Effect of Inositol Deficiency on Cellular Permeability of the Inositol Exacting Yeast

Inositol deficiency caused the abnormalities of permeability of the cell envelope of the inositol exacting yeasts. In the case of Schizosaccharomyces pombe, in which the marked leakage of cellular free-pool fraction was not detected, the uptake activity of glucose or methylglucoside decreased in inositol deficiency, especially in aerobic condition. Investigations on the compositions of lipids and fatty acids showed that the change in fatty acid composition was not so remarkable as that in phosphatides in inositol deficiency. One of the main causes of low transport activity may be due to the change in phosphatides, but not due to that in fatty acids, possibly. Intracellular contents of glucose was not less in inositol deficiency than in sufficiency. These results suggest that inositol deficiency caused the low activity of uptake, which might not be, however, the primary cause of low fermentative activity.

In the case of Saccharomyces cerevisiae Ino^– mutant A–21–20, the similar results about permeability and lipid analyses were obtained in inositol deficiency.     

Effects of Inositol Deficiency on Respiratory and Fermentative Activities of Inositol-exacting Yeast,Schizosaccharomyces pombe

Inositol deficiency of Schizosaccharomyces pombe did not induce significant change of contents of various cellular components except for phospholipids and inositol. The most remarkable decrease in inositol content by the deficiency occurred in the mitochondrial fraction. Electronmicroscopic observation of the inositol-deficient cells of Sch. pombe showed no remarkable thickening of cell wall as occurred in the inositol-exacting mutant of Saccharomyces cerevisiae Strain A–21–20.

Marked loss of fermentative activity under the aerobic condition was caused from inositol deficiency without significant change of activities of respiration and anaerobic fermentation. This seemed to indicate elevated regulatory control of the fermentative activity by oxygen in the inositol-deficient Sch. pombe.

Phosphorylative activities of intact cells and the isolated mitochondria coupled with oxidation was also remarkably suppressed by inositol deficiency.     

Phospholipid metabolism and protein kinase C mediated protein phosphorylation in dietary protein deficiency in rat lung

Nutritional deprivation of proteins decreases the protein kinase C (PKC) activity in rat lung. The activity of (PKC) is influenced by lipid metabolism. Changes in PKC activity may influence phosphorylation of its substrate proteins in the tissues. Therefore, alterations in phospholipid metabolism and PKC mediated protein phosphorylation in dietary protein deficiency in rat lung were envisaged. The study was conducted on rats fed on three different types of diet viz., casein (20% protein), deficient (4% protein, rice flour as source of protein) and supplemented (deficient diet supplemented with L-lysine and DL-threoning). Feeding of protein deficient diet caused reduction in incorporation of [3H] myo-inositol in the total phosphoinositides in lungs and an increase in total inositol phosphate pool. There was a significant reduction in the contents and turnover rate of phosphatidyl inositol and phosphatidyl inositol monophosphate. Supplementation of diet with L-lysine and DL-threonine had a reversing effect on total pool of phosphoinositides and, the metabolism of phosphatidyl inositol bisphosphate and phosphatidyl inositol. In phosphatidyl choline metabolism, the dietary protein deficiency led to a decrease in incorporation of [14C-methyl] choline-chloride in total phospholipids. In contrast, its incorporation increased in phosphatidyl choline pool. The contents of phosphatidyl choline and residue, incorporation of [14C-methyl] choline-chloride in them and their turnover rate also increased. Supplementation of diet had a reversal effect on most of these parameters. Phosphorylation of proteins of 84, 47, 35 and 16 kDa was identified to be mediated by PKC. In dietary protein deficiency, phosphorylation of all these proteins, except that of 47 kDa, increased. Supplementation of diet reversed the pattern except that of 84 kDa. The findings suggest that changes in phospholipid metabolism in dietary protein deficiency may effect the activity of PKC thereby influencing the phosphorylation of its substrate proteins and hence associated functions that may lead to pathophysiology of lung.     

Effect of inositol starvation on the in vitro syntheses of mannan and N-acetylglucosaminylpyrophosphoryldolichol in Saccharomyces cerevisiae.

An early consequence of starvation for inositol in yeast is inhibition of synthesis of the major cell wall components mannan and glucan. In looking for the mechanism of this inhibition, we found that the activity of the enzyme catalyzing the synthesis of N-acetylglucosaminylpyrophosphoryldolichol was diminished in particular membrane preparations from cells starved for inositol. This loss of reactivity was observed under a variety of in vitro assay conditions and could be restored by the addition of phosphatidylinositol but not by other phosphoinositol-containing sphingolipids known to occur in yeast. When assayed in the presence of high concentrations of Triton X-100, enzyme preparations from both control and inositol-starved cells required phosphatidylinositol for maximal activity. Since this enzyme catalyzed an early step in the synthesis of mannan that is N-linked to protein, a reasonable hypothesis is that inhibition of mannan synthesis in inositol-starved cells results from the depletion of the necessary cofactor phosphatidylinositol.     

Nitrogen chlorosis in blue-green algae

Summary Nitrogen deficient Anacystis nidulans contained normal levels of chlorophyll-a and carotenoids but did not contain any phycocyanin. These organisms also contained large amounts of polysaccharide. The addition of nitrate to a deficient culture resulted in the recovery of normal pigmentation over a period of several hours.The relation between these changes and growth was established by a kinetic study of the changes in cell composition during pigment loss and recovery. Loss of phycocyanin commenced with the cessation of growth due to nitrogen limitation and was complete after 15 hours. In contrast there were only minor changes in chlorophyll-a and carotenoid. After growth had ceased polysaccharide continued to increase and viability dropped sharply although total cell counts did not change. These trends were reversed by the addition of nitrate to deficient cultures. Phycocyanin was detected after a short lag and normal levels of phycobiliprotein were present within 8 hours. Cell division did not begin until normal levels of phycocyanin had been restored. During the recovery of normal pigmentation there was a decrease in reducing sugar content and a sharp rise in viability. Qualitative studies with 9 additional blue-green algae suggest that loss of phycocyanin is a characteristic feature of nitrogen deficiency in blue-green algae.     

Effect of metal ions in the culture medium on the stearoyl-coenzyme A desaturase activity of Mycobacterium phlei.

A particulate fraction prepared from Mycobacterium phlei grown in a metal-deficient medium exhibited a greatly reduced activity of stearoyl-CoA desaturase compared to that from normally grown cells. Metal deficiency, however, had no effect on the FAD-dependent NADPH-cytochrome C reductase activity, which has been suggested to participate in the desaturation process. When the cells were grown in the deficient medium supplemented with both Fe2+ and Mg2+, the desaturase activity was restored to the normal level. Supplementation with Mg2+ alone promoted growth but did not restore the desaturase activity, whereas Fe2+ alone did cause a significant restoration. Among the various metal ions tested, only Fe2+ and Fe3+ enhanced the formation of desaturase activity in the deficient medium. When added to the assay medium in vitro, Fe2+ and Fe3+ did not stimulate the desaturase activity of the particulate fraction from the deficient cells. Cultivation in the metal-deficient medium had essentially no effect on the levels of cytochromes in the particulate fraction, but dramatically decreased the non-heme iron content and the amount of a high-spin ferric species exhibiting an ESR signal at g=4.3. No labile sulfur could be detected in the normal or metal-deficient particulate fractions. It is concluded that the presence of iron ions in the culture medium is necessary for the synthesis and/or assembly of the terminal portion of the desaturase system.     

Membrane damage associated with inositol-less death in Saccharomyces cerevisiae.

The effect of inositol deficiency was studied on fermentation, respiration, and sugar and amino acid transport. It was found that the loss of fermetnation and respiration and sugar transport activity parallel the loss of cell viability. The loss of sugar transport activity is associated with the development of cell membrane damage. It is concluded that the ultimate cause of cell death is cell membrane leakiness.     

Physiological Properties of Yeast Cells Grown in the Proteolipid-Supplemented Media

Koji mold proteolipid (PL), a high concentration alcohol-producing factor, promoted yeast growth, and also affected the physiological properties of yeast cells: in the conditions of statical cultures, the cells grown anaerobically in PL-supplemented media showed the highest fermentative activity and the highest alcohol-durability by the dipping method in 20% alcohol for 48 hr, at 20°C, that was similar to moromi (main mash) yeasts in Japanese sake brewing; the cells grown in the stationary culture (with cotton plug) supplemented with PL showed the lowest fermentative activity and relatively low alcohol-durability, similar to moto (seed mash) yeasts; the cells grown anaerobically in PL-unsupplemented media showed high fermentative activity but the lowest alcohol-durability.     

Activation of mouse lymphocytes inhibits induction of rapid cell death by x-irradiation

A distinctive property of the resting lymphocyte is its ability to die rapidly in interphase after x-irradiation. Suspensions of thymocytes and peripheral lymphocytes from BALB/c mice were irradiated with doses ranging from 10 to 10,000 rad (0.1 to 100 Grays), and their viability was measured by eosin dye exclusion at intervals through 3 days of culture. After an initial latent period of a few hours, viability declined exponentially in a dose-dependent fashion. Doses as low as 20 rad caused some lymphocytes to die rapidly. After 1000 rad, 90% of the cells became nonviable in 15 to 20 hr and 99% in 25 to 35 hr. Peripheral lymphocytes showed a somewhat earlier loss of viability than did thymocytes, and were killed especially rapidly by 10,000 rad. Enriched T cells and B cells were killed by irradiation at equal rates, and medullary thymocytes were killed at the same rate as the whole thymocyte population. In contrast with resting cells, T and B lymphocytes activated by mitogens were not subject to such rapid induction of cell death. Irradiation with 1000 rad reduced the viability of activated cells by only 50% at a time when less than 1% of nonstimulated lymphocytes remained alive. Similarly, cloned lines of antigen-specific helper and cytotoxic T cells showed only a delayed and slow loss of viability after receiving 1000 rad. The state of activation can therefore be a significant determinant of the immunologic consequences of irradiation.     

Boron deficiency affects cell viability,phenolic leakage and oxidative burst in rose cell cultures

Despite the fact that the effect of B deficiency on cell metabolism has been studied extensively the mechanism by which B deficiency causes cell death has not been determined. Several authors have hypothesized that B deficiency leads to oxidative burst and hence cell death, though this has not been demonstrated experimentally. In the present work we utilize rose cell (Rosa damascena Mill cv Gloide de Guilan) suspension culture, maintained at the stationary growth phase to determine the effect of B deficiency on cell viability and a number of physiological and biochemical parameters including H₂O₂ production, phenolic leakage, pH of the medium, B concentration and biomass. B deficiency resulted in the death of some cells as early as 24 h following B deprivation, and continued rapidly in the following days. In B deficient cells a small oxidative burst (indicated by the production of H₂O₂) was observed coincident with first cell death and increasing thereafter. Increasing amounts of phenolics were observed in the culture medium of the deficient treatment indicating loss of membrane integrity, however results suggest this increase is a secondary consequence of cell death. The effect of B deficiency on the oxidative burst, together with the effect on cell viability is discussed.     

The Polysaccharide Releasing Phenomenon and Cell Envelope of the Inositol-Exacting Yeast in Inositol-Deficiency

Inositol-deficiency abnormalized the cell envelope, wall and membrane, and the polysaccharides were released out of the cells in a greater amount in inositol-deficiency than in inositol-sufficiency. This release was most notable in the absence of inositol under the tested conditions. The released amount decreased when the culture was provided with inositol or phospholipids, or deprived of all growth-promoting vitamins. Gel filtration of released polysaccharides showed that the lower molecular fraction increased in inositol-deficiency than in sufficiency. DEAE-Sephadex chromatography of released polysaccharides showed the presence of fractions specific for inositol-deficiency which contained mannan and phosphates. Inositol-deficiency also caused enlarging of the pore size of the cell-wall. The relationship between the release of polysaccharides and the syntheses of cell-wall polysaccharides was investigated and discussed.     

Protective effect of acidic mannan fraction of bakers' yeast on experimental candidiasis in mice

An acidic fraction of bakers' yeast mannan, WAM025, showed a significant protective effect against Candida albicans infection in mice, but a neutral fraction of the same bakers' yeast mannan, WNM, did not exhibit this effect. Moreover, pretreatment with WAM025 resulted in a marked reduction of proliferation of C. albicans cells in the organs of the infected mice. We investigated the stimulative effect of these mannan fractions on the function of mouse peritoneal phagocytes, and found that mice administered WAM025 showed a greater increase in the number of peritoneal exudate cells, macrophages and polymorphonuclear leucocytes (PMN), than the mice treated with WNM, especially in the proportion of PMN. Peritoneal phagocytes, PMN and macrophages obtained from WAM025-treated mice showed marked candidacidal activity. Of the phagocytes, PMN were responsible for the larger part of the candidacidal activity. The myeloperoxidase activities of PMN and macrophages in WAM025-treated PEC were greater than in untreated macrophages. The myeloperoxidase activity of WAM025-treated PMN was significantly greater than that of WAM025-treated macrophages. This activity paralleled the active oxygen-releasing activity of the phagocytes. On the other hand, the phagocytic activity of phagocytes from mice administered WNM or WAM025 for C. albicans cells was identical to that of untreated phagocytes. WAM025 seems to cause enhance elimination of the pathogen from mice, by increasing the number and candidacidal activity of phagocytic cells.     

Aging of Escherichia coli

Clifton, C. E. (Stanford University, Stanford, Calif.). Aging of Escherichia coli. J. Bacteriol. 92:905-912. 1966.-The rates of endogenous and exogenous (glucose) respiration decreased much more rapidly than did the viable count during the first 24 hr of aging of washed, C(14)-labeled cells of Escherichia coli K-12 suspended in a basal salt medium devoid of ammonium salts. The rates of decrease of respiration and of death approached each other as the age of the cells increased, but death was not the only factor involved in decreased respiratory activity of the suspensions. The greatest decrease in cellular contents with aging was noted in the ribonucleic acid fraction, of which the ribose appeared to be oxidized, while uracil accumulated in the suspension medium. The viable count and respiratory activities remained higher in glucose-fed than in nonfed suspensions. Proline-labeled cells fed glucose tended to lose more of their proline and to convert more proline into C(14)O(2) than in unfed controls. On the other hand, uracil-labeled cells fed glucose retained more of the uracil than did nonfed cells, but glucose elicited some oxidation of uracil. An exogenous energy source such as glucose aided in the maintenance of a population, but it was not the only factor needed for such maintenance.     

Modulation of mitogen-activated protein kinases (MAPK) activity in response to different immune stimuli in haemocytes of the common periwinkle Littorina littorea

The modulation of mitogen-activated protein kinase (MAPK) activity in haemocytes of the common periwinkle (Littorina littorea) in response to immune challenges by lipopolysaccharide from Echerichia coli (LPS), mannan from baker's yeast Saccharomyces cerevisiae and secretory-excretory products (SEP) of trematodes Himasthla elongata (Echinostomatidae) or after the treatment with phorbol ester (PMA) has been studied by Western blotting using affinity purified rabbit polyclonal antibodies. Exposure of the cells in suspension to PMA, LPS and mannan triggered an activation of p38 and ERK2. The JNK-mediated cascade was modulated differently by the elicitors examined. PMA treatment caused a transient activation of the JNK54 isoform, LPS exposure resulted in a decrease in activity of JNK46, and mannan had no effect on JNK phosphorylation status. Incubation of periwinkle haemocytes in culture medium containing trematode SEP did not affect the activity of any MAPK.     

Biosynthesis of a structurally abnormal C2 complement protein by macrophages from C2-deficient guinea pigs

In order to characterize a genetic deficiency of C2 in guinea pigs, production of C2 by peritoneal macrophage cultures derived from four normal, four heterozygous deficient, and four homozygous deficient animals was measured functionally and immunochemically after metabolic labeling with 35S-methionine. Macrophage monolayers from homozygous deficient animals failed to secrete hemolytically detectable C2 up to 74 hr in culture. A single cell hemolytic plaque assay also failed to demonstrate any functional C2 production by cells from homozygous deficient animals. No C2 protein was detected in media from three of the four homozygous deficient animals, but in one, apparent C2 fragments were present. In contrast, intracellular C2 protein was identified in all four homozygous deficient cell cultures. Its mobility on SDS-PAGE was slightly faster than normal. Much less abnormal intracellular C2 protein was recovered from homozygous deficient macrophage monolayers than intracellular C2 protein from normal macrophage monolayers. Monolayers from heterozygous animals produced functional and immunochemical C2 at approximately 30% of the normal rate. Normal rates of biosynthesis and secretion of two other MHC-linked class III antigens, C4 and factor B, were detected in macrophage cultures from homozygous and heterozygous deficient animals. These data suggest that a specific defect, i.e. a structural abnormality in C2 protein, underlies C2 deficiency in guinea pigs.     

Macrophage activation for tumor cytotoxicity: induction of tumoricidal macrophages by supernatants of PPD-stimulated Bacillus Calmette-Guérin-immune spleen cell cultures.

Resident peritoneal macrophages from normal mice were activated for tumor cytotoxicity in vitro by co-cultivation with BCG1-immune spleen cells and PPD and by incubation with supernatants of PPD-stimulated BCG-immune spleen cell cultures (lymphokine supernatants). Lymphokine activation of macrophages occurred in unfractionated PC suspensions as well as in macrophage monolayers depleted of nonadherent PC. Tumor cytotoxicity by lymphokine-activated macrophages was evident by 3 to 4 hr of culture in active supernatants, reached maximal levels by 8 to 12 hr. and was absent by 20 hr. Continued incubation in lymphokines or even re-exposure after washing did not maintain macrophage cytotoxicity. The capacity of normal resident macrophages to be activated by lymphokines in vitro progressively decreased and was absent by 20 hr in culture. This decrease did not necessarily reflect cell death; macrophage viability as estimated by exclusion of trypan blue or by phagocytic responses did not change over the 20-hr culture period. The short lived nature of both macrophage tumoricidal capacity and capacity of precursor cells to be activated by lymphokines may function as negative feedback mechanisms in immune reactions.     

Toxic effects of sulfur mustard on respiratory epithelial cells in culture

Sulfur mustard (SM) is known to induce cutaneous injury and to cause acute damage to the respiratory tract. Although skin vesication has been demonstrated on human epidermal keratinocytes in culture, no study has been carried out to analyze the effects of SM on the ultrastructural and functional activity of surface respiratory epithelial cells. To evaluate this SM toxicity, we developed an in vitro model of respiratory epithelial cells in primary culture. The study was performed on surface epithelial cells from rabbit trachea cultured according to the explant-outgrowth technique. The functional activity of the cultures was evaluated by measuring the ciliary beating frequency (CBF) of the ciliated cells with a videomicroscopic method. The morphological aspects of the cells were analyzed by light and electron microscopy. Addition of 0.1 mM SM directly into the culture medium produced a sudden and irreversible CBF inhibition, first observed after 2 hr on the ciliated cells of the outgrowth periphery. The arrest of the ciliary beating progressively reached the whole surface of the outgrowth and was simultaneously observed with a detachment of the outgrowth cells. It began at the outgrowth border, leading to the exfoliation of cell sheets, and then to the whole culture after 48 hr. Morphological damage was expressed by intense vacuolisation and disorganization of cytoplasmic and nuclear structures. These findings suggest that the detachment of the respiratory epithelial cells from the matrix represents a major toxic effect of 0.1 mM SM. SM dramatically affects the viability of respiratory epithelial cells in culture. Moreover, the sudden CBF inhibition is more likely due to the death of the ciliated cells than to a specific ciliotoxic effect of SM.Abbreviations CBF ciliary beating frequency - HEPES N2-hydroxyethylpiperazine-N'2ethanesulfonic acid - PBS phosphate buffer saline - SM sulfur mustard - TEM transmission electron microscopy     

Loss of viability correlates with altered acid phosphatase activity in suspended strawberry cell fractions after excessive agitation

Acid phosphatase (EC 3.1.3.2.) activity in all fractions and growth phases of strawberry cell culture was 3-fold higher in the excessively agitated cells compared to a control. The stressed cells lost 9% of viability in the lag phase. Concomitantly the enzyme activity ratio of the lysosome fraction to the crude organelle mixture decreased (-33%) whereas the activity ratio of the cytosol fraction to the crude organelle mixture increased (+30%). This change in the enzyme activity of the ratio of the fractions was not observed in the exponential and stationary phase where the loss of viability was only 4 to 5%.     

Human sperm acrosome reaction-initiating activity associated with the human cumulus oophorus and mural granulosa cells

This report describes the detection and partial characterization of preovulatory human cumulus oophorus and mural granulosa cell-associated activity capable of initiating the human sperm acrosome reaction (AR) in vitro. Fragments of preovulatory human cumulus (cells plus extracellular matrix) were washed 3 times, incubated for 24 hr and the spent media and washes assayed for their ability to initiate the human sperm acrosome reaction (AR) in vitro. AR activity was present in the first two washes but not the third wash; however, AR activity was recovered in the spent medium after 3 X-washed fragments were incubated for 24 hr under conditions which maintained the viability of the cumulus cells. The spent media of preovulatory human mural granulosa cells contained AR-initiating activity after 1-3, 3-6, and 6-9 days of culture. The properties of the AR activity present in spent media of human cumulus fragments included resistance to loss of activity during treatment with pronase; resistance to loss of activity during treatment with chondroitinase ABC or bacterial hyaluronidase; heat stability after overnight incubation; lack of extraction by chloroform-methanol; an apparent molecular weight (MW) of 50,000, as determined by Sephadex G-75 column chromatography; conversion to a lower apparent MW activity by incubation with pronase. These properties are also characteristic of a fraction derived by Sephadex G-75 chromatography of preovulatory human follicular fluid which also has been shown to stimulate the human sperm acrosome reaction in vitro. The AR activity from spent media of human mural granulosa cells is also found in a 50,000 MW Sephadex G-75 fraction. We propose that the sources of the 50,000 MW human follicular fluid AR activity are the cumulus oophorus and the mural granulosa cells.     

Effects of high pressure on survival and metabolic activity of Lactobacillus plantarum TMW1.460

The application of high pressure (HP) for food preservation requires insight into mechanisms of HP-mediated cell injury and death. The HP inactivation in model beer of Lactobacillus plantarum TMW1.460, a beer-spoiling organism, was investigated at pressures ranging from 200 to 600 MPa. Surviving cells were characterized by determination of (i) cell viability and sublethal injury, (ii) membrane permeability to the fluorescent dyes propidium iodide (PI) and ethidium bromide (EB), (iii) metabolic activity with tetrazolium salts, and (iv) the activity of HorA, an ATP binding cassette-type multidrug resistance transporter conferring resistance to hop compounds. HP inactivation curves exhibited a shoulder, an exponential inactivation phase, and pronounced tailing caused by a barotolerant fraction of the population, about 1 in 10(6) cells. During exponential inactivation, more than 99.99% of cells were sublethally injured; however, no sublethal injury was detected in the barotolerant fraction of the culture. Sublethally injured cells were metabolically active, and loss of metabolic activity corresponded to the decrease of cell viability. Membrane damage measured by PI uptake occurred later than cell death, indicating that dye exclusion may be used as a fail-safe method for preliminary characterization of HP inactivation. An increase of membrane permeability to EB and a reduction of HorA activity were observed prior to the loss of cell viability, indicating loss of hop resistance of pressurized cells. Even mild HP treatments thus abolished the ability of cells to survive under adverse conditions.