Dissociation of photoreceptors from whole heads of the fruit fly,Drosophila melanogaster

Photoreceptor cells that were mostly free of extracellular material and suitable for most electrophysiological study procedures were dissociated from whole heads of the fruit fly, Drosophila melanogaster, by a simple smash technique employing gentle chopping by a razor blade through Parafilm sheets. A variety of commonly available proteolytic and glycolytic digestion enzymes were tested as additions to the basic dissociation procedure described. With the aid of Nomarski interference contrast optics, periodic acid-Schiff staining, and fluorescent labeling and microscopy methods, it was determined that proteolytic enzymatic digestion does little to enhance the dissociation procedure, and instead, often damages the cells that one is attempting to recover. Unexpectedly, certain glycolytic enzymes, when added to the basic procedure, appear to enhance the recovery of intact viable Drosophila photoreceptors that are stripped of most extracellular material. Based on these results, a hypothesis concerning the biochemical nature of the extracellular matrix of the Drosophila retina is proposed. Drosophila photoreceptors are an interesting model system for the study of invertebrate phototransduction and photoreceptor cell biology because of their many well-characterized mutant strains. The technique described here should produce clean viable photoreceptors or ommatidia that respond to light, and that are suitable for patch clamping or cell culture.     

Proteolytic activity of proteasome on myofibrillar structures

The physiologic function of proteasome remains unclear. Evidence suggests a role in degradation of ubiquitin-protein conjugates, MHC antigen presentation, and some specificity of substrate within certain cell types. To explore further the properties of proteasome we have examined its effect on a well defined structure, the myofibril. We find that despite its large size (20S) proteasome is able to degrade myofibrils and intact, permeabilized muscle fibrils. The proteins degraded showed some specificity because actin, myosin and desmin were degraded faster than -actinin, troponin T and tropomyosin. Changes in ultrastructure were slow and included a general loss of structure with Z and I bands effected before the M band and costameres.     

Examination of lymphokines induced in mice following immunization with recombinant simian virus 40 large tumor antigen

Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (T-Ag) was used to immunize BALB/c mice to examine the lymphokines produced following immunization. Specifically, we examined production of interleukin-2 (IL-2), IL-4, IL-5 and interferon (IFN) from immune lymphocytes cultured with decreasing concentrations of recombinant SV40 T-Ag. We identified elevated levels of IFN and IL-2 by enzyme-linked immunosorbent assay and a murine CTLL-2 proliferation biossay respectively. We were unable to detect either IL-4 or IL-5. These data indicate the previously reported tumor immunity induced by recombinant SV40 T-Ag immunization most likely reflects a TH1-like immune response based on thein vitro production of both IFN and IL-2 by immune lymphocytes.     

Experimental analysis of some factors affecting parental expenditure and investment inCichlasoma nigrofasciatum (Cichlidae)

Synopsis Parental investment is the cost of providing parental care. The short-term cost of parental care was measured in the biparental substrate nesting cichlid,Cichlasoma nigrofasciatum, by comparing the expected future survival (measured indirectly as energy content of the body), time taken to breed again and, among females, the number of eggs produced at a subsequent spawning of parental and non-parental pairs. In comparison with non-parental pairs, parental pairs took significantly longer to respawn. Body condition and female fecundity were unaffected after a single parental cycle. The effect on parental cost and expenditure of factors likely to stress the parental fish was also investigated. Removing the male parent had no effect on female parental cost. Exposing pairs to potential predators of offspring increased the time taken by pairs to respawn. In parental males, reducing the level of feeding gave rise to a reduction in some care behaviours.     

DNA sequences of the two homoeologous SLR1 genes of Brassica napus cv Westar

The DNA sequence data reported have been lodged in the Genbank, EMBL and DDBJ databases under the accession numbers Z21609 and Z26914     

Mutations in the chemotactic response regulator, CheY, that confer resistance to the phosphatase activity of CheZ

CheY, a small cytoplasmic response regulator, plays an essential role in the chemotaxis pathway. The concentration of phospho-CheY is thought to determine the swimming behaviour of the cell: high levels of phospho-CheY cause bacteria to rotate their flagella clockwise and tumble, whereas low levels of the phos-phorylated form of the protein allow counter-ciockwise rotation of the flagella and smooth swimming. The phosphorylation state of CheY in vivo is determined by the activity of the phosphoryl donor CheA, and by the antagonistic effect of dephosphorylation of phospho-CheY. The dephosphorylation rate is controlled by the intrinsic autohydrolytic activity of phospho-CheY and by the CheZ protein, which accelerates dephosphorylation. We have analysed the effect of CheZ on the dephosphorylation rates of several mutant CheY proteins. Two point mutations were identified which were 50-fold and 5-fold less sensitive to the activity of CheZ than was the wild-type protein. Nonetheless, the phosphorylation and autodephos-phorylation rates of these mutants, CheY23ND and CheY26KE, were observed to be identical to those of wild-type CheY in the absence of CheZ. These are the first examples of CheY mutations that reduce sensitivity to the phosphatase activity of CheZ without being altered in terms of their intrinsic phosphorylation and autodephospborylation rates, interestingly, the residues Asn-23 and Lys-26 are located on a face of CheY far from the phosphorylation site (Asp-57), distinct from the previously described site of inter-action with the histidine kinase CheA, and partially overlapping with a region implicated in interaction with the flagellar switch.     

Characterization of heat-shock response of the marine bacterium Vibrio harveyi

We have investigated heat-shock response in a marine bacterium Vibrio harveyi. We have found that 39 C was the highest tempature at which V. harveyi was able to grow steadily. A shift from 30° C to 39° C caused increased synthesis of at least 10 proteins, as judged by SDS-PAGE, with molecular masses of 90, 70, 58, 41, 31, 27, 22, 15, 14.5 and 14kDa. The 70, 58, 41 and 14.5 kDa proteins were immunologically homologous to DnaK, GroEL, DnaJ and GroES heat-shock proteins of Escherichia coli, respectively. V. harveyi GroES protein had a lower molecular mass (14.5 kDa) than E. coli GroES, migrating in SDS-PAGE as 15 kDa protein. We showed that a protein of ～43 kDa, immunologically reactive with antiserum against E. coli sigma 32 subunit (σ³²) of RNA polymerase, was induced by heat-shock and co-purified with V. harveyi RNA polymerase. These results suggest that the 43 kDa protein is a heat-shock sigma protein of V. harveyi. Preparation containing the V. harveyi sigma 32 homologue, supplemented with core RNA polymerase of E. coli, was able to transcribe heat-shock promoters of E. coli in vitro.     

Influence of D-galactosamine on the synthesis of sugar nucleotides and glycoconjugates in rat hepatocytes

Rat hepatocytes were incubated in the presence of a high concentrationof the hepatopathogenic agent D-galactosamine (GalN), and theeffect on the cellular concentrations of pyrimidine nucleotidesand nucleotide sugars was determined. The UTP pool became depleted.The pools of UMP and CMP in RNA decreased to 72%, indicativefor an inhibition of RNA synthesis. UDP-HexNAc (where HexNAcis GlcNAc + GalNAc) and UDP-HexN (where HexN is GlcN + GalN)levels increased, and those of UDP-hexose and UDP-GlcA (whereGlcA is glucuronic acid) decreased. The cellular concentrationof CTP did not change, whereas that of CMP-NeuAc (where NeuAcis N-acetylneuraminic add) showed a 2-fold increase. Labellingwith [¹⁴C]orotic acid and [³H]cytidine showed that the metabolicflow via the de novo pathway was not changed. The depletionof the so-called overflow pool of UTP [Pels Rijcken et al, Biochem.J., 293, 207–213, 1993] caused a release of the feedbackinhibition by UTP and thus an increased flow through the salvagepathway. Finally, it appeared that GalN, when added to hepatocytes,gives rise to a pool of UDP-GlcNAc (where GlcNAc is N-acetylglueosamine)that is separate from the pool of UDP-GlcNAc that is derivedfrom GlcN. D-galactosamine glycosylation sugar nucleotide biosynthesis     

Mannosyltransferase activities in membranes from various yeast strains

In the yeast Golgi compartments, at least five, and potentiallyseveral additional mannosyltransferases are involved in elongatingto ‘mannan’ the core Man₈GlcNAc₂ oligosac-charidetrimmed from GlC₃Man₉GlcNAc₂ in the endoplasmic reticulum. Structuralstudies on oligosaccharides from alg3 mutant yeast, which lackthe four upper arm mannoses donated by Man-P-Dol (where Dolis dolichol), verified that the new     

Characterization of a plasma membrane-associated phosphoinositide-specific phospholipase C from soybean

Phosphoinositide-specific phospholipase C (PI-PLC) is a key signal transducing enzyme which generates the second messengers inositol trisphosphate and diacylglycerol in mammalian cells. A cDNA clone (PI-PLC1) encoding a phosphoinositide-specific phospholipase C was isolated from soybean by screening a cDNA expression library using an anti-(plasma membrane) serum. Genomic DNA gel blot analysis suggested that the corresponding gene is a member of a multigene family. The deduced amino acid sequence of the soybean PI-PLC1 isozyme contains the conserved X and Y regions, found in other PI-PLCs. It is closely related to mammalian δ-type PI-PLCs, Dictyostelium discoideum PI-PLC and yeast PI-PLC1 in terms of the arrangement of the conserved region. Unlike mammalian δ-type PI-PLCs and yeast PI-PLC1, the putative Ca²⁺-binding site of the soybean PI-PLC1 is located in the region spanning the X and Y domains, and the N-terminal region is truncated. FLAG epitope-tagged PI-PLC1 fusion protein purified from transgenic tobacco plants showed phosphoinositide-specific phospholipase C activity. Heterologous expression of the soybean PI-PLC1 cDNA in a yeast PI-PLC1 deletion mutant complemented the lethality phenotype of haploid PI-PLC1 disruptants. Immunoblot analysis of the cell fractions prepared from transgenic tobacco plants over-expressing the FLAG epitope-tagged PI-PLC1 fusion protein indicated that the protein encoded by the PI-PLC1 cDNA was localized in the cytosol and plasma membrane.