Charge balance in the alpha-hydroxyacid dehydrogenase vacuole: an acid test.

The proposal that the active site vacuole of NAD(+)-S-lactate dehydrogenase is unable to accommodate any imbalance in electrostatic charge was tested by genetically manipulating the cDNA coding for human muscle lactate dehydrogenase to make a protein with an aspartic acid introduced at position 140 instead of the wild-type asparagine. The Asn 140-Asp mutant enzyme has the same kcat as the wild type (Asn 140) at low pH (4.5), and at higher pH the Km for pyruvate increases 10-fold for each unit increase in pH up to pH 9. We conclude that the anion of Asp 140 is completely inactive and that it binds pyruvate with a Km that is over 1,000 times that of the Km of the neutral, protonated aspartic-140. Experimental results and molecular modeling studies indicate the pKa of the active site histidine-195 in the enzyme-NADH complex is raised to greater than 10 by the presence of the anion at position 140. Energy minimization and molecular dynamics studies over 36 ps suggest that the anion at position 140 promotes the opening of and the entry of mobile solvent beneath the polypeptide loop (98-110), which normally seals off the internal active site vacuole from external bulk solvent.     

Induction of gene expression in Dictyostelium by prestarvation factor, a factor secreted by growing cells

During growth, Dictyostelium cells continuously secrete a factor, PSF, that accumulates in proportion to cell density. At sufficient concentration, it triggers the production of discoidin I and certain lysosomal enzymes. Our earlier studies demonstrated these effects of PSF on protein and enzyme levels [Clarke et al., Differentiation 34:79-87, 1987; Clarke et al., Dev Genet 9: 315-326, 1988]. In the present study, we have examined whether PSF induces increased mRNA levels. By Northern blot analysis, we have found that discoidin I mRNA accumulates in exponentially growing NC4 cells as the cells reach high density; significant levels of mRNA are detectable in cells growing either on plates or in suspension, beginning about four generations before the end of exponential growth. High levels of discoidin I mRNA are also found in low-density cells grown in the presence of buffer conditioned by high-density cells. These results indicate that PSF induces the accumulation of discoidin I mRNA. Other "early developmental" genes, pCZ22 and the early I genes (16, 18, and 111), are also expressed in exponentially growing cells at high density or in the presence of conditioned buffer. We conclude that several genes previously found to be preferentially expressed very early in development are actually induced during late exponential growth by PSF.     

Binding of Epstein-Barr virus small RNA EBER-1 to the double-stranded RNA-activated protein kinase DAI.

Epstein-Barr virus encodes two small RNAs, EBER-1 and -2, that are abundantly expressed in latently infected cells. Recent evidence suggests a role for EBER-1 in regulation of translation since this RNA is able to prevent the inhibition of protein synthesis by double-stranded RNA in rabbit reticulocyte lysates. We show here that EBER-1 that has been synthesized in vitro forms a complex with the dsRNA-activated inhibitor of protein synthesis DAI, a protein kinase that specifically phosphorylates polypeptide chain initiation factor eIF-2. Gel retardation assays and UV crosslinking experiments indicate that complex formation is specific for EBER-1 and requires the presence of some secondary structure in the molecule. RNA competition studies show that EBER-1-DAI complex formation is not inhibited in the presence of other small RNA species, heparin or the synthetic double-stranded RNA, poly(I).poly(C). SDS gel analysis reveals the existence of two forms of the crosslinked complex, of 64-68kDa and 46-53kDa, both of which are recognized by anti-DAI antibodies in immunoprecipitation experiments. These data suggest that EBER-1 regulates protein synthesis through its ability to interact with DAI.     

Diazinon resistance, fluctuating asymmetry and fitness in the Australian sheep blowfly, lucilia cuprina

Genetic evidence suggests that the evolution of resistance to the insecticide diazinon in Lucilia cuprina initially produced an increase in asymmetry. At that time resistant flies were presumed to be at a selective disadvantage in the absence of diazinon. Subsequent evolution in natural populations selected modifiers to ameliorate these effects. The fitness and fluctuating asymmetry levels of resistant flies are currently similar to those of susceptibles. Previous genetic analyses have shown the fitness modifier to co-segregate with the region of chromosome III marked by the white eyes, w, locus, unlinked to the diazinon resistance locus, Rop-1, on chromosome IV. This study maps the asymmetry modifier to the same region, shows, as in the case of the fitness modifier, its effect to be dominant and presents data consistent with the fitness/asymmetry modifier being the same gene (gene complex). These results suggest changes in fluctuating asymmetry reflect changes in fitness.     

Generation of hydroxyeicosatetraenoic acids by human inflammatory cells: analysis by thermospray liquid chromatography-mass spectrometry

Arachidonic acid was converted to a series of hydroxyeicosatetraenoic acids (HETEs) by mixed human inflammatory cells following stimulation with the calcium ionophore A23187. HETEs were purified by a simple one-step extraction procedure followed by HPLC. The HPLC was coupled to a Finnigan quadrupole mass spectrometer using the now commercially available thermospray liquid chromatography-mass spectrometry interface. The HPLC eluant was monitored 'on line' by the mass spectrometer. Soft ionisation occurs, generating intense molecular ion species in the negative ion mode (M - H-:m/z 319) for each of the isomeric HETEs. The (M + H+ - H2O) ion at m/z 303 is the major species in the positive ion spectra of HETEs. Mass spectra were obtained on-line post-HPLC for HETEs formed by the human cells, and the HPLC-MS profile compared with that obtained from standards; species corresponding to the 11-, 9- and 5-HETEs were observed.     

Methodology for Estimating Numbers of Free-Living and Attached Bacteria in Estuarine Water

A fundamental problem in estuarine microbiology studies is the accurate determination of the density in the water column of both free-living bacteria and those attached to suspended particulate matter. When a water sample is filtered and the filter is viewed by epifluorescence microscopy, counts can be made of the numbers of bacteria which are seen on the filter background (free-living) and those which appear to lie on sediment particles (both free-living and attached). With only the additional knowledge of the proportion of the filter area covered by particles (a quantity that is straightforwardly determined by stereological point counting), results from geometric probability were used to determine the expected number of bacteria which are hidden by particles and hence to provide an estimation scheme for the true densities of free-living and attached bacteria. Variance equations based on a Taylor series are given, and a partial check of the method is attempted with controlled mixtures of bacteria and sediment. An alternative procedure is also proposed, in which the natural attached/free-living ratio is altered by an intervention experiment, allowing an estimation which is less model dependent but more labor intensive. Both methods are applied to a series of samples from the Tamar estuary, United Kingdom, taken in April 1985. A notable conclusion is that there are always more free-living than attached bacteria in the water column throughout the estuary.     

Platelet-activating factor induces the expression of early pregnancy factor activity in female mice

When synthetic platelet-activating factor (PAF, 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was injected into mature female mice during dioestrus, pro-oestrus or oestrus, it induced the expression of early pregnancy factor (EPF) activity in the sera of these animals within 1 h of injection. The sera of similarly injected males, metoestrous or immature females did not display any EPF activity. The results suggest that embryo-derived PAF may be the ovum factor responsible for triggering the generation of serum EPF activity during the preimplantation stages of pregnancy.