Interaction of DNA with DNA binding proteins. III. Infectivity of protein-complexed phage fd DNA in Escherichia coli spheroplasts.

Complex formation of circular, single-stranded phage fd DNA with Escherichia coli DNA binding protein HD or phage fd gene 5 protein keeps infection of E. coli spheroplasts at the level of free phage DNA, whereas complexes of this DNA with E. coli DNA unwinding protein show a strongly reduced efficiency of transfection. Displacement of the unwinding protein by HD protein or gene 5 protein also maintains the poor adsorption of the complexes to spheroplasts. Free E. coli DNA unwinding protein and residual amounts of this protein bound to the DNA may interfere with the adsorption and the uptake of the phage genome.     

Enzymatic amidation of recombinant (Leu27) growth hormone releasing hormone-Gly45

By chemoenzymatic synthesis the gene for a (Leu27) analogue of human growth hormone releasing hormone-Gly45 [(Leu27)GHRH-Gly45] was constructed, cloned and expressed in Escherichia coli as a fusion protein with beta-galactosidase under the control of the lac promoter and operator. Upon induction with isopropyl-D-thio-beta-galactopyranoside the fusion protein accumulated to a yield of 15-20% of the total cellular protein. After cyanogen bromide cleavage of the fusion protein the precursor peptide (Leu27)hGHRH-Gly45 was separated by extraction and purified by ion exchange and h.p.l.c.-RP18 chromatography. The purified peptide was analysed by sequencing, isoelectric focusing, amino acid analysis and amino acid analysis after V8 protease digestion. The carboxy-terminal glycine was subsequently amidated by PAM (peptidylglycine-alpha-amidating-monooxygenase), an enzyme which was isolated and characterized from fresh bovine pituitaries. Correct amidation of the penultimate amino acid, leucine, was verified by peptide sequencing with an authentic leucine amide reference.     

Equilibrium and stability in laboratory model of sewage ponds and polluted rivers

The persistence under controlled chemical and physical conditions and the ability to respond to defined environmental changes was follow d in extremely simple (homogenous or 2-phase) ecosystems with continuous or semicontinuous flow. In the homogenous case the Aufwuchs was removed. Primary productivity and community respiration were computed by means of continuous recording of dissolved oxygen. Under constant environmental conditions the systems usually exhibited slow oscillations of the ecosystem parameters around an equilibrium state. The temporal variations of functional criteria such as elimination rate of easily degradable organic substances or daily oxygen amplitude were slight in comparison to the significant random oscillations in individual numbers of the predominating species of phytoplankton and zooplankton. One of the mechanisms responsible for this functional stability may be the inverse relationship between biomass and activity per unit biomass as observed also in the laboratory models. If the sewage pond microecosystems after a shut down in the inflow were operated without any exchange of nutrients and gases they nevertheless maintained a very high level both of autotrophic and heterotrophic metabolism. Step forcing of nutrient (sewage) concentration, dilution rate or day length produced a new equilibrium state within 1 or 2 days, if the functional criteria mentioned above were taken as output signals. Also in the case of pulse forcing such a rapid adaptation was to be observed. This may contribute to the fact, that the turnover rate of the population was in the same range as the renewal rate of the water. The results are discussed with respect to short and long term effects of abatement of pollution from flowing and standing waters.     

Damage and partitioned mortality of teleosts discarded from two Australian penaeid fishing gears

Six field experiments were perfromed to (1) quantify the scale loss and partitioned (immediate and short-term) mortality of key teleosts discarded from a penaeid seiner and trawler operating in 2 estuaries in southeastern Australia, and (2) assess the utility of modified operational and/or onboard handling procedures for maximising survival. For both gears, several non-target species were caught, handled and discarded according to 2 general categories of treatment ('mild' and 'extreme') representing the plausible limits of severity of commercial operations. The mild treatments involved the shortest conventional deployments of the gears followed by the immediate sorting of catches in water-filled trays, while the extreme treatments comprised the longest conventional deployments and sorting in dry trays. Discards were examined for immediate mortalities and scale loss before sorting onboard, while live individuals of key species, along with appropriate numbers of controls, were released into sea cages and monitored for mortalities up to 5 d. For both gears, there was a trend of higher percentages of immediate mortalities in the extreme treatments, and more scale loss from dead than live seined and trawled silver biddy Gerres subfasciatus, seined tarwhine Rhabdosargus sarba and trawled yellowfin bream Acanthopagrus australis. Despite considerable interspecific variabilities, few intraspecific differences were detected between treatments for the short-term mortalities of live discards; however, for all species, these deaths were greater than those incurred by the controls (most of which survived). The partitioned mortality estimates were combined to provide a range of total mortalities for seined (95.97 and 99.07%, respectively) and trawled (71.74 and 97.64%) G. subfasciatus, seined R. sarba (23.95 and 100%) and trawled southern herring Herklotsichthys castelnaui (100%) and A. australis (3.40 and 35.01%). Because most deaths occurred irrespective of onboard handling procedures, we conclude that simply reducing the duration of gear deployments would provide a first step toward mitigating discard mortality in these fisheries.     

Understanding of elderly patients' resuscitation preferences by physicians and nurses.

We compared the understanding by family physicians and nurses of their elderly outpatients'' preferences for cardiopulmonary resuscitation and mechanical ventilation under 3 scenarios reflecting varying qualities of life. Physicians and nurses correctly predicted patients'' treatment preferences in from 59% to 84% and 53% to 78% of cases, respectively, for the various decisions. For most decisions, neither physicians nor nurses were significantly more accurate in their predictions than expected by chance alone. Moreover, nurses and physicians did not significantly agree with one another in their predictions of patients'' preferences for any of these decisions. These results suggest that while nurses'' and physicians'' perceptions of patients'' preferences for life-sustaining treatment are not necessarily similar, neither nurses nor physicians systematically understand their elderly patients'' resuscitation preferences.     

Expression of bovine adrenodoxin in E. coli and site-directed mutagenesis of /2 Fe-2S/ cluster ligands.

Expression systems for adrenodoxin into the periplasm and the cytoplasm of E. coli have been developed as a prerequisite for site-directed mutagenesis studies. In both systems the /2Fe-2S/ cluster of the protein was correctly assembled, the cytoplasmic one gives, however, a tenfold higher expression level. To determine which of the five cysteines at positions 46, 52, 55, 92, and 95 coordinate the /2Fe-2S/ center, they have been individually mutated into serines. From these mutants, only C95S forms a functionally active holoprotein. Thus, residues 46, 52, 55, and 92 are the cysteines that coordinate the /2Fe-2S/ cluster in adrenodoxin.     

Stereospecific production of the herbicide phosphinothricin (glufosinate) by transamination: cloning, characterization, and overexpression of the gene encoding a phosphinothricin-specific transaminase from Escherichia coli

We have cloned the gene encoding a 43-kilodalton transaminase from Escherichia coli K-12 with a specificity for L-phosphinothricin [L-homoalanine-4-yl-(methyl)phosphinic acid], the active ingredient of the herbicide Basta (Hoechst AG). The structural gene was isolated, together with its own promoter, and shown to be localized on a 1.6-kilobase DraI-BamHI fragment. The gene is subject to catabolite repression by glucose; however, repression could be relieved completely when 4-aminobutyrate (GABA) served as the sole nitrogen source. The regulation pattern obtained and a comparison of the restriction map of the initially cloned 15-kilobase SalI fragment with the physical map of the E. coli K-12 genome suggest that the cloned gene is identical with gabT, a locus on the gab gene cluster of E. coli K-12 which codes for the GABA:2-ketoglutartate transaminase (EC 2.6.1.19). A number of expression plasmids carrying the isolated transaminase gene were constructed. With these constructs, the transaminase expression in transformants of E. coli could be increased up to 80-fold compared with that in a wild-type control, and the transaminase constituted up to 20% of the total soluble protein of the bacteria. Thus, the protein crude extracts of the transformants could be used, after a simple heat precipitation step, for the biotechnological production of L-phosphinothricin in an enzyme reactor.