A new human growth hormone production process using a recombinant Bacillus subtilis strain.

We constructed a series of hybrid plasmids which directed the synthesis of different human growth hormone (hGH) precursor sequences in Bacillus subtilis. In addition to the 191 amino acids of the hormone, the precursors had in common an amino-terminal extension characterized by the presence of a methionine at position 1 and of the tetrapeptide Ile-Glu-Gly-Arg preceding the first residue (Phe) of hGH. The sequence between the methionine and the tetrapeptide was specific for each precursor and, because of the presence of charged residues, conferred particular properties to the molecules. Long homopolymeric tail-containing precursors such as MRRRRRRIILM-IEGR appeared insoluble whereas shorter sequences of the type MRR-IEGR and MEELM-IEGR augmented the solubility of the precursors with respect to Met-hGH. The soluble precursors could be easily purified from the bulk proteins taking advantage of the charged residues present on the N-terminal tail. After purification, the natural hGH was obtained by treating the precursors with the protease Factor Xa which cleaves after the arginine residue of the tetrapeptide IEGR. A protocol for the production and purification of authentic hGH from a strain expressing one of these soluble precursors is reported.     

On possible different mechanisms subserving the release of arachidonic and di-homo-gamma-linolenic acids for the formation of monoenoic and bisenoic prostaglandins in uteri from ovariectomized rats

The uptake of arachidonic acid (AA) and of di-homo-gamma-linolenic acid (DGLA) and their incorporations into phospholipids (PLs) and into neutral lipids (NLs) of uteri isolated from spayed rats and the effect of inhibiting triglyceride (TG) metabolism with 4-pentenoic acid (4-PEA) on tissue TG levels and the output of prostaglandins (PGs), were explored. Attempts were also made to determine whether the acylation of labelled AA and of labelled DGLA into PLs and TGs is different and to confirm possible correlations between the synthesis of PGE1 and the degradation of TGs. Uterine PLs incorporated significantly less DGLA than AA (P less than 0.05). AA was acylated mainly into the phosphatidylinositol (PI) and into phosphatidylcholine (PC) subfractions of rat uteri, whereas the incorporation of DGLA into these two subfractions was significantly smaller than that of AA. The acylation of labelled DGLA into NL fractions, mainly into triacylglycerol, almost doubled that of labelled AA. The levels of TGs in isolated rat uteri suspended in glucose-free medium during a period of 60 minutes were significantly less than immediately after isolation (P less than 0.001). PGE1 released from uteri into the incubating solution, was significantly higher than that of PGE2. Moreover, the presence of 4-PEA (1.0 mM), added after tissue isolation, prevented the decrement of TGs observed following 60 minutes of incubation and simultaneously diminished significantly (P less than 0.001) the enhanced output of PGE1, without altering that of PGE2. Results presented herein suggest that PLs are not normal precursors for the synthesis of PGE1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bi-directional transfer of nitrogen between alfalfa and bromegrass: Short and long term evidence

Transfer of N from legumes to associated non-legumes has been demonstrated under a wide range of conditions. Because legumes are able to derive their N requirements from N₂ fixation, legumes can serve, through the transfer of N, as a source of N for accompanying non-legumes. Studies, therefore, are often limited to the transfer of N from the legume to the non-legume. However, legumes preferentially rely on available soil N as their source of N. To determine whether N can be transferred from a non-legume to a legume, two greenhouse experiments were conducted. In the short-term N-transfer experiment, a portion of the foliage of meadow bromegrass (Bromus riparius Rhem.) or alfalfa (Medicago sativa L.) was immersed in a highly labelled ¹⁵N-solution and following a 64 h incubation, the roots and leaves of the associated alfalfa and bromegrass were analyzed for ¹⁵N. In the long-term N transfer experiment, alfalfa and bromegrass were grown in an ¹⁵N-labelled nutrient solution and transplanted in pots with unlabelled bromegrass and alfalfa plants. Plants were harvested at 50 and 79 d after transplanting and analyzed for ¹⁵N content. Whether alfalfa or bromegrass were the donor plants in the short-term experiment, roots and leaves of all neighbouring alfalfa and bromegrass plants were enriched with ¹⁵N. Similarly, when alfalfa or bromegrass was labelled in the long-term experiment, the roots and shoots of neighbouring alfalfa and bromegrass plants became enriched with ¹⁵N. These two studies conclusively show that within a short period of time, N is transferred from both the N₂-fixing legume to the associated non-legume and also from the non-legume to the N₂-fixing legume. The occurrence of a bi-directional N transfer between N₂-fixing and non-N₂-fixing plants should be taken into consideration when the intensity of N cycling and the directional flow of N in pastures and natural ecosystems are investigated.     

Environmental and developmental regulation of carotenogenesis in the dimorphic fungus Mucor rouxii

Aerobic mycelium of wild-type Mucor rouxii accumulated about ten times higher amounts of the carotenoid pigment -carotene when grown continuously in the presence of light than the corresponding cultures grown in the dark. Carotenoid accumulation was dependent on light intensity, with the threshold located at about 10^-2 W.m^-2. Photocarotenogenesis in complex medium was more efficient with glucose as a carbon source. Carotenoid synthesis by M. rouxii mycelium was unaffected by both retinol acetate and retinal, which are stimulators of carotenogenesis in other zygomycetes. Carotenogenesis was significant in aerobic mycelium but was almost undetectable in anaerobic mycelium as well as in aerobic or anaerobic yeast cells. This suggested an involvement of oxygen in carotenoid synthesis by M. rouxii and the existence of developmental regulation of the expression or operation of the pathway.     

Restriction analysis of the Streptomyces glaucescens genome by agarose gel electrophoresis

A method for the analysis of total DNA of Streptomyces glaucescens is described. The relevant steps are (a) extraction and purification of DNA, (b) restriction of DNA samples with type II restriction enzymes, (c) one dimensional separation of restriction fragments by agarose gel electrophoresis. A typical banding pattern was obtained for each wild type strain, independant of growth conditions or age of the culture. Mutant strains exhibited in most cases the same banding pattern as the parent wild type strain. Only in one specific mutant class a fragment of about 9 megadalton was missing.     

Effect of chronic alcohol consumption on rat uterine spontaneous motility, prostaglandin production and triglyceride metabolism

The spontaneous isometric developed tension (IDT), the synthesis and release of prostaglandins (PGs) into the incubating medium and the metabolism of triglycerides (TGs) in uterine strips isolated from controls and chronic ethanol fed rats, were studied. In order to observe how the uterus of rats fed alcohol reacts during a situation of metabolic emergency, the above mentioned studies were done in the presence or in the absence of glucose in the incubating medium. The decrement of IDT as time progressed was significantly greater in strips obtained from rats which had been drinking 20% ETOH than in controls. Nevertheless, the absolute magnitude of the initial IDT was similar in both groups. On the other hand, the decline of the frequency of contractions (FC) of uterine strips isolated from controls and from ETOH-exposed rats, after 60 min of spontaneous activity was similar. When the uterine strips isolated from ETOH-exposed and from control rats were suspended in glucose-free solution they exhibited the same decrement of IDT and FC after 60 min of activity. The basal release of PGE1 and PGE2 was similar in control tissues incubated in medium containing glucose, but the output of PGE2 was significantly smaller than that of PGE1 in uterine strips isolated from ETOH-exposed rats. The production of PGE1 and PGE2 by uteri suspended in glucose-free medium was similar in control preparations. On the contrary the release of both PGs differs in uterine strips from ETOH-exposed rats, i.e. the output of PGE2 was significantly smaller than in controls and the release of PGE1 increased around 4-fold in comparison with controls.(ABSTRACT TRUNCATED AT 250 WORDS)