Chemically defined media for growth and sporulation of Entomophthora virulenta

Amino acids, salts, and vitamins were combined with dextrose to test their effect on growth and sporulation of Entomophthora virulenta in liquid shake culture. The addition of a vitamin solution to the tested media did not enhance growth or sporulation. MgSO₄·7H₂O was the only salt individually tested that allowed for good growth and sporulation. MgSO₄·7H₂O concentrations exceeding 250 mg/liter in media lacking other salts inhibited sporulation. A simple medium of l-arginine, l-leucine, glycine, and mineral salts allowed high growth and sporulation.     

Sporulation in Hansenula wingei Is Induced by Nitrogen Starvation in Maltose-Containing Media

The sexually agglutinative yeast Hansenula wingei lives in association with bark beetles that inhabit coniferous trees. This yeast was induced to sporulate by malt extract, which contains a high percentage of maltose (50%) and a low percentage of nitrogen (0.5%). A solution of 1.5% maltose without any growth factors also induced ascosporogenesis in H. wingei. Thus, only a carbon source is required for sporulation as in Saccharomyces. However, potassium acetate did not induce sporulation in H. wingei as it does in S. cerevisiae. Instead, disaccharides (such as maltose, sucrose, or cellobiose) promote sporulation better than either monosaccharides (such as dextrose, fructose, or mannose) or respiratory substrates (such as ethanol or glycerol). The specificity of disaccharides in promoting sporulation in H. wingei may be considered an adaptation since these disaccharides are present in the natural environment of this yeast. In addition, the specificity of disaccharides may be related to the induction of the disaccharidase because cells precultured on dextrose sporulate well on maltose, but cells precultured on maltose sporulate poorly on maltose. When (NH₄)₂SO₄ was added at a low concentration (3 mM) to synthetic sporulation medium (1.5% maltose solution), sporulation was abolished, whereas other salts and nitrogen sources inhibited to a lesser extent and vitamins and trace elements had no effect. Oxygen was required for sporulation, as expected for an obligate aerobe. Maximal sporulation was achieved in 2% malt extract broth at high cell density (10⁹ cells per ml), pH 5, and 25°C. By using these optimal physiological conditions and hybrid strains selected from an extensive genetic breeding program, about 30% asci (10% tetrads) were obtained routinely. Thus, the genetics of cell recognition in this yeast can now be studied.     

Effects of Cadmium on Aquatic Hyphomycetes

Two kinds of experiments, sporulation and growth experiments, were carried out to demonstrate the effect of cadmium on aquatic hyphomycetes. Oak (Quercus petraea L.) leaves were exposed in a hard-water stream (Lüssel, Swiss Jura) and a soft-water stream (Ibach, Black Forest) for 2 months. In the laboratory, fungal sporulation on the leaves in stream water enriched with cadmium (as CdCl₂) was studied. A measurable effect was found when the cadmium concentration exceeded 0.1 ppm (0.1 mg/liter). Concentrations higher than 100 ppm inhibited conidium production completely. This toxic effect of cadmium was species dependent and much higher in soft water (water with low concentrations of calcium and magnesium) than in hard water. Growth experiments with Alatospora acuminata Ingold, Clavariopsis aquatica De Wildeman, Flagellospora curvula Ingold, Heliscus lugdunensis Saccardo and Therry, and Tetracladium marchalianum De Wildeman showed the same pattern of cadmium sensitivity as that seen in the sporulation experiments. Mycelial growth was less sensitive to cadmium than was fungal sporulation. High concentrations of competing cations (e.g., calcium and zinc) or potential ligands could reduce cadmium toxicity. Calcium content seems to be the most important factor responsible for the different sensitivity of aquatic hyphomycetes in hard and soft water.     

Nitrate salts suppress sporulation and production of enterotoxin in Clostridium perfringens strain NCTC8239

Clostridium perfringens type A is a common source of food‐borne illness in humans. Ingested vegetative cells sporulate in the small intestinal tract and in the process produce C. perfringens enterotoxin (CPE). Although sporulation plays a critical role in the pathogenesis of food‐borne illness, the molecules triggering/inhibiting sporulation are still largely unknown. It has previously been reported by our group that sporulation is induced in C. perfringens strain NCTC8239 co‐cultured with Caco‐2 cells in Dulbecco's Modified Eagle Medium (DMEM). In contrast, an equivalent amount of spores was not observed when bacteria were co‐cultured in Roswell Park Memorial Institute‐1640 medium (RPMI). In the present study it was found that, when these two media are mixed, RPMI inhibits sporulation and CPE production induced in DMEM. When a component of RPMI was added to DMEM, it was found that calcium nitrate (Ca[NO₃]₂) significantly inhibits sporulation and CPE production. The number of spores increased when Ca(NO₃)₂‐deficient RPMI was used. The other nitrate salts significantly suppressed sporulation, whereas the calcium salts used did not. qPCR revealed that nitrate salts increased expression of bacterial nitrate/nitrite reductase. Furthermore, it was found that nitrite and nitric oxide suppress sporulation. In the sporulation stages, Ca(NO₃)₂ down‐regulated the genes controlled by Spo0A, a master regulator of sporulation, but not spo0A itself. Collectively, these results indicate that nitrate salts suppress sporulation and CPE production by down‐regulating Spo0A‐regulated genes in C. perfringens strain NCTC8239. Nitrate reduction may be associated with inhibition of sporulation.     

Calcium ions and osteoclastogenesis initiate the induction of bone formation by coral‐derived macroporous constructs

Coral-derived calcium carbonate/hydroxyapatite macroporous constructs of the genus Goniopora with limited hydrothermal conversion to hydroxyapatite (7% HA/CC) initiate the induction of bone formation. Which are the molecular signals that initiate pattern formation and the induction of bone formation? To evaluate the role of released calcium ions and osteoclastogenesis, 7% HA/CC was pre-loaded with either 500 μg of the calcium channel blocker, verapamil hydrochloride, or 240 μg of the osteoclast inhibitor, biphosphonate zoledronate, and implanted in the rectus abdominis muscle of six adult Chacma baboons Papio ursinus. Generated tissues on days 15, 60 and 90 were analysed by histomorphometry and qRT-PCR. On day 15, up-regulation of type IV collagen characterized all the implanted constructs correlating with vascular invasion. Zoledronate-treated specimens showed an important delay in tissue patterning and morphogenesis with limited bone formation. Osteoclastic inhibition yielded minimal, if any, bone formation by induction. 7% HA/CC pre-loaded with the Ca⁺⁺ channel blocker verapamil hydrochloride strongly inhibited the induction of bone formation. Down-regulation of bone morphogenetic protein-2 (BMP-2) together with up-regulation of Noggin genes correlated with limited bone formation in 7% HA/CC pre-loaded with either verapamil or zoledronate, indicating that the induction of bone formation by coral-derived macroporous constructs is via the BMPs pathway. The spontaneous induction of bone formation is initiated by a local peak of Ca⁺⁺ activating stem cell differentiation and the induction of bone formation.     

Essential role of PI-3K, ERKs and calcium signal pathways in nickel-induced VEGF expression

Exposure to a highly nickel-polluted environment has the potential to cause a variety of adverse health effects, such as the respiratory tract cancers. Since numerous studies have demonstrated that nickel generally has weak mutagenic activity, research focus had turned to cell signalling activation leading to gene modulation and epigenetic changes as a plausible mechanism of carcinogenesis. Previous studies have revealed that nickel compounds can induce the expression of vascular endothelial growth factor (VEGF), which is a key mediator of angiogenesis both in physiological and pathologic conditions. In the present study, we investigated the potential roles of PI-3K, ERKs, p38 kinase and calcium signalling in VEGF induction by nickel in Cl 41 cells. Exposure of Cl 41 cells to nickel compounds led to VEGF induction in both time- and dose-dependent manners. Pre-treatment of Cl 41 cells with PI-3K inhibitor, wortmannin or Ly294002, resulted in a striking inhibition of VEGF induction by nickel compounds, implicating the role of PI-3K in the induction. However, mTOR, one of downstream molecules of PI-3K, may not contribute to the induction because pre-treatment of Cl 41 cells with its inhibitor, rapamycin, did not show obvious decrease in nickel-induced VEGF expression. Furthermore, pre-treatment of Cl 41 cells with MEK1/2-ERKs pathway inhibitor, PD98059, significantly inhibited VEGF induction by both NiCl₂ and Ni₃S₂, whereas p38 kinase inhibitor, SB202190, did not impair the induction. Pre-treatment of Cl 41 cells with intracellular calcium chelator, but not calcium channel blocker, inhibited VEGF induction by nickel. Collectively these data demonstrate that PI-3K, ERKs and cytosolic calcium, but not p38 kinase, play essential roles in VEGF induction by nickel compounds. The first two authors have contributed equally to this work.     

Regulation of ovarian ornithine decarboxylase: Role of calcium ions in enzyme induction in isolated swine granulosa cells in vitro

When swine granulosa cells were cultured in chemically defined medium selectively deficient in Ca²⁺, the dose-dependent stimulation of ornithine decarboxylase (EC 4.1.1.17) activity in response to prostaglandin E₂, l-epinephrine or the somatomedin, multiplication-stimulating activity, was attenuated markedly. Putative calcium influx blockers, verapamil and diltiazem, also inhibited hormone-stimulated enzymic activity. Similar inhibitory effects were exerted by divalent (cobalt) or trivalent (lanthanum) cations believed to compete with calcium for extracellular binding sites. The suppressive effects of extracellular calcium deprivation were time-dependent (suggesting gradual depletion of intracellular calcium stores), and could be mimicked by the intracellular antagonist of calcium action, trifluoperazine. The mechanism(s) subserving diminished hormonal induction of enzyme activity could not be accounted for by alterations in cell viability, general protein synthesis, half-life of decay of enzyme activity (measured in the presence of cycloheximide), or apparent K_m of ornithine decarboxylase. Ca²⁺ and/or calcium antagonists did not modify enzyme activity in cell-free preparations. These observations implicate Ca²⁺ in the hormonal induction of a discrete cytosolic enzyme in isolated intact ovarian cells.     

Role of Ca2+ in induction and secretion of dengue virus-induced cytokines

The present study was undertaken to investigate the role of calcium ions (Ca₂₊) in the induction and secretion of the dengue type 2 virus induced cytotoxic factor and the cytotoxin. This was done by using calcium channel blocking drugs such as verapamil, nifedipine or diltiazem hydrochloride. The production of cytotoxic factor was significantly reduced by treatment of dengue type 2 virus infected mice with verapamil. Similarly, a dosedependent inhibition of the secretion of cytotoxic factor was observed, when spleen cells of the virus-primed mice were treatedin vitro with the 3 calcium channel blockers. The production of cytotoxin by macrophages was abrogated by pretreatment with calcium channel blockers but had little effect on its secretion as shown by treatment of macrophages with verapamil at 1 h after the induction to later periods up to 18 h. The findings thus show that in the induction of both the cytokines Ca₂₊ plays a critical role; on the other hand it is required for the secretion of the cytotoxic factor but not for that of the cytotoxin.     

Formation of Crystalline δ-Endotoxin or Poly-β-Hydroxybutyric Acid Granules by Asporogenous Mutants of Bacillus thuringiensis

Parental strains and asporogenous mutants of Bacillus thuringiensis subspp. kurstaki and aizawai produced high yields of δ-endotoxin on M medium, which contained 330 μg of potassium per ml, but not on ST and ST-a media, each of which contained only 11 μg of potassium per ml. On ST and ST-a media, refractile granules were formed instead. These granules had no insecticidal activity against silkworms and were isolated and identified as poly-β-hydroxybutyric acid. Supplementation of the potassium-deficient ST-a medium with 0.1% KH₂PO₄ (3.7 mM) led to the formation of crystalline δ-endotoxin. The replacement of KH₂PO₄ with equimolar amounts of KCl, KNO₃, and potassium acetate or an equivalent amount of K₂SO₄ had a similar effect, whereas the addition of an equimolar amount of NaH₂PO₄ or NH₄H₂PO₄ did not cause the endotoxin to form. An asporogenous mutant, B. thuringiensis subsp. kurstaki strain 290-1, produced δ-endotoxin on ST-a medium supplemented with 3 mM or more potassium but formed only poly-β-hydroxybutyric acid granules on the media containing ≤1 mM potassium. These results clearly indicate that a certain concentration of potassium is essential for the fermentative production of δ-endotoxin by these isolates of B. thuringiensis. Manganese could not be substituted for potassium. Phosphate ions stimulated poly-β-hydroxybutyric acid formation by strain 290-1. The sporulation of B. thuringiensis and several other Bacillus strains was suppressed on the potassium-deficient ST medium. This suggests that potassium plays an essential role not only in Bacillus cell growth and δ-endotoxin formation but also in sporulation.     

Glucose Decouples Intracellular Ca2+ Activity from Glucagon Secretion in Mouse Pancreatic Islet Alpha-Cells

The mechanisms of glucagon secretion and its suppression by glucose are presently unknown. This study investigates the relationship between intracellular calcium levels ([Ca²⁺]_i) and hormone secretion under low and high glucose conditions. We examined the effects of modulating ion channel activities on [Ca²⁺]_i and hormone secretion from ex vivo mouse pancreatic islets. Glucagon-secreting α-cells were unambiguously identified by cell specific expression of fluorescent proteins. We found that activation of L-type voltage-gated calcium channels is critical for α-cell calcium oscillations and glucagon secretion at low glucose levels. Calcium channel activation depends on K_ATP channel activity but not on tetrodotoxin-sensitive Na⁺ channels. The use of glucagon secretagogues reveals a positive correlation between α-cell [Ca²⁺]_i and secretion at low glucose levels. Glucose elevation suppresses glucagon secretion even after treatment with secretagogues. Importantly, this inhibition is not mediated by K_ATP channel activity or reduction in α-cell [Ca²⁺]_i. Our results demonstrate that glucose uncouples the positive relationship between [Ca²⁺]_i and secretory activity. We conclude that glucose suppression of glucagon secretion is not mediated by inactivation of calcium channels, but instead, it requires a calcium-independent inhibitory pathway.     

Relation between inhibition of Bacilli sporulation and synthesis of lytic enzymes.

In two strains of Bacillus, the synthesis of two specific lytic enzymes was studied concomitantly with an inhibition of the sporulation: LD-carboxypeptidase synthesis was unaffected whereas γ-D-glutamyl-(L)meso-diaminopimelyl endopeptidase synthesis was shown to be closely related to sporulation. The endopeptidase production is totaly inhibited when netropsin inhibits sporulation in B. sphaericus and is low in B. subtilis Thy^?A when sporulation is inhibited by thymidine starvation. This enzyme seems directly connected with the sporulation sequence.     

A Regulatory Mutant of Hansenula polymorpha Exhibiting Methanol Utilization Metabolism and Peroxisome Proliferation in Glucose

Mutant LGM-128 of Hansenula polymorpha harbors the recessive mutation glr2-1 which confers a complex pleiotropic phenotype, the major feature of which is the metabolically unnecessary induction of methanol utilization metabolism (C₁ metabolism) during growth on glucose, whether or not methanol is in the medium. Therefore, in this mutant, peroxisomes are formed and proliferate upon cultivation in glucose-containing media. In these media, LGM-128 shows induction levels of C₁ metabolism that are similar to those observed in methanol-containing media. This indicates that GLR2 controls the repression-derepression process stimulated by glucose and that the induction process triggered by methanol plays only a minor role in activating C₁ metabolism. Cultivating LGM-128 in methanol and then transferring it to glucose media revealed that active degradative processes occur, leading to the disappearance of C₁ metabolism. This observation suggests that, although stimulated by glucose, the two processes are controlled by elements which are, at least in part, distinct. Finally, glr2-1 does not affect ethanol repression, suggesting that in H. polymorpha the two repressing circuits are separated.     

Technetium-99m labeled 50H.19 antibody fragments: Interaction of the antibody with platelets

The monoclonal antibody 50H.19 recognized three antigens (M_r = 31-, 40-, 45-K) on normal and thromboasthenic platelets, but only one (M_r = 31-K) on Bernard-Soulier platelets. The intact antibody and its F(ab′)₂ fragments, had direct platelet-aggregating activity, and induced the platelet release reaction. The intact antibody potentiated platelet aggregation induced by platelet-activating factor or thrombin. Additions of indomethacin did not inhibit aggregation: addition of PGI₂, or a calcium channel blocker completely inhibited aggregation. A reduced amount of platelet-aggregating activity was observed with antibody fragments prepared for labeling with ^99mTc by pre-exposure to stannous ions, and herein used in biodistribution studies and elsewhere in thrombus imagining studies (J. Nucl. Med. 27: 1315; 1986). Antibody fragments radiolabeled with ^99mTc bound to isolated platelets and to clots containing platelets.     

灰斑古毒蛾口腔反吐物诱导沙冬青细胞Ca2+内流及H2O2积累

植物对昆虫取食活动进行成功防御的关键,取决于对昆虫口腔反吐物的激发子的快速识别。实验利用无损伤微测系统及激光共聚焦显微镜,研究了沙冬青细胞经灰斑古毒蛾口腔反吐物诱导后Ca2+流及H2O2的变化。结果发现:灰斑古毒蛾口腔反吐物诱导沙冬青细胞Ca2+内流及H2O2的积累,表明Ca2+内流及H2O2的积累是沙冬青细胞对口腔反吐物产生应答的早期响应事件;Ca2+钙通道阻断剂仅部分抑制Ca2+内流,说明Ca2+内流除经过质膜上的Ca2+通道进入细胞外,尚存在其他的内流途径;灰斑古毒蛾口腔反吐物中的某些成分与沙冬青细胞的质膜结合后,诱导质膜上形成允许Ca2+通过的孔道,而GdCl3不能抑制这类孔道的活性。胞外Ca2+螯合剂EGTA完全抑制H2O2的积累,GdCl3预处理仅部分抑制了H2O2的积累,说明灰斑古毒蛾诱导的沙冬青细胞内H2O2的积累依赖于Ca2+内流;抑制剂实验表明,H2O2的积累主要来源于质膜上NADPH氧化酶的作用。     

Effects of nutrients present in Bold’s basal medium on the green algaStigeoclonium pascheri

The effects of varying concentrations of nutrients present in Bold’s basal medium on the extent of colony formation from vegetative fragments, sporulation and spore germination of the green algaStigeoclonium poscheri were studied. A decrease of colony formation was observed in media deficient in MgSO₄, NaNO₃, phosphates, and containing a 10-fold increase of H₃BO₃. Sporulation decreased in the same media. However, sporulation was greater in an increasing order in media containing 2- to 10-fold increase in MgSO₄. There was a decrease in spore germination in media deficient in phosphates, MgSO₄, containing 5- or 10-fold MgSO₄, or containing 2- to 10-fold of CaCl₂, H₃BO₃ or microelements. Spore germination increased in media containing 2-fold MgSO₄, deficient in H₃BO₃ or microelements or containing none of the three micronutrient solutions.     

A Secreted Factor Coordinates Environmental Quality with Bacillus Development

Entry into sporulation is governed by the master regulator Spo0A. Spo0A accumulates in its active form, Spo0A-P, as cells enter stationary phase. Prior reports have shown that the acute induction of constitutively active Spo0A during exponential growth does not result in sporulation. However, a subsequent study also found that a gradual increase in Spo0A-P, mediated through artificial expression of the kinase, KinA, during exponential growth, is sufficient to trigger sporulation. We report here that sporulation via KinA induction depends on the presence of an extracellular factor or factors (FacX) that only accumulates to active levels during post-exponential growth. FacX is retained by dialysis with a cutoff smaller than 500 Dalton, can be concentrated, and is susceptible to proteinase K digestion, similar to described quorum-sensing peptides shown to be involved in promoting sporulation. However, unlike previously characterized peptides, FacX activity does not require the Opp or App oligopeptide transporter systems. In addition, FacX activity does not depend on SigH, Spo0A, or ComX. Importantly, we find that in the presence of FacX, B. subtilis can be induced to sporulate following the artificial induction of constitutively active Spo0A. These results indicate that there is no formal requirement for gradual Spo0A-P accumulation and instead support the idea that sporulation requires both sufficient levels of active Spo0A and at least one other signal or condition.     

Induction of sporulation in Saccharomyces cerevisiae leads to the formation of N6-methyladenosine in mRNA: a potential mechanism for the activity of the IME4 gene

N⁶-Methyladenosine (m⁶A) is present at internal sites in mRNA isolated from all higher eukaryotes, but has not previously been detected in the mRNA of the yeast Saccharomyces cerevisiae. This nucleoside modification occurs only in a sequence- specific context that appears to be conserved across diverse species. The function of this modification is not fully established, but there is some indirect evidence that m⁶A may play a role in the efficiency of mRNA splicing, transport or translation. The S.cerevisiae gene IME4, which is important for induction of sporulation, is very similar to the human gene MT-A70, which has been shown to be a critical subunit of the human mRNA [N⁶-adenosine]-methyltransferase. This observation led to the hypothesis that yeast sporulation may be dependent upon methylation of yeast mRNA, mediated by Ime4p. In this study we show that induction of sporulation leads to the appearance of low levels of m⁶A in yeast mRNA and that this modification requires IME4. Moreover, single amino acid substitutions in the putative catalytic residues of Ime4p lead to severe sporulation defects in a strain whose sporulation ability is completely dependent on this protein. Collectively, these data suggest very strongly that the activation of sporulation by Ime4p is the result of its proposed methyltransferase activity and provide the most direct evidence to date of a physiologic role of m⁶A in a gene regulatory pathway.     

TRPA1 Has a Key Role in the Somatic Pro-Nociceptive Actions of Hydrogen Sulfide

Hydrogen sulfide (H₂S), which is produced endogenously from L-cysteine, is an irritant with pro-nociceptive actions. We have used measurements of intracellular calcium concentration, electrophysiology and behavioral measurements to show that the somatic pronociceptive actions of H₂S require TRPA1. A H₂S donor, NaHS, activated TRPA1 expressed in CHO cells and stimulated DRG neurons isolated from Trpa1^+/+ but not Trpa1^−/− mice. TRPA1 activation by NaHS was pH dependent with increased activity at acidic pH. The midpoint of the relationship between NaHS EC₅₀ values and external pH was pH 7.21, close to the expected dissociation constant for H₂S (pK_a 7.04). NaHS evoked single channel currents in inside-out and cell-attached membrane patches consistent with an intracellular site of action. In behavioral experiments, intraplantar administration of NaHS and L-cysteine evoked mechanical and cold hypersensitivities in Trpa1^+/+ but not in Trpa1^−/− mice. The sensitizing effects of L-cysteine in wild-type mice were inhibited by a cystathionine β-synthase inhibitor, D,L-propargylglycine (PAG), which inhibits H₂S formation. Mechanical hypersensitivity evoked by intraplantar injections of LPS was prevented by PAG and the TRPA1 antagonist AP-18 and was absent in Trpa1^−/− mice, indicating that H₂S mediated stimulation of TRPA1 is necessary for the local pronociceptive effects of LPS. The pro-nociceptive effects of intraplantar NaHS were retained in Trpv1^−/− mice ruling out TRPV1 as a molecular target. In behavioral studies, NaHS mediated sensitization was also inhibited by a T-type calcium channel inhibitor, mibefradil. In contrast to the effects of NaHS on somatic sensitivity, intracolonic NaHS administration evoked similar nociceptive effects in Trpa1^+/+ and Trpa1^−/− mice, suggesting that the visceral pro-nociceptive effects of H₂S are independent of TRPA1. In electrophysiological studies, the depolarizing actions of H₂S on isolated DRG neurons were inhibited by AP-18, but not by mibefradil indicating that the primary excitatory effect of H₂S on DRG neurons is TRPA1 mediated depolarization.