Characterization of polygalacturonase-overproducing Aspergillus niger transformants

Summary Aspergillus niger pyrA co-transformants with multiple copies of the gene (pgaII) encoding the major endopolygalacturonase were characterized in detail. Typically, these transformants produced tenfold or more polygalacturonase from plasmids that had integrated in most cases at ectopic sites, in comparison to the untransformed strain. Some mitotic instability was observed upon application of a positive selection procedure for reversion of the pyrA marker. Analysis of these strains indicated that the most frequent event involved is the excision of part of the array of tandemly integrated plasmids, without scrambling of the plasmids remaining in the chromosome. From promoter deletion analysis it was concluded that the pgaII gene is subject to positive control. The putative positive regulatory protein appears not to be limiting for overexpression of the pgaII gene. Correspondence to: J. Visser     

Isolation and characterization of beta- and gamma-caseins from horse milk.

Three groups of casein components were isolated from horse milk. Group I is almost insoluble at acid and neutral pH, and is rather heterogeneous on alkaline gels with or without sodium dodecyl sulphate. Group II shows strong similarity to beta-casein from other species, as concluded from its amino acid composition and its N- and C-terminal sequences. This group consists of five electrophoretically distinguishable forms, all containing ester phosphate groups but no carbohydrate. Group III is composed of C-terminal fragments of the beta-like (group II) fraction and probably arises from the action of a plasmin-like enzyme present in horse milk. It does not contain phosphate or carbohydrate. Homology of this group with bovine gamma-caseins is demonstrated. Both beta- and gamma-like caseins are more soluble at 4 degrees C than at room temperature.     

Discontinuation syndrome after continuous infusion of clonidine in the spontaneously hypertensive rat

In conscious spontaneously hypertensive rats prepared with permanent indwelling aortic catheters the continuous infusion of clonidine (500 μg/kg/day) via an ALZET miniosmopump induced significant reductions in blood pressure and heart rate. These effects were well sustained during 12 days of treatment. A marked overshoot in heart rate was observed following withdrawal of clonidine administration. The tachycardia persisted for more than 36 hours. Mean arterial pressure exceeded control level slightly in the immediate withdrawal period only, whereas significant blood pressure lability was observed for more than 36 hours. These withdrawal symptoms were accompanied by an elevation of plasma noradrenaline concentration. The present study shows the consistent antihypertensive and bradycardic activities of clonidine during 12 days of infusion in spontaneously hypertensive rats. Furthermore, this model may provide a useful tool in the study of withdrawal phenomena of antihypertensive drugs.     

The enzymes of the ammonia assimilation in Pseudomonas aeruginosa

Glutamine synthetase from Pseudomonas aeruginosa is regulated by repression/derepression of enzyme synthesis and by adenylylation/deadenylylation control. High levels of deadenylylated biosynthetically active glutamine synthetase were observed in cultures growing with limiting amounts of nitrogen while synthesis of the enzyme was repressed and that present was adenylylated in cultures with excess nitrogen.NADP-and NAD-dependent glutamate dehydrogenase could be separated by column chromatography and showed molecular weights of 110,000 and 220,000, respectively. Synthesis of the NADP-dependent glutamate dehydrogenase is repressed under nitrogen limitation and by growth on glutamate. In contrast, NAD-dependent glutamate dehydrogenase is derepressed by glutamate. Glutamate synthase is repressed by glutamate but not by excess nitrogen.     

Use of Transposons in Cloning Poorly Selectable Genes of Escherichia coli: Cloning of uvrA and Adjacent Genes

A transposon was introduced close to a poorly selectable gene. This gene could be cloned by using selection for the antibiotic resistance marker of the transposon.     

Life span changes in the presence of alpha-melanocyte-stimulating-hormone-containing cells in the human pituitary.

Since recent circumstantial evidence has suggested possible functions of alpha-MSH in intrauterine growth and labour, the presence of this hormone in the human pituitary was determined by means of the indirect immunofluorescence procedure during development and adulthood. Cross reaction of the antibodies with other peptides was measured after which they were purified by solid phase absorption. Experiments on the rat pituitary showed that staining of alpha-MSH- and ACTH-containing cells could be obtained well until 48 h after death. In the pars distalis the ability of ACTH-containing cells to take up stain increased during the period of post-mortem storage. In the youngest human fetus studied (15 weeks) only alpha-MSH-containing cells were found in the pars intermedia and no ACTH-containing cells were observed. In the other fetal pituitaries a distinct pars intermedia containing more alpha-MSH cells than ACTH cells was found. In the pars distalis of the fetuses more ACTH- than alpha-MSH-containing cells were observed. From birth to 19 years, progressively fewer alpha-MSH containing cells could be detected in the 'zona intermedia' and pars distalis, while in adults only a few such cells were found in either area. Irrespective of age, sex, cause of death or therapy, alpha-MSH-containing cells were found in all pituitaries throughout life. The number of ACTH containing cells gradually increased in the zona intermedia and pars distalis and reached a high adult level in the latter structure. In the pituitaries of seven anencephalics, no alpha-MSH-containing cells were present. The presence of alpha-MSH in the fetal pars intermedia, the change in the ratio of the alpha-MSH/ACTH cells during the course of development, and the absence of alpha-MSH in anencephaly all support the possibility that human fetal pituitary alpha-MSH is involved in both intrauterine growth and fetal adrenal function and thus also in parturition.     

The measurement of subnanosecond fluorescence decay of flavins using time-correlated photon counting and a mode-locked Ar ion laser.

A system is described consisting of a mode-locked Ar ion laser and time-resolved photon-counting electronics. The system is capable of measuring fluorescence lifetimes in the subnanosecond time domain. The Ar ion laser is suitable for the excitation of flavins, since the available laser wavelengths encompass the first absorption band of the yellow chromophore. Due to the high radiation density and the short pulse, both the time and wavelength resolution of the fluorescence of very weakly emitting compounds can be measured. Experiments have been described for flavin models exhibiting single and multiple modes of decay. In these examples lifetimes were determined both from deconvolved decay curves and from direct analysis of the tail of the curve, where no interference of the exciting pulse is encountered. Both determinations showed very good agreement. Due to the highly polarized laser light the decay of the emission anisotropy could be measured directly after the exciting pulse. In principle, fast rotational motions might be detected. An anisotropy measurement conducted with a flavoprotein with a noncovalently attached FAD is presented.     

Binding of radioiodinated propylthiouracil to rat liver microsomal fractions. Stimulation by substrates for iodothyronine 5''-deiodinase.

Previous studies have shown that 2-thiouracil derivatives are uncompetitive inhibitors of iodothyronine 5'-deiodinase activity of rat liver microsomal fraction. Therefore the interaction of radioiodinated 6-propyl-2-thiouracil with rat liver microsomal fraction and the effect of substrate, cofactor and other inhibitors of 5'-deiodinase activity activity were investigated. It was found that micromolar concentrations of, in order of increasing potency, 3,5-diiodotyrosine, thyroxine, 3,3',5'-tri-iodothyronine and 3',5'-di-iodothyronine significantly enhanced binding of 5-[125I]iodo-6-propyl-2-thiouracil to the enzyme preparation. This stimulation was not seen in the presence of 1 mM dithiothreitol, 0.1 mM-6-propyl-2-thiouracil, 0.1 mM-6-propyl-2-thiouracil, 0.1 M-2-mercapto-1-methylimidazole or 1 mM-sodium sulphite. These results support the hypothesis that thiouracil derivatives inhibit 5'-deiodinase activity by forming a mixed disulphide with an intermediate enzyme complex, probably a sulphenyl iodide.