Screening of thermotolerant yeasts as producers of superoxide dismutase

Abstract A screening procedure for highly thermostable yeast superoxide dismutase was developed. Growth yields at various temperatures were estimated for ten mesophilic and thermotolerant strains, belonging to the genera Saccharomyces, Kluyveromyces and Pichia . Higher yields at 45°C were obtained for K. lactis 90-3 and 90-4. A correlation between the ability to grow at higher temperature and the thermostability of the superoxide dismutase enzyme synthesized was observed. A comparison of the operational stability of the superoxide dismutase of all tested strains suggests that the enzyme of K. lactis strains was more thermostable than that of the other tested microorganisms.     

The effect of a long-term water stress on the metabolism and emission of terpenes of the foliage of Cupressus sempervirens

The effects of long-term water stress on water and terpene contents of the foliage of Cupressus sempervirens were studied. A great deal of water was lost over 2 months before a remarkable stabilization. A strong decrease of all the classes of terpenes accompanied this dehydration. Mono- and sesquiterpene hydrocarbons and free terpenols were almost entirely metabolized, whereas esters and terpene glycosides rose slightly and remained at a constant level when the water content had stabilized. Although a significant part of the mono- and sesquiterpene hydrocarbons was emitted in the early stage of stress application, the major part was used by the plant in response to the drought conditions.     

Tumorigenic transformation of NIH 3T3 cells by the autocrine synthesis of transforming growth factor alpha

A variety of cancer cells overexpress transforming growth factor alpha (TGF alpha), a mitogenic peptide. A cDNA sequence coding for the full-length human TGF alpha precursor protein was subcloned into a retroviral expression vector and introduced into clone 7 NIH 3T3 cells, which have low numbers of endogenous epidermal growth factor receptors (EGFRs). The autocrine synthesis of TGF alpha by these cells resulted in their focal transformation. In contrast, control NIH 3T3 cells treated in a paracrine manner with exogenous, saturating concentrations of the mature form of TGF alpha, though stimulated to divide, remained morphologically untransformed. The addition of saturating quantities of soluble, mature TGF alpha to NIH 3T3 cells expressing the transferred TGF alpha gene actually suppressed their growth and focal transformation. The transformation induced by the TGF alpha gene remained an EGFR-dependent process, since the degree of transformation was correlated with EGFR expression in NIH 3T3 cells and since NR6 cells, which are Swiss 3T3 cells devoid of endogenous EGFRs, were transformed by the TGF alpha vector only when exogenous EGFR genes were also introduced. When inoculated into nude mice, the TGF alpha-expressing cells rapidly gave rise to tumors that grew progressively, whereas control cells did not form tumors. We conclude that in certain circumstances autocrine TGF alpha can be more oncogenic than paracrine and that paracrine TGF alpha can suppress this effect.     

Neutral alpha-mannosidase in human B- and T-lymphoid cells]

The presence of neutral alpha-mannosidase activity in normal and pathological lymphoid cells has been demonstrated. The specific activities of the enzyme in different cell types were similar with the exception of B-cells from B-CLL patients when it was a little higher. The activity of acid alpha-mannosidase was also determined in these lymphoid cells. The neutral to acid alpha-mannosidase activity ratio was different in B- and T-cells: in the former neutral alpha-mannosidase activity prevailed, whereas in the latter the predominance of acid alpha-mannosidase activity was apparent. Neutral alpha-mannosidases from pathological B- and T-cells were partially purified and their properties were investigated. In both cell types the enzyme was localized in the cytosol, was very labile and could be stabilized with Mn2+ and dithiothreitol. The enzyme was activated by Co2+ and inhibited by Zn2+ and EDTA. Swainsonine inhibited the B-cell neutral alpha-mannosidase somewhat more strongly in comparison with the T-cell enzyme.     

Conservation in structure of TOC1 transposons from Chlamydomonas reinhardtii.

TOC1 transposons from Chlamydomonas reinhardtii have an unusual arrangement of long terminal repeats. Polymorphic regions between TOC1 transposons were identified by restriction mapping on Southern blots. The variation in size of an internal MluI fragment defines two subclasses of TOC1 elements. Full-length cloned members of each subclass of TOC1 element were compared by electron microscope heteroduplex analysis. The cloned elements were co-linear over their entire length with no large sequence discontinuities. Base substitutions and small insertion/deletion events of less than 50 bp are responsible for forming the two subclasses of TOC1 elements.     

Regulation of apolipoprotein expression]

Recent data and concepts on the structure and functioning of apolipoprotein genes as well as on the role of hereditary factors in pathogenesis of atherosclerosis are reviewed. The most important variants of inherited disorders in the system of apolipoproteins promoting the development of atherosclerosis are considered. Special attention is paid to the RFLP of apolipoprotein genes which serves as a peculiar genetic background, increasing probability of the atherosclerosis development in certain individuals.     

Outer membrane translocation of the extracellular enzyme pullulanase in Escherichia coli K12 does not require a fatty acylated N-terminal cysteine.

Site-directed mutagenesis was used to construct three mutant derivatives of the extracellular, cell surface lipoprotein pullulanase (PulA) in which the normally fatty acylated cysteine of the signal peptide-bearing precursor was replaced by other amino acids. When produced in Escherichia coli expressing all genes required for pullulanase secretion, approximately 90% of the PulA derivatives persisted as cell-associated precursors, indicating inefficient signal peptide processing. Processed (intermediate-sized) forms of the two derivatives that were studied in detail were found to result from proteolytic cleavage at different sites within the signal peptide. Both were further processed to smaller polypeptides by cleavage at an undetermined site that is presumably close to their C termini. The intermediate-sized pullulanase derived from prepullulanase in which Cys+1 had been replaced by Leu and Gly-1 by Glu (PulA:C1L/G-1E) appeared rapidly, was apparently entirely extracellular, and accounted for approximately 10% of synthesized PulA. Prolonged incubation did not result in further conversion of the precursor to the intermediate form, and the precursor remained anchored to the cytoplasmic membrane. The smaller processed form was also found extracellularly. The active form of the extracellular enzyme was monomeric, which is again in contrast to the fatty acylated, wild-type enzyme. Taken together, these results indicate that replacement of Cys+1 of prePulA eliminates processing by lipoprotein signal peptidase and does not permit processing by leader peptidase, but allows inefficient, aberrant processing by an unknown peptidase and immediate secretion of the resulting polypeptide, which retains most of its signal peptide. Processing and secretion only occur when the pullulanase secretion functions are expressed.     

Evidence indicating that pig renal phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane

Phosphate-activated glutaminase in intact pig renal mitochondria was inhibited 50-70% by the sulfhydryl reagents mersalyl and N-ethylmaleimide (0.3-1.0 mM), when assayed at pH 7.4 in the presence of no or low phosphate (10 mM) and glutamine (2 mM). However, sulfhydryl reagents added to intact mitochondria did not inhibit the SH-enzyme beta-hydroxybutyrate dehydrogenase (a marker of the inner face of the inner mitochondrial membrane), but did so upon addition to sonicated mitochondria. This indicates that the sulfhydryl reagents are impermeable to the inner membrane and that regulatory sulfhydryl groups for glutaminase have an external localization here. The inhibition observed when sulfhydryl reagents were added to intact mitochondria could not be attributed to an effect on a phosphate carrier, but evidence was obtained that pig renal mitochondria have also a glutamine transporter, which is inhibited only by mersalyl and not by N-ethylmaleimide. Mersalyl and N-ethylmaleimide showed nondistinguishable effects on the kinetics of glutamine hydrolysis, affecting only the apparent Vmax for glutamine and not the apparent Km calculated from linear Hanes-Woolf plots. Furthermore, both calcium (which activates glutamine hydrolysis), as well as alanine (which has no effect on the hydrolytic rate), inhibited glutamine transport into the mitochondria, indicating that transport of glutamine is not rate-limiting for the glutaminase reaction. Desenzitation to inhibition by mersalyl and N-ethylmaleimide occurred when the assay was performed under optimal conditions for phosphate activated glutaminase (i.e. in the presence of 150 mM phosphate, 20 mM glutamine and at pH 8.6). Desenzitation also occurred when the enzyme was incubated with low concentrations of Triton X-100 which did not affect the rate of glutamine hydrolysis. Following incubation with [14C]glutamine and correction for glutamate in contaminating subcellular particles, the specific activity of [14C]glutamate in the mitochondria was much lower than that of the surrounding incubation medium. This indicates that glutamine-derived glutamate is released from the mitochondria without being mixed with the endogenous pool of glutamate. The results suggest that phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane.