Mapping of the constitutive lysyl-tRNA synthetase gene of Escherichia coli K-12.

The constitutive lysyl-tRNA synthetase gene (lysS) was mapped at 62.1 min on the Escherichia coli chromosome by a combination of conjugation and transduction, with physical confirmation by two-dimensional gel electrophoresis. Revertant analysis suggests that the altered isoelectric point and the low amount of the mutant LysS protein may be due to a single mutational event.     

Signaling Defect in the Activation of Caspase-3 and PKCδ in Human TUR Leukemia Cells Is Associated with Resistance to Apoptosis

Exposure of the two related human leukemic cell lines U937 and TUR to chemotherapeutic compounds resulted in opposite effects on induction and resistance to apoptosis. Incubation of U937 cells with 1-β- -arabinofuranosylcytosine or the etoposide VP-16 was accompanied by growth arrest in G₀/G₁of the cell cycle and an accumulation of a population in the sub-G₁phase which exhibited characteristics typical for the apoptotic pathway. In contrast, human TUR leukemia cells demonstrated no significant effects after a similar treatment with Ara-C and VP-16. Thus, TUR cells continued to proliferate in the presence of these anti-cancer drugs and the number of apoptotic cells as evaluated by propidium iodide staining and the detection of internucleosomal DNA fragmentation was significantly reduced when compared to the parental U937 cells. Similar effects were observed upon serum-starvation demonstrating resistance to apoptosis in TUR cells. Whereas induction of apoptosis is regulated by a network of distinct factors including the activation of proteolytically active caspases, we investigated these pathways in both cell lines. U937 cells demonstrated activation of the 32-kDa caspase-3 upon drug treatment by cleavage into the 20-kDa activated form. However, there was no 20-kDa caspase-3 fragment detectable in TUR cells. Simultaneously, the enzymatic activity of caspase-3 was significantly increased in drug-treated U937 cells as measuredin vitroby enhanced metabolization of a fluorescence substrate andin vivoby cleavage of an appropriate substrate for caspase-3, namely, protein kinase Cδ. In contrast, there was little if any caspase-3 activation detectable in drug-treated TUR cells. Taken together, these data suggest a signaling defect in the activation of the caspase-3 proteolytic system in TUR cells upon treatment with chemotherapeutic compounds which is associated with resistance to apoptosis in these human leukemia cells.     

Immunohistochemical localisation of ecdysteroids in the follicular epithelium of locust oocytes

Summary Frozen sections of growing terminal follicles of the locust ovary were incubated with an ecdysteroid-specific rabbit antibody and the bound antibody visualised by the use of FITC-labelled goat-anti-rabbit antiserum. A bright fluorescence was seen in the cytoplasm of the follicle cells in terminal follicles with a length between 4.0 and 6.0mm with a maximum intensity at 5.5mm, indicating the presence of ecdysteroids in these cells in this particular developmental stage.     

Excretion of juvenile hormone and its metabolites in the locust, Locusta migratoria

Racemic synthetic ³HC₁₈ juvenile hormone, dissolved in paraffin oil, was injected into adult Locusta migratoria and the excreted radioactive material in the faeces was determined. Within 48 hr two-thirds of the injected radioactivity can be recovered in the frass, half of it within 3 hr. The remaining one-third of the injected label is incorporated or is released as water. Adult locusts of either sex or of different ages show no difference in the metabolic pathways of the JH and its excretion rate.The excreta contain as a degradation product 7-ethyl-3,11-dimethyl-cis-10,11-epoxy-trans, trans-2,6 trideca-dienoic acid, the corresponding dioldienoic acid and the dioldienoic methyl ester. Unchanged Cecropia JH was also found in the frass. The radioactive hormone, as well as the metabolites, were excreted mainly by the Malpighian tubules; smaller amounts of the radioactive material were also found in the fore-, mid, and hindgut.     

A radioimmunoassay for arthropod moulting hormones, introducing a novel method of immunogen coupling

Inokosteron-26-oic acid was coupled to thyroglobulin in aqueous pyridine by a water-soluble carbodiimide. After exhaustive dialysis and gel filtration on Sephadex G-25 in the presence of sodium dodecylsulfate, a coupling ratio of 164 haptens per molecule of thyroglobulin was determined. In all three animals injected with the conjugate, ecdysone-binding antibodies were detected. After one booster injection the antiserum could be diluted 1 : 5000 (1 : 4000, or 1 : 2000) in order to get a 50% binding of [3H]ecdysone. The dissociation constant was calculated as 5.8 X 10(-10) MOL/L. The antiserum has a greater affinity for ecdysone and 22-isoecdysone than for all other ecdysteroids and steroids tested.     

Absence of humic substance reduction by the acidophilic Fe(III)-reducing strain Acidiphilium SJH: implications for its Fe(III) reduction mechanism and for the stimulation of natural organohalogen formation

A vast amount of volatile organohalogens (VOX) has natural origins. Both soils and sediments have been shown to release VOX, which are most likely produced via redox reactions between Fe(III) and quinones in the presence of halide anions, particularly at acidic pH. We tested whether acidophilic Fe(III)-reducers might indirectly stimulate natural VOX formation at acidic pH by providing reactive Fe and quinone species. However, it is unknown whether acidophilic Fe(III)-reducers can reduce humic acids (HA) or fulvic acids (FA). We therefore tested the ability of the acidophilic Fe(III)-reducer Acidiphilium SJH to reduce macromolecular, suspended HA and dissolved FA at pH 3.1–3.3. We found that (i) SJH can neither reduce HA/FA nor the humic model quinone anthraquinone-2,6-disulfonic-acid (AQDS) nor stimulate the formation of FA radicals, (ii) at acidic pH, significantly more electrons are transferred abiotically both from native and reduced FA to dissolved Fe(III) than from native or reduced HA, and (iii) the presence of strain SJH does not stimulate VOX formation. Our results imply that the acidophilic Fe(III)-reducer SJH either uses an enzyme for Fe(III) reduction that can neither be used for HA/FA nor for AQDS reduction or that the location of Fe(III) reduction is inaccessible for these compounds. We further conclude that microorganisms such as strain SJH probably do not indirectly stimulate natural VOX formation at acidic pH via the formation of reactive quinone species.     

Catalytical oxidation of ecdysteroids to 3-dehydro products and their biological activities

Oxidation of ecdysone and ecdysterone with platinum as catalyst gives rise to several polar and 4–5 apolar substances, among them 3-dehydroecdysone and 3-dehydroecdysterone. Highest yields in the 3-dehydro products were reached after 4 to 8 hr of oxidation. 3-dehydroecdysone and 3-dehydroecdysterone are less active in inducing ecdysone specific puffs in Drosophila hydei salivary gland giant chromosomes but still contain a remarkable biological activity when compared with the original ecdysteroids. The activity of these two compounds is about one tenth that of ecdysone in the Calliphora assay.