The effects of different concentrations of glycerol and dimethylsulfoxide on the metabolic activities of kidney slices

Kidney slices either were exposed to the cryoprotectants for 1 hr at room temperature and subsequently washed and incubated in fresh KR buffer containing only the radioactive metabolic tracers, or were immediately incubated for 2 hr at 37 °C in KR buffer containing the cryoprotectant and the tracers. Exposure to glycerol by incubation of kidney slices in Krebs-Ringer bicarbonate buffer containing varying concentrations of glycerol from 0 to 70% ($\text{v}\text{v}$) resulted in a pronounced inhibitory effect on the protein synthesizing activity, while thymidine incorporation into DNA and the α-aminoisobutyric acid uptake through the cell membranes were less affected. Exposure of the tissue to buffer containing dimethylsulfoxide (Me₂SO) in concentrations of 10 to 20% ($\text{v}\text{v}$) resulted in a stimulatory effect on metabolism. At higher concentrations, Me₂SO was toxic resulting in damaging effects on protein and DNA synthesis as well as on membrane integrity. The stimulatory effects of exposure to low concentrations of Me₂SO on protein and DNA synthesis in kidney slices were concluded to be the result of an increased transport of precursors through the cell membranes.     

Agrobacterium-mediated transformation of apple (Malus x domestica Borkh.): an assessment of factors affecting regeneration of transgenic plants

We have previously developed a protocol for efficient gene transfer and regeneration of transgenic calli following cocultivation of apple (cv. Jonagold) explants with Agrobacterium tumefaciens (De Bondt et al. 1994, Plant Cell Reports 13: 587–593). Now we report on the optimization of postcultivation conditions for efficient and reproducible regeneration of transgenic shoots from the apple cultivar Jonagold. Factors which were found to be essential for efficient shoot regeneration were the use of gelrite as a gelling agent and the use of the cytokinin-mimicing thidiazuron in the selective postcultivation medium. Improved transformation efficiencies were obtained by combining the hormones thidiazuron and zeatin and by using leaf explants from in vitro grown shoots not older than 4 weeks after multiplication. Attempts to use phosphinothricin acetyl transferase as a selectable marker were not successful. Using selection on kanamycin under optimal postcultivation conditions, about 2% of the leaf explants developed transgenic shoots or shoot clusters. The presence and expression of the transferred genes was verified by -glucuronidase assays and Southern analysis. The transformation procedure has also been succesfully applied to several other apple cultivars.Abbreviations BAP benzylaminopurine - CTAB hexadecyltrimethylammoniumbromide - Na₂EDTA ethylenediamine-tetra-acetate ferric-sodium salt - FeNaEDTA ethylenediamine-tetra-acetate ferric-sodium salt - GA₃ gibberellic acid 3 - GusA -glucuronidase - gusA -glucuronidase gene of Escherichia coli - IAA indole acetic acid - IBA indole butyric acid - 2iP N6-2-isopentenyl adenine - NAA naphthalene acetic acid - nptII neomycinphosphotransferase II gene - bar phosphinothricin acetyl transferase gene - PCR polymerase chain reaction - PPT phosphinothricin - STS silver thiosulphate - T-DNA transferred DNA - TDZ thidiazuron - X-Gluc 5-bromo-4-chloro-3-indolyl -D-glucuronide - Zea trans-Zeatin     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Pathogen-induced systemic activation of a plant defensin gene in Arabidopsis follows a salicylic acid-independent pathway.

A 5-kD plant defensin was purified from Arabidopsis leaves challenged with the fungus Alternaria brassicicola and shown to possess antifungal properties in vitro. The corresponding plant defensin gene was induced after treatment of leaves with methyl jasmonate or ethylene but not with salicylic acid or 2,6-dichloroisonicotinic acid. When challenged with A. brassicicola, the levels of the plant defensin protein and mRNA rose both in inoculated leaves and in nontreated leaves of inoculated plants (systemic leaves). These events coincided with an increase in the endogenous jasmonic acid content of both types of leaves. Systemic pathogen-induced expression of the plant defensin gene was unaffected in Arabidopsis transformants (nahG) or mutants (npr1 and cpr1) affected in the salicylic acid response but was strongly reduced in the Arabidopsis mutants eln2 and col1 that are blocked in their response to ethylene and methyl jasmonate, respectively. Our results indicate that systemic pathogen-induced expression of the plant defensin gene in Arabidopsis is independent of salicylic acid but requires components of the ethylene and jasmonic acid response.     

Characterization of the multiple forms of β-xylanase produced by a Streptomyces sp. growing on lignocellulose

Summary Streptomyces sp. strain EC10 degraded efficiently the hemicellulose fraction of wheat straw. Three forms of -xylanases detected in the culture filtrate were purified by precipipation with ammonium sulphate, chromatography on DEAE-Sephadex A-50 and gel filtration on Sephadex G-100. The three purified enzymes (X _ia , X _ib and X _ii ) were homogeneous by polyacrylamide gel electrophoresis. The enzymes were typical non-debranching endo--xylanases (1,4--d-xyla xylanohydrolases; E.C.3.2.1.8) with respective relative molecular weights of 32,000, 22,000 and 21,000 and isoelectric points of 6.8, 8.9 and 5.2. The enzymes were highly specific for xylans and showed optimal activity at pH 7.0–8.0 and 60°C. The preparations were completely free from cellulolytic activity (endoglucanase) and showed high thermal stability. No synergy between the three enzymes was detected for complete xylan hydrolysis of deacetylated arabino- and glucuronoxylans.Offprint requests:to: M. J. Penninckx     

Affinity of arginase for ornithine carbamoyltransferase in Saccharomyces cerevisiae.

The association between the two trimeric enzymes ornithine carbamoyltransferase and arginase, which is under the control of arginine and ornithine, is endothermic (ΔH° = + 14.6 kcal mol^?1). The process is clearly entropy-driven (ΔS° = + 94.7 cal mol^?1 deg.^?1) allowing a dissociation constant of 0.1 nm for the complex at optimal pH 8, 30 °C and an ionic strength of 0.025. The stability of the complex is moderately sensitive to pH and ionic strength. The dissociation constant of the complex was measured in a medium at cellular pH and salt concentration and found to be close to the constant expected for operative inhibition of ornithine Carbamoyltransferase in the yeast cell. The importance of the presence of arginine as an effector for the formation of the complex under the above conditions is clearly demonstrated.     

Metabolic changes in human skin preserved at −3 and at −196 °C

Full-thickness human skin samples from living donors or split-thickness cadaver skin samples were stored at ?3 °C and at ?196 °C. In both circumstances, additions to the storage medium of cortisol, free l-amino acids, or a combination of both, resulted in an increased protection of the stored tissue. A most striking protective effect on protein synthesizing activity and on the membrane integrity of cutaneous cells was obtained by the addition of adenosine -5′-triphosphate (ATP) to the various storage media.     

Pathways of glutathione degradation in the yeast Saccharomyces cerevisiae

The degradation of glutathione (GSH) in the yeast Saccharomyces cerevisiae appears to be mediated only by γ-glutamyltranspeptidase and cysteinylglycine dipeptidase. Other enzymes of the γ-glutamyl cycle, γ-glutamyl cyclotransferase and 5-oxo-l-prolinase, are not present in the yeast. In vivo transpeptidation was shown in the presence of a high intracellular level of γ-glutamyltranspeptidase, but only when the de-repressing nitrogen source was a suitable acceptor of the transferase reaction. In contrast, when the de-repressing source was not an acceptor of the transferase reaction (e.g. urea), only glutamate was detected. Intracellular GSH is virtually inert when the level of γ-glutamyltranspeptidase is low. Possible roles for in vivo transpeptidation are discussed.     

The role of snail aestivation in transmission of schistosomiasis in changing climatic conditions

Schistosomiasis vector snails are subjected to extreme seasonal changes, particularly in ephemeral rivers and lentic waterbodies. In the tropics, aestivation is one of the adaptive strategies for survival and is used by snails in times of extremely high temperatures and desiccation. Aestivation therefore plays an important role in maintaining the transmission of schistosomiasis. This review assesses the possible impacts of climate change on the temporal and spatial distribution of schistosomiasis-transmitting snails with special emphasis on aestivation, and discusses the effect of schistosome infection on aestivation ability. The impacts of parasite development on snails, as well as physiological changes, are discussed with reference to schistosomiasis transmission. This review shows that schistosome-infected snails have lower survival rates during aestivation, and that those that survive manage to get rid of the infection. In general, snail aestivation ability is poor and survival chances diminish with time. Longer dry periods result in fewer, as well as uninfected, snails. However, the ability of the surviving snails to repopulate the habitats is high.