Metabolic correlates to glycerol biosynthesis in a freeze-avoiding insect,Epiblema scudderiana

Summary The course of glycerol biosynthesis, initiated by exposure to –4°C, was monitored in larvae of the goldenrod gall moth,Epiblema scudderiana, and accompanying changes in the levels of intermediates of glycolysis, adenylates, glycogen, glucose, fructose-2,6-bisphosphate, and fermentative end products were characterized. Production of cryoprotectant was initiated within 6 h after a switch from +16° to –4°C, with halfmaximal levels reached in 30 h and maximal content, 450–500 mol/g wet weight, achieved after 4 days. Changes in the levels of intermediates of the synthetic pathway within 2 h at –4°C indicated that the regulatory sites involved glycogen phosphorylase, phosphofructokinase, and glycerol-3-phosphatase. A rapid increase in fructose-2,6-bisphosphate, an activator of phosphofructokinase and inhibitor of fructose-1,6-bisphosphatase, appeared to have a role in maintaining flux in the direction of glycerol biosynthesis. Analysis of metabolite changes as glycerol production slowed suggested that the inhibitory restriction of the regulatory enzymes was slightly out of phase. Inhibition at the glycerol-3-phosphatase locus apparently occurred first and resulted in a build-up of glycolytic intermediates and an overflow accumulation of glucose. Glucose inhibition of phosphorylase, stimulating the conversion of the activea to the inactiveb forms, appears to be the mechanism that shuts off phosphorylase function, counteracting the effects of low temperature that are the basis of the initial enzyme activation. Equivalent experiments carried out under a nitrogen gas atmosphere suggested that the metabolic make-up of the larvae in autumn is one that obligately routes carbohydrate flux through the hexose monophosphate shunt. The consequence of this is that fermentative ATP production during anoxia is linked to the accumulation of large amounts of glycerol as the only means of maintaining redox balance.Abbreviations G6P glucose-6-phosphate - F6P fructose-6-phosphate - F1, 6P fructose-1,6-bisphosphate - F2,6P ₂ fructose-2,6-bisphosphate - G3P grycerol-3-phosphate - DHAP dinydroxyacetonephosphate - GAP glyceraldehyde-3-phosphate - PEP phosphoenolpyruvate - PFK phosphofructokinase - FBPase fructose-1,6-bisphosphatase - PK pyruvate kinase     

Expression of a muscle-type alpha-actinin cDNA clone in non-muscle cells

We have previously isolated a chick smooth muscle-type alpha-actinin cDNA clone (C17) from a chick embryo fibroblast cDNA library. As part of an investigation into a possible role for a muscle isoform of alpha-actinin in non-muscle cells, we have cloned C17 into a eucaryotic expression vector, pKCR3, and examined the distribution of the expressed protein in non-muscle, monkey COS cells. We report here that the muscle isoform of chick alpha-actinin encoded by C17, was found in focal contacts and periodically distributed along actin filaments.     

Carbon-13 NMR of glycogen: hydration response studied by using solids methods

The carbon-13 NMR spectra of glycogen are reported by using the methods of magic-angle sample spinning and high-power proton decoupling to provide a dynamic report on the glucose monomer behavior as a function of hydration. Although the glycogen behaves as a typical polymer in the dry state, addition of water makes a significant difference in the spectral appearance. Water addition decreases the carbon spin-lattice relaxation times by 2 orders of magnitude over the range from 7% to 70% water by weight. The proton-carbon dipole-dipole coupling, which broadens the carbon spectrum and permits cross-polarization spectroscopy, is lost with increasing hydration over this range. By 60% water by weight, scalar decoupling methods are sufficient to achieve a reasonably high-resolution spectrum. Further, at this concentration, the carbon spin-lattice relaxation times are near their minimum values at a resonance frequency of 50.3 MHz, making acquisition of carbon spectra relatively insensitive to intensity distortions associated with saturation effects. Though motional averaging places the spectrum in the solution phase limit, the static spectrum shows a residual broader component that would not necessarily be detected readily by using high-resolution liquid-state experiments.     

An assessment of short-term depletion of stream macroinvertebrate benthos by drift

A study was conducted to determine if emigration of drifting macroinvertebrates from a stream riffle which was blocked for one week from immigration by upstream colonists significantly reduced the abundance of drift collected from the tail of the riffle. The head of a 9 m long riffle of a 2nd order stream in Maryland (USA) was blocked from incoming drift by a 250 m mesh weir. Upstream immigration of invertebrates into the riffle was largely prevented by a partition placed at the tail of the riffle which held the drift nets. Benthos and drift samples were collected from the riffle prior to weir placement and following its removal, and drift was collected at dusk on each day. No difference in drift or in benthic abundance between the beginning and end of the study was observed. This is largely attributed to recruitment of immature insects (primarily hatching of eggs present at the outset), particularly of Dolophilodes distinctus and species of Tanytarsini, from within the riffle. Results suggest that recruitment of riffle species is of sufficient magnitude to more than compensate for short-term riffle depletion due to drift. Samples of drifting and non-drifting (benthic) animals were held without food for 12 h after collection and mortality within each group was determined. The mortality of drifting animals was three-fold that of benthic animals.     

Courtship in Saccharomyces cerevisiae: an early cell-cell interaction during mating.

During conjugation in Saccharomyces cerevisiae, two cells of opposite mating type (MATa and MAT alpha) fuse to form a diploid zygote. Conjugation requires that each cell locate an appropriate mating partner. To investigate how yeast cells select a mating partner, we developed a competition mating assay in which wild-type MAT alpha cells have a choice of two MATa cell mating partners. We first demonstrated that sterile MAT alpha 1 cells (expressing no a- or alpha-specific gene products) do not compete with fertile MATa cells in the assay; hence, wild-type MATa and MAT alpha cells can efficiently locate an appropriate mating partner. Second, we showed that a MATa strain need not be fertile to compete with a fertile MATa strain in the assay. This result defines an early step in conjugation, which we term courtship. We showed that the ability to agglutinate is not necessary in MATa cells for courtship but that production of a-pheromone and response to alpha-pheromone are necessary. Thus, MATa cells must not only transmit but must also receive and then respond to information for effective courtship; hence, there is a "conversation" between the courting cells. We showed that the only alpha-pheromone-induced response necessary in MATa cells for courtship is production of a-pheromone. In all cases tested, a strain producing a higher level of a-pheromone was more proficient in courtship than one producing a lower level. We propose that during courtship, a MAT alpha cell selects the adjacent MATa cell producing the highest level of a-pheromone.     

Roles of proteins in cation/membrane interactions of isolated rat cardiac sarcolemmal vesicles

To ascertain the roles of the membrane proteins in cation/sarcolemmal membrane binding, isolated rat cardiac sarcolemmal vesicles were extensively treated with Protease (S. aureus strain V.8). SDS-gel electrophoresis, protein and phosphate analysis confirmed that at least 20–22% of the protein, but none of the phospholipid, was solubilized by this procedure, and that the remaining membrane proteins were extensively hydrolyzed into small fragments. The cation binding properties of the treated vesicles were then examined by analyzing their aggregation behavior. The results demonstrate that this procedure had no effect on the selectivity series for di- and trivalent cation binding, or the divalent cation-induced aggregation behavior of the sarcolemmal vesicles at different pHs, indicating that proteins are probably not involved in these interactions and cannot be the low affinity cation binding sites previously observed [21, 22]. It did, however, change the pH at which protons induced sarcolemmal vesicle aggregation, suggesting a possible role for proteins in these processes. Protease treatment also modified the effects of fluorescamine labelling on divalent cation-induced vesicle aggregation, indicating that the NH, groups being labelled with fluorescamine are located on the sarcolemmal proteins. Together, these results support the hypothesis that di- and trivalent cation binding to the sarcolemmal membrane is largely determined by lipid/lipid and/or lipid/carbohydrate interactions within the plane of the sarcolemmal membrane, and that membrane proteins may exert an influence on these interactions, but only under very specialized conditions.Abbreviations MES 2-(N-morpholino)ethanesulfonic acid - MOPS 3-(N-morpholino) propanesulfonic acid - HEPES N-2-Hydroxyethylpiperizine-N-2- ethanesulfonic acid - CHES 2(N-Cyclohexylamino) ethanesulfonic acid - DTT DL-Dithiothreitol - PMSF Phenylmethyl-sulfonyl fluoride     

Comparative studies of hepatotoxicity and fumonisin B1 and B2 content of water and chloroform/methanol extracts of Fusarium moniliforme strain MRC 826 culture material

Fusarium moniliforme has been associated with several diseases including equine leukoencephalomalacia, human esophageal cancer and hepatotoxicity/hepatocarcinogenicity in laboratory animals. The potential health risks to animals and humans posed by F. moniliforme contaminated grains cannot be assessed until the toxins are identified and toxicologically evaluated. As part of a systematic approach to identifying the hepatotoxins produced by F. moniliforme, diets containing aqueous and chloroform/methanol (11) extracts of F. moniliforme strain MRC 826 culture material (CM) and/or the extracted CM residues were fed to male Sprague-Dawley rats for four weeks. Serum alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase activities were increased after two and four weeks and microscopic liver lesions were found in those animals fed aqueous CM extract and the CM residue after chloroform/ methanol extraction. Fumonisins B₁ and B₂ were extracted from the CM by water, but not chloroform/ methanol, and were present in the toxic diets at concentrations of 93–139 and 82–147 ppm, respectively. Nontoxic diets contained 22 ppm fumonisin B₁ and 65 ppm fumonisin B₂.Abbreviations CM culture material - ELEM equine leukoencephalomalacia Mention of a trademark, proprietory name or vendor does not imply its approval by the US Department of Agriculture to the exclusion of others that may also be suitable.     

The 76 kD cell-adhesion factor from crayfish haemocytes promotes encapsulation in vitro

Summary Semigranular cells from the crayfish, Pacifastacus leniusculus, were separated by Percoll gradient centrifugation and were used to study the encapsulation of foreign particles. The semigranular cells were found strongly to encapsulate glass beads coated with haemocyte lysate in which the prophenoloxidase-activating system had been activated with laminarin or with a low concentration of calcium ions. The granular cells only weakly encapsulated these particles. The encapsulationpromoting factor was purified from haemocyte lysates and found to be a 76 kD protein which was recognized by an antiserum to the previously described 76 kD cell-adhesion factor. After the last step in purification (Con A-Sepharose chromatography), the flowthrough consisted of several proteins, which had some, but less, encapsulation-promoting activity and contained a 30 kD band that was also recognized by the antiserum to the 76 kD cell-adhesion factor. If the haemocyte lysate prepared in low [Ca²⁺] was incubated with a -1,3-glucan prior to purification, no 76 kD protein could be isolated but only a 30 kD protein. The 30 kD protein thus seems to be a degradation product of the 76 kD cell-adhesion factor. We conclude that the 76 kD protein which is released from degranulating haemocytes, and to a lesser extent its 30 kD fragment, can promote encapsulation. Phenoloxidase did not have any encapsulation-promoting activity.     

Rhodobacter capsulatus strain BK5 possesses a membrane bound respiratory nitrate reductase rather than the periplasmic enzyme found in other strains

Rhodobacter capsulatus strain BK5 possesses a membrane bound respiratory nitrate reductase rather than the periplasmic enzyme found in other strains. The enzyme in strain BK5 is shown to be both functionally and structurally related to the nitrate reductase of Paracoccus denitrificans and Escherichia coli.Abbreviation TMAO trimethylamine-N-oxide