations of inositol half of that of the guinea pig. (b) Incubated rat brain cortex slices are depleted of inositol by 80%. (c) The slices require 10 mM inositol supplementation to restore in vivo concentrations. We now show that in monkey brain cortex slices, therapeutic concentrations of Li+ increase accumulation of inositol 1,4,5-trisphosphate. The inositol 1,3,4,5-tetrakisphosphate level is not increased. Neither inositol nor an agonist is required. The same effects are seen whether inositol 1,4,5-trisphosphate is quantified by the [3H]inositol prelabeling technique or by mass assay, although mass includes a pool of inositol 1,4,5-trisphosphate that is metabolically inactive. Thus, in a therapeutically relevant model for humans, Li+ increases inositol 1,4,5-trisphosphate levels in brain cortex slices, as was previously seen in lower mammals at non-rate-limiting concentrations of inositol.