The isolation of glucose tolerance factors from brewer's yeast and their relation to chromium

A new dietary factor, the glucose tolerance factor (GTF), was reported in 1957 that improved impaired glucose tolerance in rats. Most studies on GTF have used brewer's yeast as the starting material, and it has been postulated that the active material is a low-mol wt organic complex containing Cr³⁺. It seemed thus important to isolate an active GTF from chromium-rich yeast (228 ppm Cr) obtained by incubation with chromium and to compare each fraction with corresponding ones from untreated yeast (0.48 ppm Cr). We developed an isolation and purification procedure by fractionation of yeast extract on an anion and cation exchange resin, and tested the GTF activity (glucose oxidation) on rat adipocytes. PIXE (proton-induced X-ray emission) was used to measure the chromium content of the individual fraction. Individual fractions with GTF activity did not differ between Cr-rich and Cr-deficient yeast, and there was no relationship between Cr content and GTF activity. This does not support the hypothesis that chromium is an obligatory constituent of the GTF, assuming that GTF is a unique substance.     

Selection of lactic yeast producing glucosylceramide from cheese whey

From 2150 isolates from raw milk and milk products, yeast strains were surveyed to produce glucosylceramide from cheese whey. Most of the 54 strains that had accumulated a detectable amount of glucosylceramide were identified as Kluyveromyces lactis var. lactis. The cells of K. lactis var. lactis strain M-11 derived from domestic raw milk accumulated glucosylceramide 2.5-fold higher than K. lactis var. lactis NBRC 1267, the reference strain selected from the culture collections. Strain M-16 of K. lactis var. lactis derived from the same origin was found to synthesize a considerable amount of steryl glucoside in addition to glucosylceramide. Sequence analysis of ribosomal DNA intergenic spacer two regions revealed that strains M-11 and M-16 were diverged from a type strain of K. lactis var. lactis in the same species.     

Presence of Aspergillus flavus in developing cotton bolls and its relation to contamination of mature seeds

Aspergillus flavus spores were dusted onto the involucral nectaries of cotton flowers. The fungus was present in 20 to 58% of the immature bolls harvested 25 or 35 days after anthesis. Among similarly inoculated bolls fully matured either in the field or under sterile conditions at ambient temperatures after excision from the plants, only 3 to 14% contained A. flavus in the seeds. There was no significant difference in the numbers of contaminated bolls between the excised and field-matured treatments. It is concluded that A. flavus is present in developing cotton bolls before dehiscence, but its presence does not ensure infection of mature seeds, and that excision does not reduce A. flavus contamination if the bolls are maintained at ambient temperatures.     

Glycosphingolipids in Plasmodium falciparum. Presence of an active glucosylceramide synthase.

Malaria remains a major health problem especially in tropical and subtropical regions of the world, and therefore developing new antimalarial drugs constitutes an urgent challenge. Lipid metabolism has been attracting a lot of attention as an application for malarial chemotherapeutic purposes in recent years. However, little is known about glycosphingolipid biosynthesis in Plasmodium falciparum. In this report we describe for the first time the presence of an active glucosylceramide synthase in the intraerythrocytic stages of the parasite. Two different experiments, using UDP-[(14)C]glucose as donor with ceramides as acceptors, or UDP-glucose as donor and fluorescent ceramides as acceptors, were performed. In both cases, we found that the parasitic enzyme was able to glycosylate only dihydroceramide. The enzyme activity could be inhibited in vitro with low concentrations of d,l-threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP). In addition, de novo biosynthesis of glycosphingolipids was shown by metabolic incorporation of [(14)C]palmitic acid and [(14)C]glucose in the three intraerythrocytic stages of the parasite. The structure of the ceramide, monohexosylceramide, trihexosylceramide and tetrahexosylceramide fractions was analysed by UV-MALDI-TOF mass spectrometry. When PPMP was added to parasite cultures, a correlation between arrest of parasite growth and inhibition of glycosphingolipid biosynthesis was observed. The particular substrate specificity of the malarial glucosylceramide synthase must be added to the already known unique and amazing features of P. falciparum lipid metabolism; therefore this enzyme might represent a new attractive target for malarial chemotherapy.     

Mechanical stimulation-induced chilling tolerance in tobacco suspension cultured cells and its relation to proline

Mechanical stimulation (MS), widely existing but usually ignored in nature, is one of the major environmental stress factors. MS by increasing the rotational speed of shaker incubator could alleviate a decrease in vitality of tobacco (Nicotiana tabacum L.) suspension cultured cells and reduce the accumulation of MDA under chilling stress at 1°C, which in turn improved survival percentage under chilling stress and regrowth ability of tobacco suspension cells after chilling stress. In addition, MS could increase the activity of Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and induce the accumulation of endogenous proline in tobacco cells; exogenously applied proline also could enhance its endogenous level under normal culture conditions and survival percent-age of the cells under chilling stress. These results suggest that MS could improve chilling tolerance of tobacco suspension cells and the acquisition of this chilling tolerance was related to proline.     

Characteristics of mechanically disrupted bakers' yeast in relation to its separation in industrial centrifuges

The viscosity, density, and sedimentation characteristics of suspensions of whole and mechanically disrupted yeast cells were measured. Mechanical disruption increases the suspension viscosity and its non-Newtonian behavior. Experiments showed a good correlation between laboratory- and industrial-scale centrifugation results.     

Carbohydrate metabolism in the developing and maturing wheat embryo in relation to its desiccation tolerance

Embryos of wheat (Triticum aestivum L. cv. Sappo) were studiedthroughout their development and maturation to investigate therelationships among starch, sucrose and raffinose and the onsetof desiccation tolerance. Starch accumulated in axes and scutellafrom about 20 d post anthesis (dpa) to reach a maximum at approximately35 d. The starch content then declined to a very low value inlate maturation. Extractable -amylase activity increased inembryos throughout the period of starch deposition and showeda substantial rise coincident with starch breakdown. In earlymaturation (approximately 26 dpa) sucrose and raffinose appeared,and continued to increase. The rise in the amount of sucroseparalleled the accumulation of starch, but the major increasein raffinose approximated to the fall in starch content. Embryoswere desiccation intolerant prior to the age when free sucroseand raffinose accumulated: the development of desiccation tolerancewas associated with increasing raffinose: sucrose ratios. Possiblemetabolic and physiological relationships among starch, raffinose,sucrose and the onset of desiccation tolerance are discussed. Key words: Wheat embryos, development, maturation, starch, raffinose, sucrose, desiccation tolerance     

Diversity and mechanisms of alkali tolerance in lactobacilli

We determined the maximum pH that allows growth (pHmax) for 34 strains of lactobacilli. High alkali tolerance was exhibited by strains of Lactobacillus casei, L. paracasei subsp. tolerans, L. paracasei subsp. paracasei, L. curvatus, L. pentosus, and L. plantarum that originated from plant material, with pHmax values between 8.5 and 8.9. Among these, L. casei NRIC 1917 and L. paracasei subsp. tolerans NRIC 1940 showed the highest pHmax, at 8.9. Digestive tract isolates of L. gasseri, L. johnsonii, L. reuteri, L. salivarius subsp. salicinius, and L. salivarius subsp. salivarius exhibited moderate alkali tolerance, with pHmax values between 8.1 and 8.5. Dairy isolates of L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, and L. helveticus exhibited no alkali tolerance, with pHmax values between 6.7 and 7.1. Measurement of the internal pH of representative strains revealed the formation of transmembrane proton gradients (DeltapH) in a reversed direction (i.e., acidic interior) at alkaline external-pH ranges, regardless of their degrees of alkali tolerance. Thus, the reversed DeltapH did not determine alkali tolerance diversity. However, the DeltapH contributed to alkali tolerance, as the pHmax values of several strains decreased with the addition of nigericin, which dissipates DeltapH. Although neutral external-pH values resulted in the highest glycolysis activity in the presence of nigericin regardless of alkali tolerance, substantial glucose utilization was still detected in the alkali-tolerant strains, even in a pH range of between 8.0 and 8.5, at which the remaining strains lost most activity. Therefore, the alkali tolerance of glycolysis reactions contributes greatly to the determination of alkali tolerance diversity.     

Presence of Aspergillus flavus in developing cotton bolls and its relation to contamination of mature seeds.

Aspergillus flavus spores were dusted onto the involucral nectaries of cotton flowers. The fungus was present in 20 to 58% of the immature bolls harvested 25 or 35 days after anthesis. Among similarly inoculated bolls fully matured either in the field or under sterile conditions at ambient temperatures after excision from the plants, only 3 to 14% contained A. flavus in the seeds. There was no significant difference in the numbers of contaminated bolls between the excised and field-matured treatments. It is concluded that A. flavus is present in developing cotton bolls before dehiscence, but its presence does not ensure infection of mature seeds, and that excision does not reduce A. flavus contamination if the bolls are maintained at ambient temperatures.     

Provenance variation in morphology and leaflet anatomy of Parkia biglobosa and its relation to drought tolerance

 Measurements of morphological and anatomical characteristics made on 4-month-old seedlings from five provenances of Parkia biglobosa (Jacq.) Benth., grown in a tropical greenhouse under controlled conditions, showed significant differences between the northern provenances, which represented the Sudan savanna zone, and the southern provenances which represented the forest zone of the species distribution. Seedlings of provenances from the savanna zone showed more xerophytic characteristics than those from the forest zone. They were smaller in height, with a greater number of smaller leaflets per pinnae, a lower specific leaflet mass, smaller palisade and guard cells and a lower ratio of palisade:non-palisade tissue. There were also significant differences in the same characteristics, except palisade cell length, between provenances from the easterly and westerly extremes of the species range. Stomatal conductance measured after 2 weeks of droughting also showed that provenances from the savanna zone had higher values, indicating greater physiological activity and reduced stress, compared with provenances from the forest zone. Multivariate discriminant analysis of the morphological and anatomical characteristics showed that 98% of the seedlings were classified correctly with respect to provenance. The squared distances between provenances were closely related to the geographical distances between locations. Yet, not all variation in leaf anatomy could be explained by the difference in climatic conditions from north to south. Also drought tolerance was not always related to morphological and anatomical features. It is possible that clinal variation within the species from east to west may be interacting with climatic differences from north to south. Received: 2 June 1998 / Accepted: 29 June 1998     

Immunologic tolerance to heterologous immunoglobulin: its relation to in vitro filtration by macrophages.

The cellular and molecular basis for the difference in ability of BCG to induce tolerance in BALB/c and DBA/2 mice has been examined by in vitro biofiltration. It was found that incubation with the adherent cells from BALB/c but not DBA/2 spleens could remove the material from BGG which inhibited tolerance induction in BALB/c mice. This material was shown to represent only a trace component in BGG, was present in only certain commercial batches of BGG, and was apparently unrelated to the presence of aggregates or endotoxin.     

Induction of heat tolerance in wheat coleoptiles by calcium ions and its relation to oxidative stress

Effects of Ca²⁺ ions on the intensity of lipid peroxidation, activities of guaiacol peroxidase, superoxide dismutase (SOD), and catalase, as well as on heat resistance of winter wheat (Triticum aestivium L.) coleoptiles were examined. A preliminary incubation of coleoptile segments in a 5 mM CaCl₂ solution was shown to improve their survival rates after an injuring heat treatment (43.5°C). The effect of Ca²⁺ was suppressed by the inhibitor of Ca²⁺ channels (1 mM LaCl₃). An incubation of coleoptiles in the presence of 5 mM CaCl₂ prior to the stress treatment elevated the content of lipid peroxidation product, malondialdehyde (MDA) and stimulated the activities of guaiacol peroxidase, SOD, and catalase. After the heat exposure of untreated and Ca²⁺-treated seedlings, differential changes in MDA content and in activities of guaiacol peroxidase, SOD, and catalase were observed. It is concluded that a short-term oxidative stress arising in Ca²⁺-enriched plant tissues after the heat treatment is unrelated to their irreversible damage.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 227–232.Original Russian Text Copyright © 2005 by Kolupaev, Akinina, Mokrousov.This revised version was published online in April 2005 with a corrected cover date.     

Microtubule function and its relation to cellular development and the yeast cell cycle in Wangiella dermatitidis

The microtubule inhibitor nocodazole {methyl-5-[2-(thienylcarbonyl)-1H-benzimidazol-2-yl]-carbamate} prevented nuclear migration and nuclear division in yeasts and developing multicellular forms of the polymorphic fungus Wangiella dermatitidis. It did not prevent yeast bud formation during at least two or three budding cycles, and caused yeasts to accumulate as premitotic forms with one to three buds. The effects of the drug suggested that at least three control pathways were involved in the yeast cell cycle; that the nocodazole block point was separate from the execution points of two temperature-sensitive mutations which lead to multicellularity; and that microtubules were controlling neither the yeast budding process nor the development of multicellular forms.Non-standard Abbreviations DMSO dimethylsulfoxide; nocodazole, methyl-5-[2-(thienylcarbonyl)-1H-benzimidazol-2-yl]-carbamate     

Uptake by neuroblastoma cells of glucosylceramide, glucosylceramide glucosidase, its stimulator protein, and phosphatidylserine

Serum-free cultured neuroblastoma cells (clone NlE-115) have been shown to absorb emulsified glucosylceramide, glucosylceramide glucosidase, an activator protein for the enzyme, and phosphatidylserine from a synthetic medium. Uptake of the enzyme was augmented by phosphatidylserine, and vice versa. Uptake of the enzyme-lipid complex was further augmented by the activator protein. It appears likely that the activator forms a complex only with the enzyme-lipid complex, not with the individual components. Two uptake mechanisms for the enzyme seem to be involved, one of which (the complex with activator proteins and acidic lipid) is sensitive to mannosyl phosphate groups. Hydrolysis of absorbed glucosylceramide was slow unless the medium was supplemented with the acidic phospholipid or glucosidase. The most rapid disappearance of stored glycolipid took place when the ternary mixture was added to the cell medium, enzyme + activator protein + phosphatidylserine. These findings may be relevant to enzyme replacement therapy for Gaucher disease.     

Ethanol tolerance of yeast

The ethanol tolerance mechanism in yeasts is not very well understood. This may result from the complex inhibitory mechanisms of ethanol, the lack of a universally accepted definition and method to measure ethanol tolerance, and its complex polygenic characteristic. Recently, there has been some progress in understanding ethanol tolerance. Plasma membrane phospholipids have been shown to play an important role in the ethanol tolerance mechanism. Increases in membrane unsaturated fatty acids result in increased yeast ethanol tolerance. Supplementation of growth media with combinations of unsaturated fatty acids, vitamins and proteins also enhance ethanol tolerance. Physiological factors such as mode of substrate feeding, intracellular ethanol accumulation, temperature and osmotic pressure all contribute to the ethanol tolerance of yeast. The complex nature of ethanol toxicity suggests that a number of different genes are likely to be involved in the ethanol tolerance mechanism. Genetic approaches such as spheroplast or protoplast fusion, hybridization and continuous culture selection have been used to obtain ethanol tolerant yeasts. Isolation and characterization of such strains may provide a better understanding of ethanol tolerance.     

Structure of the cell surface of the yeast Candida tropicalis and its relation to hydrocarbon transport

The surface structure of the hypdrocarbon-utilizing yeast Candida tropicalis was investigated by scanning and transmission electron microscopy (SEM and TEM respectively). The sample preparation technique was based on a rapid cryofixation without any addition of cryoprotectants. In subsequently freeze-dried samples the surface structure was analysed by scanning electron microscopy. Thin sections were prepared from freeze substituted samples. Both techniques revealed hair-like structures at the surface of hydrocarbon-grown cells. The hairy surface structure of the cells was less expressed in glucose-grown cells and it was absent completely after proteolytic digestion of the cells. When cells were incubated with hexadecane prior to cyryofixation a contrast-rich region occured in the hair fringe of thin sections as revealed by TEM. Since these structures were characteristic for hexadecane-grown cells and could not be detected in glucose-grown or proteasetreated cells it was concluded that they originate from hexadecane adhering to the cell surface and are functionally related to hexadecane transport. The structure of the surface and its relation to hydrocarbon transport are discussed in view of earlier results on the chemical composition of the surface layer of the cell wall.Abbreviations SEM Scanning electron microscopy - TEM transmission electron microscopy     

The binding of glucosylceramidase to glucosylceramide is promoted by its activator protein

A protein activator of glucosylceramidase (EC 3.2.1.45) has been previously identified by us in human placenta [(1985) Biochim. Biophys. Acta 836, 157-166]. In the present paper we report that its function in vitro is to stimulate the binding of the enzyme to its substrate, glucosylceramide. After the purification step which frees the enzyme of most of its activator protein (octyl-Sepharose 4B chromatography), the capacity of glucosylceramidase to bind to the glucosylceramide micelles is dramatically decreased. The addition of the activator protein to the purified enzyme restores this binding.