Effect of Growth Conditions and Trehalose Content on Cryotolerance of Bakers' Yeast in Frozen Doughs

The cryotolerance in frozen doughs and in water suspensions of bakers' yeast (Saccharomyces cerevisiae) previously grown under various industrial conditions was evaluated on a laboratory scale. Fed-batch cultures were very superior to batch cultures, and strong aeration enhanced cryoresistance in both cases for freezing rates of 1 to 56°C min⁻¹. Loss of cell viability in frozen dough or water was related to the duration of the dissolved-oxygen deficit during fed-batch growth. Strongly aerobic fed-batch cultures grown at a reduced average specific rate (μ = 0.088 h⁻¹ compared with 0.117 h⁻¹) also showed greater trehalose synthesis and improved frozen-dough stability. Insufficient aeration (dissolved-oxygen deficit) and lower growth temperature (20°C instead of 30°C) decreased both fed-batch-grown yeast cryoresistance and trehalose content. Although trehalose had a cryoprotective effect in S. cerevisiae, its effect was neutralized by even a momentary lack of excess dissolved oxygen in the fed-batch growth medium.     

Chromatin reconstitution on small DNA rings. I

Chromatin was reconstituted using the four core histones on 359 base-pair nicked and closed rings by salt dialysis and/or at physiological ionic strength by means of polyglutamic acid. The products, which consisted of mono- and dinucleosomes, were characterized by gel electrophoresis, sedimentation in sucrose gradients and high-resolution electron microscopy. The results were as follows. (1) The efficiency of the reconstitution was found first to increase with the negative linking difference of the closed rings relative to their relaxed configuration to reach a maximum for -2 turns, and then to decrease for the largest difference of -3 turns. Discrepancies between topoisomers were also observed with regard to differential formation of mono- and dinucleosomes. Topoisomer -1 reconstituted monomers easily but reconstituted dimers with difficulty, whilst this discrimination was virtually absent in the case of topoisomers -2 and -3. Moreover, mononucleosomes on the nicked ring were, with respect to their electrophoretic mobility, similar to mononucleosomes formed on topoisomer -1 but not to those on the other topoisomers, whose mobilities were greater. These features were interpreted in terms of the linking number change associated with the formation of a nucleosome monomer and dimer, approximately -1 and -2 turns, respectively. (2) Two dinucleosome subtypes were found to form in a sequential manner. Their different electrophoretic mobilities and sedimentation coefficients suggested that the early subtype is lighter, probably because of an incomplete histone complement in the second nucleosome of that subtype as a result of an impaired co-operativity in octamer assembly due to the small ring size. (3) An electron microscopic examination of the chromatin reconstituted on topoisomer -2 revealed that both mono- and dinucleosomes adopt two different, salt-dependent, morphologies each: in type I, entering and exiting DNAs do not cross, whilst they do in type II. Type I configuration is favoured in lower salt, whereas type II is favoured in higher salt. Such behaviour explains why nucleosomes in dimers were found to be always diametrically opposed on the rings rather than sometimes apposed, as would have been expected from a random deposition of the histone cores.     

Chromatin reconstitution on small DNA rings. II. DNA supercoiling on the nucleosome

DNA supercoiling on the nucleosome was investigated by relaxing with topoisomerase I mono- and dinucleosomes reconstituted on small DNA rings. Besides 359 base-pair (bp) rings whose linking differences were integers, two additional series of rings with fractional differences, 341 and 354 bp in size, were used. Mononucleosomes reconstituted on 359 bp rings were found to relax into a single mononucleosome form. In contrast, 341 and 354 bp mononucleosomes relaxed into a mixture of two forms, corresponding to two adjacent topoisomers. The observation that the ratio between these two forms was, within each ring series, virtually independent of the initial linking number of the topoisomer used for the reconstitution suggested that each partition reflected an equilibrium. Comparison with the equilibria observed for the same rings in the absence of histones showed that the formation of a single nucleosome is associated with a linking number change of -1.1(+/-0.1) turn. Dinucleosomes, in contrast, were not relaxed to completion and do not reach equilibria. The corresponding linking number change per nucleosome was, however, estimated to be similar to the above figure, in agreement with previous data from the literature obtained with circular chromatins containing larger numbers of nucleosomes. DNA structure in mononucleosomes was subsequently investigated by means of high-resolution electron microscopy and gel electrophoresis. It was found that the above linking number reduction could be ascribed to a particle with a large open extranucleosomal DNA loop and with no more than 1.5 turns of a superhelix around the histone core. A theoretical model of a nucleosome on a small ring was constructed in which one part of the DNA was wrapped around a cylinder and the other part was free to vary both in torsion and flexion. The linking number reduction predicted was found to be most consistent with experimental data when the twist of the DNA in the superhelix was between 10.5 and 10.65 pb per turn, suggesting that wrapping on the nucleosome does not alter the twist of the DNA significantly. A lower estimate of the linking number reduction associated with a two-turn nucleosome was also derived, based on an analysis of recent data obtained upon treatment of reconstituted minichromosomes with gyrase. The value, 1.6 turns, set a lower limit of 10.44 bp per turn for the twist of nucleosomal DNA, in agreement with the above estimate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Control ofThrips tabaci [Thysanoptera: Thripidae] on glasshouse cucumber using large introductions of predatory mitesAmblyseius barkeri [Acarina: Phytoseiidae]

The phytoseiid miteAmblyseius barkeri (Hughes) (=Amblyseius mckenziei Sch. & Pr.) was used for biological control ofThrips tabaci Lind. in 7 commercial glasshouses with cucumber (a total of 5780 m²). Predatory mites were introduced 3–4 times in densities ranging from 40 to 300/m² at each release. In 6 of the 7 glasshouses, control of thrips was satisfactory throughout the growing season. Thrips densities were kept below 15 individuals per leaf. In 1 glasshouse, thrips damage was seen on the fruits at densities of 25 thrips per leaf, but the thrips population was quickly reduced and remained at low densities for the next 3 months.      

Highly regioselective hydrolysis of tricarballylates (“retro-fats”) and citrates by subtilisin

Trimethyl and triethyl esters of tricarballylic acid and citric acid were hydrolysed with porcine liver esterase(PLE) to the isomeric diesters. In all cases the hydrolysis took place with poor regioselectivity (maximum 50% excess). However, the hydrolysis of trimethyl and triethyl esters of tricarballylic acid and of the triethyl ester of citric acid with subtilisin was absolutely regioselective and the symmetric 1,5-diester was obtained.     

Fluorescence studies on aged and young erythrocyte populations

Structural changes in red blood cell (RBC) membrane are investigated by fluorescence techniques. Results obtained with three probes (DPH, 3-PM and fluorescamine) indicate a significant increase in membrane rigidity associated with aging of RBCs. Discrepancies between our observations and published data could arise from utilization of experimental conditions closer to physiological conditions in our study. Use of intact RBCs continuously manipulated in a 37 degrees C environment could represent experimental conditions favourable to the identification of rheologic membrane changes in senescent RBCs.     

Construction of immunogens for synthetic malaria vaccines

The immunogenicity of a peptide consisting of eight repeats of the tetrapeptide sequence NANP (Asn-Ala-Asn-Pro) contained in the circumsporozoite protein of Plasmodium falciparum was investigated in mice under different modes of presentation. This peptide was able to produce biologically active antibodies when administered with adjuvant and linked to a protein carrier. However, a (NANP) peptide polymerized by carbodiimide was found to be immunogenic in the absence of protein carrier in H-2b mice. In contrast, the (NANP)8 peptide polymerized by glutaraldehyde was not immunogenic in the same strain. Furthermore, the efficacy of murabutide in saline, as an immunological adjuvant, was compared to the efficacy of Freund's complete adjuvant.     

A Study of the Chronic Effects of Insecticides in Man

Signs of possible chronic intoxication due to insecticides were sought among 441 apple-growers. A group of 170 persons living in the same environment and 162 other persons having no contact with insecticides were used as controls. It was the first attempt to determine signs of chronic intoxication by organic phosphate and chlorinated hydrocarbon insecticides. The greater incidence of leukopenia and of neurologic manifestations found among the subjects suggest that insecticides may have some chronic effects in man. This assumption is reinforced by the similarities in the findings between the subjects and the environments, and by the parallelism between the duration of exposure to insecticides and the number of affected subjects. The repetition of this study at five-year intervals seems desirable until a sufficient number of apple-growers have been studied who have been exposed to large doses of insecticides for 10, 15 or 20 years.     

RFLP of RT-PCR products: Application to the expression ofCHS multigene family in poplar

A novel method for studying differential expression of multigene family members based on the high sensitivity of RT-PCR completed by restriction site polymorphism of DNA is described. This method allows the identification of specific patterns of expression of fourchalcone synthase genes in a Hunnegem poplar clone (Populus trichocarpa ×Populus deltoides).