Double-stranded RNA unwinding and modifying activity is detected ubiquitously in primary tissues and cell lines.

A double-stranded RNA unwinding and modifying activity was found to be present in a wide range of tissues and cell types. The level of activity did not vary significantly with respect to the state of cell differentiation, cell cycle, or transformation. Thus, the unwinding and modifying activity, localized in the nucleus in somatic cells and capable of converting many adenosine residues to inosine, appears to be one of the housekeeping genes.     

Recombinant human prorenin from CHO cells: Expression and purification

The gene for human preprorenin was obtained from total RNA prepared from primary human chorion cells. An expression vector was constructed containing an SV40 early promoter, a human preprorenin cDNA, bovine growth hormone poly-A addition signal, and a dihydrofolate reductase (dhfr) expression cassette. This vector was inserted into the DXB-11 Chinese hamster ovary (CHO) cell line. The recombinant protein was exported by CHO cells into the tissue culture media. At harvest the prorenin levels ranged from 1–5 mg/L. For prorenin isolation the cell culture supernatants were processed by filtration, concentration, dialysis, and batch extraction. Preparative-scale isolation of prorenin was accomplished using blue-dye chromatography and size-exclusion chromatography. The isolated prorenin yielded a single SDS-gel band with Mr 40,000. The proprotein was characterized with respect to N-terminal sequence and N-linked sugar composition. Trypsin-activated renin prepared from the proprotein was characterized with respect to N-terminal sequence andpH-activity profile. Enzyme activity was measured with a newly developed fluorogenic peptide substrate containing the P₆-P₃ sequence of human angiotensinogen.     

The fluorescence induction kinetics as a non-destructive tool for investigating spruce treated with ozone

Summary The scattering coefficient of yellow spruce needles exceeds that of green needles by a factor of 2, whereas the fluorescence efficiency is approximately equal for both needle colours. As shown by the angular distribution the fluorescence light is diffusely emitted. However, the scattered light consists of a diffuse and a reflecting portion below 20° with a ratio of the intensities of 1 : 2 at perpendicular observation (0°). Control measurements show that in the rejection region the effective transmission of cut-off-filters commonly used to separate fluorescence light and excitation light exceeds the value calculated from the filter specifications by a factor of 100. Therefore, the portion of the scattered light in the measuring signal must be controlled if the fluorescence induction kinetics is measured from specimen of different colour. A device for the determination of the fluorescence induction kinetics is described which employs a He-Ne laser, a mechanically working shutter with an opening time of 4 ms for the excitation, and a computer for data storage and device control. Two filters select the fluorescence components at 685 nm and 730 nm and they reduce the portion of the scattered light in the measuring signal to 0.18% and 0.55%, respectively. In order to consider the temporal development of the fluorescence kinetics the sampling rate is reduced from 2 kHz to 1 Hz. From the data stored in the computer maximum valueF _P, and steady-state-valueF _S are determined for both fluorescence components. Measurements on 4-year-old spruce exposed to ozone-concentrations of 0, 300 ppb, 600 ppb, and 1000 ppb were repeated every week. With increasing concentration and duration of treatmentR _fd =(F _P-F_s)/F _S was decreased for both fluorescence components. With the highest ozone concentration a reduction ofR _fd of 23% and 24%, respectively, was obtained for the two fluorescence components after three weeks.     

Effects of trypsin on secretion stimulated by micromolar Ca2+ and phorbol ester in digitonin-permeabilized adrenal chromaffin cells

1. Catecholamine secretion from digitonin-treated chromaffin cells is stimulated directly by micromolar Ca2+ in the medium. The permeabilized cells are leaky to proteins. 2. In this study trypsin (30-50 micrograms/ml) added to cells after digitonin treatment completely inhibited subsequent Ca2+-dependent catecholamine secretion. The same concentrations of trypsin did not inhibit secretion from permeabilized cells if trypsin was present only prior to cell permeabilization. 3. The data indicate that trypsin entered digitonin-treated chromaffin cells which were capable of undergoing secretion and that an intracellular, trypsin-sensitive protein is involved in secretion. Chymotrypsin was less potent but had effects similar to those of trypsin. 4. The enhancement of Ca2+-dependent secretion from permeabilized chromaffin cells induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was inhibited by trypsin added simultaneously with Ca2+ to permeabilized cells at concentrations (3-10 micrograms/ml) which had little or no effect on Ca2+-dependent secretion from cells untreated with TPA. Ca2+-dependent secretion in TPA-treated cells was reduced by trypsin only to the level that would have occurred in cells not treated with TPA. Trypsin reduced the large TPA-induced increment of membrane-bound protein kinase C.     

Further localization of ETS1 indicates that the chromosomal rearrangement in Ewing sarcoma does not occur at fra(11)(q23)

Summary A genomic probe homologous to 5.4 kb of the c-ets-1 gene was hybridized in situ to chromosomes expressing fra(11)(q23). This probe hybridized distal to the fragile site, which is just distal to the midpoint of band 11q23.3. This result localizes ETS1 from the FRA11B locus to 11q24. The result also distinguishes the FRA11B locus from the site of translocation at 11q23-q24 in the Ewing sarcoma- and peripheral neuroepithelioma-specific t(11;22), indicating that the chromosomes of a previously reported patient heterozygous for fra(11)(q23) did not rearrange at this fragile site to give rise to Ewing sarcoma. This adds to the mounting evidence against individuals with fragile sites being predisposed to developing cancer.     

Production and purification of Escherichia coli fimbrial antigen F165

Abstract The production of fimbrial antigen F165 by Escherichia coli strains was found to be dependent on the composition of the culture medium and was repressed in the presence of alanine or high levels of glucose, in anaerobic conditions or at growth temperatures of lower than 37°C. Optimal F165 production was found on a minimal medium containing 1% (w/v) casamino acids (MD-1). F165 antigen was isolated from bacteria by mechanical shearing, precipitated with ammonium sulfate, and purified by deoxycholate treatment and gel filtration on Superose 12. The purified fimbriae retained their native morphology as observed by electron microscopy and consisted of two separate protein subunits with apparent molecular weights of 17 500 and 19 000 on sodium dodecyl sulfate-polyacrylamide gels.     

Physiological control and process kinetics of clavine alkaloid production byClaviceps purpurea

Summary Kinetic parameters of production of clavine alkaloids were evaluated in twoClaviceps purpurea strains. Mutagenesis brought about enhanced resistance of the biosynthetic system towards alkaloids. Addition of glucose into the fermentation medium altered the zero order kinetics of production to activation-inhibition kinetics. The glucose treatment allowed performance of both elymoclavine-inhibitionless and clavine alkaloid-decompositionless fermentations if a combination of fermentation and separation units in a closed loop was used.Nomenlacture k ₁ rate constant of agroclavine synthesis (mg Agro · mg Elymo/l·g DW·day for stage 1, mg Agro/g DW·day for stage 2) - k ₂ parameter describing inhibition of agroclavine formation rate by elymoclavine (mg Elymo/l) - k ₃ specific rate of agroclavine decay (l/g DW·day) - k ₄ maximal specific rate of elymoclavine synthesis (stage 1, 1/g DW·day, stage 2, mg Elymo/g DW·day) - k ₄ ^– maximal specific rate of elymoclavine synthesis in stage 1 (inhibition-activation mechanism) (mg Elymo/g DW·day) - k ₅ physiological constant describing the elymoclavine decay rate (l²/g DW·day·mg Elymo) - k ₅ ^– physiological constant describing the activation of elymoclavine biosynthesis by elymoclavine (mg Elymo/l) - k ₆ physiological constant describing the repression of elymoclavine biosynthesis by elymoclavine (mg Elymo/l) - k ₇ maximal specific growth rate (1/day) - k ₈ specific rate of biomass decay (l/g DW·day) - A agroclavine concentration (mg/l) - E elymoclavine concentration (mg/l) - r _A specific rate of agroclavine biosynthesis (mg Agro/g DW·day) - r _E specific rate of elymoclavine biosynthesis (mg Elymo/g DW·day) - r _i specific rate of alkaloid biosynthesis (mg alkaloid/g DW·day) - X dry biomass concentration (g/l) - specific growth rate (1/day) Abbreviations Agro agroclavine - Elymo elymoclavine - Chano chanoclavine - DW dry weight of biomass     

Autoradiographic Analysis of [3H]Imipramine Binding in the Human Brain Postmortem: Effects of Age and Alcohol

In vitro quantitative autoradiography of high-affinity [3H]imipramine binding sites was performed on 16 human brains postmortem. The densities of binding sites were highest in the hypothalamus. Next, in descending order, were the basal and lateral nuclei of the amygdala; substantia innominata; insular cortex; the central nucleus of the amygdala; the anterior nucleus of the thalamus; the head of the caudate nucleus; portions of the frontal, parietal, and temporal cortex; claustrum; the granular layer of the dentate gyrus; substantia nigra; the pyramidal layer of CA fields; globus pallidus; red nucleus; and white matter. Imipramine binding was found to increase with age in a region-specific manner. The presence of alcohol had a similar effect, which was most pronounced in the hippocampus. Sex and time from death to autopsy did not affect imipramine binding, in our sample.     

High Ammonia Levels in Brain Induce Tubulin in Cerebrum but Not in Cerebellum

Ingestion of large amounts of ammonium increases markedly the content of tubulin in brain. The effect on tubulin induction of ammonium ingestion for up to 100 days was investigated. Brain tubulin content showed a rapid initial increase (28%) at 2 days and reached 50% after 100 days on the diet. To discern if ammonia, the increase in urea synthesis, or both was responsible for tubulin induction, rats were maintained at several levels of uremia (by administering diets containing 0 to 80% protein) or in hyperammonemia (by urease treatment). Only ammonium administration in the diet and urease injection induced tubulin in brain. Tubulin was quantified in three different brain regions. There was a regional selectivity of tubulin induction by ammonia in rat brain. Whereas the cerebellum remained unaltered, the paleencephalon showed the highest increase, and the cerebral cortex exhibited only a modest increase.