Acid-Soluble Nucleotides in an Asporogenous Mutant of Bacillus subtilis

An asporogenous mutant of Bacillus subtilis Sp(-)H12-3, which is considered to have a block at stage 0, showed general growth characteristics similar to those of sporulating cultures. However, a sudden increase in the total amount of acid-soluble nucleotides observed at t(2) in sporulating bacteria was completely absent in this mutant. In sporulating cells, a marked increase in two nucleotides which were identified to be uridine diphosphate (UDP)-galactose and UDP-N-acetylglucosamine was noted, whereas UDP-glucose appeared to be accumulated in the mutant cells at t(2). No unusual nucleotides were found in the strains of B. subtilis examined. The possible role of these UDP derivatives in early stages of sporulation in B. subtilis is discussed.     

Two active forms of valyl-tRNA synthetase from Bacillus subtilis: alteration related to the early stages of sporulation

Two enzymatically active forms, E-I and E-II, of valyl-tRNA synthetase [EC 6.1.1.9] from cells at various stages in the life cycle of Bacillus subtilis 168 LTT, germinated cells, vegetative cells (t-0.5), sporulating cells (t0, t1, t3, and t4), forespores and mature spores, were analyzed by hydroxyapatite column chromatography. The E-II activity was detected in the main fraction of valyl-tRNA synthetase during the life cycle of B. subtilis 168 LTT. The high activity of E-II at t0 decreased rapidly in the stationary and sporulating phases. On the other hand, the E-I activity increased in the early sporulating stage and was about twofold higher at t3 than at t0. After t3, this activity also decreased rapidly and was not detected in forespores and mature spores. The relative amount of E-I at t0 was 3.4% of the total valyl-tRNA synthetase activity eluted from the hydroxyapatite column, 12.9% at t1 and 29.2% at t3, but it was less than 10% at t4 and in germinated cells. The alteration in E-I and E-II activities was also observed in cells of B. subtilis NIG 1121 (spo+), W23 and 168W, but not in any asporogenous mutant strain studied. These results show that the alteration in the valyl-tRNA synthetase activity appears only during the early stages of sporulation and is closely related to the sporulation of B. subtilis.     

Occurrence of polysaccharide granules in sporulating hyphae of Streptomyces viridochromogenes.

Evidence of the presence of polysaccharide polymers is shown in Streptomyces sp. for the first time. Cytochemical methods revealed the occurrence of polysaccharide granules in sporulating hyphae of Streptomyces viridochromogenes. Onset of the sporogenesis coincided with the appearance of the granules, which reached a maximum number during the early stages of maturation. The later stages of maturation showed a decrease of these granules, and in mature spores no granules were observed.     

Energy-dependence of calcium accumulation during sporulation of Bacillus megaterium KM.

Ca2+ accumulation and endogenous respiration of sporulating Bacillus megaterium are inhibited to the same extent by electron-transport of inhibitors and the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone, suggesting that Ca2+ is accumulated by an active transport process. Forespores isolated in stage V of sporulation demonstrated Ca2+-specific carrier-mediated Ca2+ uptake, consistent with downhill transfer [Hogarth & Ellar (1978) Biochem. J. 176, 197-203]. In the present studies forespore Ca2+ uptake was unaffected by carbonyl cyanide p-trifluoromethoxyphenylhydrazone and by concentrations of respiratory inhibitor that inhibited forespore endogenous respiration by 85%. These data suggest that Ca2+ enters the isolated forespore by facilitated diffusion. Ca2+ uptake into sporulating protoplasts was completely inhibited by concentrations of respiratory inhibitors that had no effect on either Ca2+ uptake or respiration of stage-V forespores, but which resulted in inhibition of mother-cell membrane NADH oxidase. These results indicate that the mother-cell membrane is a site for active transport of Ca2+ into the sporulating cell. The effects of the adenosine triphosphatase inhibitor dicyclohexylcarbodi-imide on mother-cell membrane adenosine triphosphatase, NADH oxidase and protoplast Ca2+ uptake were examined.     

Isolation of human salivary polymorphonuclear leukocytes and their stimulation-coupled responses.

A simple method was developed to isolate viable human salivary polymorphonuclear leukocytes (SPMN) from the oral cavity, and stimulation-coupled responses of these cells were examined. From morphological characteristics and the presence of neutrophil-specific annexin protein (39-kDa protein), we found that these cells seemed to be very similar to human peripheral polymorphonuclear leukocytes (PPMN), although they were in rather young stages. Stimulation-coupled responses of these cells were observed in terms of superoxide (O2.-) genration, luminol chemiluminescence response (LCL), membrane depolarization, and changes in intracellular calcium ion concentration ([Ca2+]i). The rates of superoxide generation by various stimuli, such as formylmethionylleucylphenylalanine (FMLP), phorbol 12-myristate 13-acetate (PMA) and opsonized zymosan (OZ) were different. Superoxide generation and strong chemiluminescence response were observed without addition of any stimuli. This endogenous LCL was inhibited by azide and superoxide dismutase (SOD), but not by uric acid (UA). The intensity of the endogenous LCL decreased with time after isolation from the oral cavity. This decrease was accompanied by the appearance of a FMLP-coupled response. Furthermore, the endogenous activity which produced active oxygen species was maintained in the medium at 4 degrees C for a long period after isolation. From these results, it is suggested that SPMN have the ability to show characteristic responses to various stimuli, and that SPMN play important roles in the defense mechanisms in the oral cavity.     

Site and stage specific action of endogenous nuclease and micrococcal nuclease on histone genes of sea urchin embryos

The early histone genes of sea urchin embryos are expressed exclusively during cleavage stages of embryogenesis. The chromatin containing these genes was examined by nuclease sensitivity. An endogenous nuclease active during cleavage, produces 1300-bp segments containing early histone genes. The cutting sites have been mapped; there are very sensitive sites close to the cap site for H1, H2A, H2B, and H4. Chromatin obtained from embryos of later stages, when the genes are not expressed, do not display this pattern of nuclease sensitivity. Micrococcal nuclease produces nucleosomes that contain histone genes when used with nuclei from later stages, but not with nuclei from cleavage stages.     

Role of glutamate in the sporogenesis of Bacillus cereus.

Bacillus cereus T, sporulating in a chemically defined medium under optimum conditions, requires substrate quantities of glutamate during the first 4 h of sporogenesis. Seventy percent of the glutamate utilized was catabolized to CO2 during this period, with the remaining glutamate carbon assimilated into various spore constituents, principally protein and nucleic acid. The importance of glutamate as the primary source of reducing potential and energy for early stages of spore formation was investigated. Although the relative efficiency at which tricarboxylic acid cycle intermediates substituted for glutamate was suggestive of oxidation via the tricarboxylic acid cycle, only partial inhibition of glutamate oxidation by fluoroacetate was observed.     

Cannibalism: a social behavior in sporulating Bacillus subtilis

A social behavior named cannibalism has been described during the early stages of sporulation of the Gram-positive Bacillus subtilis. This phenomenon is based on the heterogeneity of sporulating populations, constituted by at least two cell types: (1) sporulating cells, in which the master regulator of sporulation Spo0A is active, and (2) nonsporulating cells, in which Spo0A is inactive. Sporulating cells produce two toxins that act cooperatively to kill the nonsporulating sister cells. The nutrients released by the dead cells into the starved medium are used for growth by the sporulating cells that are not yet fully committed to sporulate, and as a result, sporulation is arrested. This review outlines the molecular mechanisms of the killing and immunity to the toxins, the regulation of their production and other examples of killing of siblings in microorganisms. The biological significance of this behavior is discussed.     

Protease and peptidase activities in growing and sporulating cells and dormant spores of Bacillus megaterium.

Peptidase and protease activities on many different substrates have been determined in several stages of growth of Bacillus megaterium. Extracts of log-phase cells, sporulating cells, and dormant spores of B. megaterium each hydrolyzed 16 different di- and tripeptides. The specific peptidase activity was highest in dormant spores, and the activity in sporulating cells and log-phase cells was about 1.2-fold and 2- to 3-fold lower, respectively. This peptidase acticity was wholly intracellular since extracellular peptidase activity was not detected throughout growth and sporulation. In contrast, intracellular protease activity on a variety of common protein substrates was highest in sporulating cells, and much extracellular activity was also present at this time. The specific activity of intracellular protease in sporulating cells was about 50- and 30-fold higher than that in log-phase cells and dormant spores, respectively. However, the two unique dormant spores proteins known to be the major species degraded during spore germination were degraded most rapidly by extracts of dormant spores, and slightly slower by extracts from log-phase or sporulating cells. The specific activities for degradation of peptides and proteins are compared to values for intracellular protein turnover during various stages of growth.     

Changes in Cytokinin Distribution in the Ovule and Ovary of Taraxacum officinale Web. in Early Stages of Embryogenesis

Quantitative changes in the hormonal status of the ovules and ovaries were first studied in Taraxacum officinale Web. at the early stages of embryogenesis. The plant material was analyzed by ELISA using labeled anti-rabbit antibodies. A new procedure for differential and quantitative determination of the main endogenous cytokinins based on the estimation of the effective zeatin and zeatin riboside concentrations from calibration curves constructed using zeatin and zeatin riboside as standard antigens was developed. It was shown that, at the three initial stages of embryogenesis examined, the concentration of zeatin uniformly increased in T. officinale ovules. The concentration of zeatin riboside, conversely, uniformly decreased. However, their total concentration changed insignificantly. A gradual increase in the concentration of the active and storage hormone forms from the ovary to the ovule was shown.     

SSEA-1 marks regionally restricted immature subpopulations of embryonic retinal progenitor cells that are regulated by the Wnt signaling pathway

Identification and expansion of retinal progenitor cells are critical issues from both scientific and clinical aspects. Here, we identified SSEA-1 (CD15) as a novel surface antigen that can be used to define immature retinal progenitor cells. SSEA-1-expressing retinal cells were found in the peripheral region of the early embryonic mouse retina, and then their number dramatically disappeared along with retinal development. FACS analysis showed that the cells strongly positive for SSEA-1 co-expressed Ki67 proliferation antigen in all the developmental stages examined. The SSEA-1-expressing cells formed larger colonies than the non-expressing ones in retinal re-aggregation cultures. Moreover, late onset of rhodopsin expression was observed in SSEA-1-positive progenitor cells, supporting the idea that these cells have an intrinsically immature character. Differential expression of Wnt signal-related genes between SSEA-1-positive and -negative subpopulations of retina cells was revealed, and the expression of constitutively active forms of Wnt signaling molecules resulted in a greater number of SSEA-1-positive cells. In light of all of the data taken together, we propose SSEA-1 to be a surface marker to define a regionally restricted immature subset of progenitor cells of mouse neural retina, with SSEA-1 expression by them positively regulated by Wnt signals.     

Changes in Acetylcholinesterase Molecular Forms During the Embryonic Development of Torpedo marmorata

Abstract: Multiple molecular forms of acetylcholinesterase from electric organ and electric lobe of Torpedo marmorata were examined at various developmental stages by sucrose density sedimentation. Four major forms were characterized by their apparent sedimentation coefficients of 6 S, 11 S, 13 S, and 17 S. Embryonic lobe possessed at early stages predominantly the 11 S form. With maturation the 17 S form became the most abundant. The early embryonic stages of the electric organ were characterized by predominating amounts of 6 S and 11 S forms. With differentiation of the postsynaptic membrane of the developing electrocytes, 13 S and 17 S forms replaced the slower-sedimenting forms. Concomitant with the formation of synaptic contacts, a transient increase in the 13 S form was followed by a dramatic accumulation of rapid-sedimenting 17 S form. The establishment of fully functional synapses was accompanied by an increase in the amount of the hydrophobic 6 S form. At birth, equal amounts of 6 S and 17 S form were found, with the other forms present in only trace amounts. The observed characteristic changes correlated with morphological and physiological events, indicating a close functional relationship between the accumulation of the 17 S form and synapse formation and the accumulation of the 6 S form and onset of function.     

Changes in the hormonal status of the Taraxacum officinale Web. ovary at early stages of embryogenesis

The hormonal status of the Taraxacum officinale Web. ovary was quantitatively assayed for the first time during early stages of embryogenesis. Apparent concentrations of endogenous cytokinins were measured using two systems of enzyme-linked immunosorbent assay (ELISA). The ELISA systems differed from one another by the specificity for the main endogenous forms of zeatin. The specificity of two heterological ELISA systems based on zeatin- and kinetin-specific antisera was studied. A new immunochemical approach to the problem of differential quantitative determination of natural zeatin forms is suggested. This approach does not require preliminary separation of experimental samples into individual fractions. True concentrations of zeatin and zeatin riboside in the T. officinale ovary were calculated based on the average values of apparent concentrations of endogenous cytokinins. When the embryo sac maturation had been completed, there was a threefold increase in the zeatin riboside concentration within the following 12 h. By the time of the first division of an unfertilized ovicell (i.e., within the next 12 h), there had been a twofold decrease in the zeatin riboside concentration. Therefore, at early stages of division of the unfertilized ovicell the zeatin riboside concentration virtually returned to the initial level. In contrast to zeatin riboside, there was a steady trend toward an increase in the zeatin concentration in the T. officinale ovary. Within the first 12 h and the next 12 h after completion of the embryo sac maturation, the zeatin concentration was increased 1.5-fold and 2-fold, respectively. The results of this work provide a pioneering insight into the dynamics of various natural forms of zeatin during the reproductive process. The immunochemical approach to quantitative monitoring of various natural forms of zeatin and their dynamics during embryogenesis suggested in this work can be extended to similar biological, medical, and agricultural problems of differential determination of low-molecular-weight agents of similar structure but different biological activity.     

Changes in penicillin-binding proteins during sporulation of Bacillus subtilis

The penicillin-binding proteins (PBPs) of Bacillus subtilis were examined in samples collected at various times from sporulating cultures and compared with the PBPs in a presporulation sample. Large increases in vegetative PBPs 2B and 3 and the appearance of at least one new PBP (42,000 daltons) occurred at reproducible times during sporulation. In some strains a second new PBP (60,000 daltons) was also produced. By comparing the PBP activities in sporulating cells and two spo0 mutants we have classified these changes as sporulation-related events rather than the consequences of stationary-phase aging. The other vegetative PBPs (PBPs 1, 2A, 4, and 5) decreased during sporulation, but not in sufficient amount or at the appropriate time to account for the appearance of the new proteins. A possible connection between specific PBP changes and the penicillin-sensitive stages of sporulation is suggested.     

The program of protein synthesis during sporulation in Bacillus subtilis

The program of protein synthesis was examined during sporulation in Bacillus subtilis as an index of the control of gene expression. At various stages of growth and spore formation, cells of B. subtilis were pulse-labeled with ³⁵S-methionine. Protein was extracted from the radioactively labeled bacteria and then subjected to high resolution one-dimensional and two-dimensional slab gel electrophoresis. We report that sporulating cells restricted or “turned off” the synthesis of certain polypeptides characteristic of the vegetative phase of growth. In certain cases, this “turn off” was prevented in a mutant (SpoOa-5NA) blocked at the first stage of spore formation. Sporulating bacteria also elaborated new polypeptide species that could not be detected in vegetatively growing cells or in cells of the asporogenous mutant SpoOa-5NA in sporulation medium. The synthesis of these sporulation-specific proteins was “turned on” in a temporally defined sequence throughout the period of spore formation. Spore coat protein, for example, was first synthesized at 4 hr after the onset of sporulation, the time at which refractile prespores appeared. Certain sporulation-specific polypeptides including the coat protein were among the most actively produced polypeptides in sporulating cells.     

Two polypeptides associated with the ribonucleic acid polymerase core of Bacillus subtilis during sporulation.

The ribonucleic acid (RNA) polymerase from log-phase and sporulating cells of Bacillus subtilis was analyzed to determine whether any structural changes occurred during sporulation. The elution pattern of RNA polymerase from a deoxyribonucleic acid (DNA)-cellulose column revealed that sporulating cells at stages III and IV contained a new RNA polymerase fraction in addition to the vegetative holoenzyme (alpha2betabeta'sigma). Stage III cells contained the vegetative holoenzyme and a new enzyme with the composition alpha2betabeta'delta1; the molecular weight of delta1 was 28,000. Stage IV cells contained the vegetative holoenzyme, the delta1-containing enzyme, and another enzyme with the composition alpha2betabeta'delta2. The delta2 factor had a molecular weight of around 20,000. The delta-containing enzymes have a higher affinity for the DNA-cellulose column and a higher specific activity on various templates than vegetative holoenzyme. The simultaneous appearance of these enzymes with vegetative holoenzymes in sporulating cells is consistent with the data found previously with DNA-RNA hybridization studies, which showed that sporulating cells contained both vegetative and sporulation messenger RNAs.     

Evidence of a competitive hierarchy among coprophilous fungal populations.

Evidence is presented that interference competition may be important in later states of fungal colonization of cattle feces from a semiarid grassland in Colorado. Cultural antagonism was examined among fungal isolates representing early sporulating colonists (Ascobolus furfuraceus and Saccobolus truncatus), later sporulating colonists (Iodophanus carneus, Coniochaeta discospora, Hypocopra merdaria, and Poronia punctata), and one early successional species that is able to persist (sporulate) through later stages (Podospora decipiens). Poronia punctata, a comparatively slower-growing and later-appearing colonist (18- to 54-month-old fecal pats), is uniformly antagonistic to all of seven earlier-appearing and co-occurring fungal species. Antibiosis is believed to account for the observed antagonism. The authors suggest that the evolutionary product of interference competition among coprophilous fungal populations may be a pattern of competitive hierarchy in which certain slower-growing, later-successional species can limit the reproductive potential of other fungal colonists on fecal substrates.     

A new micromethod for differential quantitative assay of zeatin and zeatin riboside

A new method is proposed for differential quantitative assay of two major endogenous cytokinin forms. It is based on determination of two effective parameters-concentrations of zeatin and zeatin riboside--with the use of appropriate antigens as standards. The method can be used for determining cytokinins in small samples of plant tissues without extract fractionation. This study pioneers in quantitation of changes in the hormonal status of ovules and ovaries of Triticum aestivum L. at early stages of embryogeny. A gradual increase in the content of the active and storage forms of the hormones from the ovary to the ovule was revealed.     

Activated carbon from olive kernels in a two-stage process: industrial improvement

Activated carbons have been prepared from olive kernels and their adsorptive characteristics were investigated. A two stage process of pyrolysis-activation has been tested in two scales: (a) laboratory scale pyrolysis and chemical activation with KOH and (b) pilot/bench scale pyrolysis and physical activation with H(2)O-CO(2). In the second case, olive kernels were first pyrolysed at 800 degrees C, during 45 min under an inert atmosphere in an industrial pyrolyser with a throughput of 1t/h (Compact Power Ltd., Bristol, UK). The resulting chars were subsequently activated with steam and carbon dioxide mixtures at 970 degrees C in a batch pilot monohearth reactor at NESA facility (Louvain-la Neuve, Belgium). The active carbons obtained from both scales were characterized by N(2) adsorption at 77 K, methyl-blue adsorption (MB adsorption) at room temperature and SEM analysis. Surface area and MB adsorption were found to increase with the degree of burn-off. The maximum BET surface area was found to be around 1000-1200 m(2)/g for active carbons produced at industrial scale with physical activation, and 3049 m(2)/g for active carbons produced at laboratory with KOH activation. The pores of the produced carbons were composed of micropores at the early stages of activation and both micropores and mesopores at the late stages. Methylene blue removal capacity appeared to be comparable to that of commercial carbons and even higher at high degrees of activation.     

Model-based definition of population heterogeneity and its effects on metabolism in sporulating Bacillus subtilis

The soil bacterium Bacillus subtilis forms dormant, robust spores as a tactic to ensure survival under conditions of starvation. However, the sporulating culture includes sporulating and non-sporulating cells, because a portion of the cell population initiates sporulation in wild-type strain. We anticipated that the population effect must be considered carefully to analyse samples yielding population heterogeneity. We first built a mathematical model and simulated for signal transduction of the sporulation cue to see what mechanisms are responsible for generating the heterogeneity. The simulated results were confirmed experimentally, where heterogeneity is primarily modulated by negative feedback circuits, resulting in generation of a bistable response within the sporulating culture. We also confirmed that mutants relevant to negative feedback yield either sporulating or non-sporulating subpopulations. To see the effect of molecular mechanism between sporulating and non-sporulating cells in distinct manner, metabolome analysis was conducted using the above mutants. The metabolic profiles exhibited distinct characteristics with time regardless of whether sporulation was initiated or not. In addition, several distinct characteristics of metabolites were observed between strains, which was inconsistent with previously reported data. The results imply that careful consideration must be made in the interpretation of data obtained from cells yielding population heterogeneity.