Influence of Methanogenic Populations in Holocene Lacustrine Sediments Revealed by Clone Libraries and Fatty Acid Biogeochemistry

Methanogenic populations were investigated in subsaline Laguna Potrok Aike sediments, southern Argentina. Microbial density and activity were assessed via cell count and in situ ATP detection for the last ～11K years. Methanogen phylogenetics highlighted species stratification throughout depth, whereas CO₂ reduction was the major pathway leading to methane production. Organic substrates, characterized using pore water analysis, bulk organic fractions and saturated fatty acids, showed a clear link between sediment colonization and initial organic sources. Concentrations and δ¹³C compositions of methane and fatty acids provided final evidence of a microbial imprint on Holocene organic proxies in the most colonized intervals.     

Anterograde Axonal Transport of Peptidylglycine α-Amidating Monooxygenase in Rat Sciatic Nerves

Axonal transport of peptidylglycine alpha-amidating monooxygenase (PAM) activity was studied in rat sciatic nerves from 12 to 120 h after double ligations. The anterograde axonal transport increased and reached a plateau between 48 and 72 h and then decreased. The flow rate was 100 mm/day, and the molecular mass of the active entity was 70 kDa, which was determined by gel filtration. In contrast, there was no evidence for significant retrograde axonal transport. Anterograde axonal transport of immunoreactive cholecystokinin, a carboxy-terminal-amidated putative neuropeptide, was also found. These results suggest that PAM is transported by a rapid axonal flow and may play a role as a processing enzyme during transport or in the terminals of rat sciatic nerves.     

Protein kinase A inhibitors enhance radiation-induced apoptosis

In addition to a role for de novo protein synthesis in apoptosis we have previously shown that activation of a protein phosphatase or loss of activity of a kinase is also important in radiation-induced apoptosis in human cells [Baxter, and Lavin (1992): J Immunol 148:149–1954]. We show here that some inhibitors of protein kinases exacerbate radiation-induced apoptosis in the human cell line BM13674. The specific protein kinase A inhibitor isoquinoline sulfonamide (20 μM) gave rise to significantly increased levels of apoptosis at 2–6 h postirradiation compared to values after radiation exposure only. The same concentration of isoquinolinesulfonamide, which was effective in increasing apoptosis, reduced activity markedly. A 66% inhibition of cyclic AMP-dependent protein kinase A activity occurred in unirradiated cells at this concentration of H89 and activity was reduced to 58% in irradiated cells. Calphostin C, a specific inhibitor of protein kinase C, at a concentration of 0.1 μM, which caused 68% inhibition of enzyme activity in irradiated cells, failed to enhance the level of radiation-induced apoptosis. Other kinase inhibitors did not lead to an additional increase in apoptosis over and above that observed after irradiation. The results obtained here provide further support for an important role for modification of existing proteins during radiation-induced apoptosis.     

Fatty Acid Specificity in Lipase-Catalyzed Synthesis of Glucoside Esters

The fatty acid specificity of the B-lipase derived from Candida antarctica was investigated in the synthesis of esters of ethyl D-glucopyranoside. The specificity was almost identical with respect to straight-chain fatty acids with 10 to 18 carbon atoms. However, lower fatty acids such as hexanoic and octanoic acid and the unsaturated 9-cis-octadecenoic acid were found to be poor substrates of the enzyme. As a consequence of this selectivity, these fatty acids were accumulated in the unconverted fraction when ethyl D-glucopyranoside was esterified with an excess of a mixture of fatty acids. This accumulation can reduce the overall effectiveness of the process as the activity of the lipase was found to be reduced when exposed to high concentrations of short-chain fatty acids. Finally, using a simplified experimental set-up, the specificity of the C. antarctica B-lipase was compared to the specificity of lipases derived from C. rugosa, Mucor miehei, Humicola, and Pseudomonas. Apart from the C. rugosa lipase, which exhibited a very poor performance, all the enzymes showed a very similar specificity with respect to fatty acids longer than octanoic acid while only the C. antarctica B-lipase showed activity towards sort-chain fatty acids.     

Ala258Phe substitution in Bacillus sp. YX-1 glucose dehydrogenase improves its substrate preference for xylose

Bacillus sp. YX-1 glucose dehydrogenase (BsGDH) with good solvent resistance catalyzes the oxidation of β-d-glucose to d-glucono-1,5-lactone. Xylose is a recyclable resource from hemicellulase hydrolysis. In this work, to improve the preference of BsGDH for xylose, we designed seven mutants inside or adjacent to the substrate binding pocket using site-directed mutagenesis. Among all mutants, Ala258Phe mutant displayed the highest activity of 7.59 U mg⁻¹ and nearly 8-folds higher k_cat/K_m value towards xylose than wild-type BsGDH. The kinetic constants indicated that the A258F mutation effectively altered the transition state. By analysis of modeled protein structure, Ala258Phe created a space to facilitate the reactivity towards xylose. A258F mutant retained good solvent resistance in glycol, ethyl caprylate, octane, decane, cyclohexane, nonane, etc. as with BsGDH. This work provides a protein engineering approach to modify the substrate stereo-preference of alcohol dehydrogenase and a promising enzyme for cofactor regeneration in chiral catalysis.     

Localization of Endo-Oligopeptidase (EC 3.4.22.19) in the Rat Nervous Tissue

The subcellular and regional distribution of endo-oligopeptidase (EC 3.4.22.19), an enzyme capable of generating enkephalin by single cleavage from enkephalin-containing peptides, was determined by an enzymatic assay using metorphamide and by immunochemical techniques in the CNS of the rat. The rat CNS contains a membrane-associated form of endo-oligopeptidase, an enzyme predominantly associated with the soluble fraction of brain homogenates. Subcellular fractionation showed that approximately 17% of the total activity of the enzyme is associated with membrane fractions including synaptosomes. Synaptosomal membranes were prepared from neocortex, striatum, hypothalamus, medulla, spinal cord, and cerebellum. The amount of EC 3.4.22.19 activity solubilized by 3-[( 3-cholamidopropyl]dimethylammonio)-1-propanesulfonate from synaptosomal membranes was similar in neocortex, striatum, and hypothalamus, being three- to 10-fold greater than in spinal cord, cerebellum, and medulla. A polyclonal antibody exhibiting high affinity for endo-oligopeptidase was raised in rabbits against the purified rat brain enzyme and used to localize endo-oligopeptidase by Western blotting and by immunoperoxidase techniques. A strong band corresponding to the Mr of EC 3.4.22.19 was found in solubilized proteins obtained from synaptosomal membranes prepared from hypothalamus, neocortex, and striatum when subjected to Western blotting. The immunohistochemical localization of endo-oligopeptidase indicated that the immunoreactivity was confined to gray matter in regions known to be rich in peptide-containing neurons such as the striatum. In the cerebellum, a region poor in peptides, no staining could be detected. The nonuniform distribution of endo-oligopeptidase in rat brain suggests a role in neurotransmitter processing in the CNS.     

Biology and host preference of Selitrichodes neseri: A potential biological control agent of the Eucalyptus gall wasp,Leptocybe invasa

Selitrichodes neseri (Hymenoptera: Eulophidae) is a parasitoid of the invasive gall-forming wasp Leptocybe invasa (Hymenoptera: Eulophidae), which has caused serious damage to Eucalyptus plantations in many parts of the world. S. neseri is a recently discovered parasitoid considered to be a potentially important biological control agent of L. invasa. The aim of this study was to provide the first basic data on the biology of S. neseri, which is essential for its application in biological control. S. neseri was shown to be a biparental ectoparasitoid. Observation from dissected galls indicated that the parasitoid developed on late larvae, pupae and callow adults, although development did occur in a range of gall ages. Observed nominal parasitism in captivity ranged from 9.7% to 71.8%. Adult S. neseri specimens, fed with honey-water and galled Eucalyptus leaves, survived an average of 26 days at 26 °C. The average developmental time from oviposition to emergence was 19.3 days ± 0.2 days. There was no pre-oviposition period. A single female produced a maximum of thirty-nine offspring, with a maximum of ten per day. Dissection of the ovaries showed that twelve ovarioles were present. The sex ratio of S. neseri observed in this study was 1:3.43 males:females. Galls of native insects most closely related to L. invasa and to galls of similar morphology to L. invasa-induced galls, were not suitable for S. neseri oviposition. S. neseri showed considerable potential as a biological control agent of L. invasa due to its relatively short developmental time, long adult life span when supplemented with carbohydrates, ability to utilize a range of gall ages and the fact that it has a high level of host specificity.     

Development of a one-step enzyme immunoassay for the quantitation of gibberellin A3 in barley seeds

The development of a sensitive and specific enzyme immunoassay for GA₃ is reported. This method was based on the use of peroxidase labelled GA₃ and immobilized antibodies. In order to obtain a rapid immunoassay, several steps of purification were analyzed to show their necessity. Barley seed extracts were assayed at different steps of purification to exhibit the effect of extract components on the assay. It was demonstrated that HPLC had to be performed when a selective quantitation of GA₃ was required. This assay allowed GA₃ to be measured with reproducibility as its unmethylated form and the quantitation of GA₃ in barley seeds with this enzyme immunoassay was correlated to a GC-MS method.Abbreviations GA₃ gibberellin A₃ - EIA enzyme immunoassay - DMF dimethylformamide - TEA tri(n)ethylamine - BSA bovine serum albumin - OVA ovalbumine - ECF ethylchloroformate - PB phosphate buffer     

Substrate specificity of the erythrocyte Ca2+-ATPase

In the absence of Mg²⁺, the observed activity of the erythrocyte plasma membrane Ca²⁺-ATPase is due to the hydrolysis of CaATP at a low rate. In the presence of Mg²⁺, the activity of the enzyme is much higher, but it is inhibited by high levels of free Mg²⁺. This inhibition appears to be due to competition of Mg²⁺ and Ca²⁺ for a site on the enzyme, rather than for ATP.     

Transmission characteristics of Spiroplasma citri and its effect on leafhopper vectors from the Circulifer tenellus complex

Several leafhopper variants of the Circulifer tenellus complex were collected in “citrus stubborn” affected areas in Israel. Two of these variants transmitted the Spiroplasma citri to Matthiola incana after being injected with the disease agent. The variant from Atriplex halimus was designated Circulifer tenellus-A (CTA) and the variant from Portulaca oleracea was designated Circulifer tenellus-? (CTP). Transmission characteristics were determined for both leafhoppers. A high rate of transmission (43.3%) was obtained by single CTA leafhoppers that were injected with the Amiad S. citri isolate from the Upper Galilee, compared with 7% transmission obtained with the CTP leafhoppers. The Gilgal S. citri isolate from the Jordan Valley, was not transmitted by either. Injection was more effective than acquisition access feeding to render the leafhopper infective for both CTA and CTP. The minimum acquisition access period needed for the CTA variant to transmit the Amiad isolate was 1 h. Longer AAPs did not necessarily result in a higher rate of transmission. The minimum incubation period was 6 days and the maximum was 32 days. The LP₅₀ calculated from the logarithmic curve y = 45.74Ln(x)–53.68 was 9.64 days. The minimum inoculation access period (IAP) was lh. The same transmission parameters for the CTP variant could not be determined, as no transmission was obtained even when groups of five-six insects were placed on a single plant.