The relationship between cell size and viability of soil bacteria

The number of bacterial cells in soil that form colonies on nutrient agar represent a small fraction of the direct microscopic counts (DMC). The colony-forming cells have larger cell dimensions than the very small (dwarf) cells which represent the majority of the DMC. This may indicate that the dwarf cells are species unable to form visible colonies on agar, or that they swell to normal dimensions when growing. Indigenous bacterial cells were separated from soil by density gradient centrifugation and fractionated according to diameter by filtration through polycarbonate filters. Each filtrate was studied with respect to DMC, cell dimensions, colony-forming cells (visible colonies and microcolonies), and cell dimensions during growth on the agar. The calculated average percent viability was only 0.2% for cells with diameters below 0.4m, about 10% for cells with diameters between 0.4 and 0.6m, and 30–40% for cells with diameters above 0.6m. Only 10–20% of the viable cells with diameters <0.4m increased their diameter to >0.4m prior to growth. Thus, size change during starvation and growth cycles did not explain the high numbers of dwarf cells observed by microscopy. The results show that despite the relatively low number of colony-forming bacteria in soil, the species that form colonies may be fairly representative for the medium size and large cells, which constitute a major part of the bacterial biovolume. Thus plate counting could be a useful method to count and isolate the bacteria accounting for much of the biovolume in soil. The origin of the dwarf cells is still unclear, but the low number of small cells that increased in size seems to indicate that the majority of these bacterial cells are not small forms of ordinary sized bacteria.     

Increase of steroid-producing cells in interrenal tissue and masculinization of gonads after long-term treatment of juvenile rainbow trout with cyanoketone

Summary Cyanoketone administered via the food (0.1, 0.2 and 2 mg/g) for 8 weeks from the first feeding (day 46 after fertilization) or via the aquarium water (3 and 30 mg/100 l) for 4 weeks from day 41 does not influence the activity of 3-hydroxysteroid dehydrogenase (3-HSD) in the interstitial cells of the gonads or interrenal cells of juvenile trout in vivo. However, the number of 3-HSD-positive interrenal cells was strongly increased by administration of the highest dose of cyanoketone via both routes. These high doses furthermore affect the sex ratio in favor of males. It is concluded that interrenal tissue is responsible for the masculinizing effect of cyanoketone via increased production of androgens and/or corticosteroids. Cyanoketone at concentrations of 0.01 to 100 g/ml causes a dose-response inhibition of 3-HSD activity in the interrenal cells, when the substance is administered to an incubation medium for demonstration of this enzyme in tissue sections. The controversial in-vivo and in-vitro effects of cyanoketone on 3-HSD activity are discussed.     

Role of guanosine tetraphosphate in gene expression and the survival of glucose or seryl-tRNA starved cells of Escherichia coli K12

The concentration of guanosine 3,5-bispyrophosphate (ppGpp) increases in bacteria in response to amino acid or carbon/energy source starvation. An Escherichia coli K12 relAspoT mutant lacking the ability to synthesize ppGpp lost viability at an increased rate during both glucose and seryl-tRNA starvation. Also, the deleterious effect of chloramphenicol on starved wild-type cells could be overcome by inducing expression of RelA from a plasmid carrying the relA gene transcribed from a tac promoter, prior to starvation and chloramphenicol treatment. As demonstrated by two dimensional gel electrophoresis, this induction of the RelA protein resulted in global alterations in gene expression including increased synthesis of some rpoS-dependent proteins. The relAspoT mutant maintained high expression of several ribosomal proteins during starvation and appeared to exhibit significantly decreased translational fidelity, as demonstrated by an unusual heterogeneity in the isoelectric point of several proteins and the failure to express higher molecular weight proteins during starvation. Moreover, both rpoS-dependent and independent genes failed to exhibit increased expression in the mutant. It is suggested that the deleterious effects on the cells of the relA, spoT deletions are not due solely to the inability of these cells to induce the sigma factor ^s, but also to deficiencies in translational fidelity and failure to exert classical stringent regulation.     

Effect of Osmolality and myo-Inositol Deprivation on the Transport Properties of myo-Inositol in Primary Astrocyte Cultures

myo-Inositol uptake measured in primary astrocyte cultures was saturable in the presence of Na⁺ with a Km of 13–18 M and a Vmax of 9.4 nmoles/mg protein/hour in myo-inositol-fed cells, indicating a high affinity transport system. In myo-inositol-deprived cells, Km was about 53 M with a Vmax of 13.2 nmoles/mg protein/hour. Decreasing osmolality decreased the Vmax to about 1.9 nmoles/mg protein/hour whereas increasing osmolality increased Vmax about 5-fold, while Kms were essentially unchanged in myo-inositol fed cells. In cells deprived of myo-inositol, Vmax decreased in hypotonic medium and increased in hypertonic medium almost 10-fold, but with more than a doubling of the Km regardless of the osmolality. Glucose (25 mM) inhibited myo-inositol uptake 51% whereas the other hexoses used inhibited uptake much less. Our findings indicate that myo-inositol uptake in astrocytes occurs through an efficient carrier-mediated Na⁺-dependent co-transport system that is different from that of glucose and its kinetic properties are affected by myo-inositol availability and osmotic stress.     

Effect of cell turgor on hydraulic conductivity and elastic modulus of Elodea leaf cells

Water relation parameters of leaf cells of the aquatic plant Elodea densa have been measured using the pressure probe. For cells in both the upper and lower epidermis it was found that the elastic modulus () and the hydraulic conductivity (Lp) were dependent on cell turgor (P). Lp was (7.8±5.5)·10^-7 cm s^-1 bar^-1 (mean±SD; n=22 cells) for P>4 bar in cells of the upper epidermis and was increasing by a factor of up to three for P0 bar. No polarity of water movement or concentration dependence of Lp was observed. For cells of the lower epidermis the Lp-values were similar and the hydraulic conductivity also showed a similar dependence on turgor. No wall ingrowth or wall labyrinths (as in transfer cells) could be found in the cells of the lower epidermis. The elastic modulus () of cells of the upper epidermis could be measured over the whole pressure range (P=0–7 bar) by changing the osmotic pressure of the medium. increased linearly with increasing turgor and ranged between 10 and 150 bar. For cells of the lower epidermis the dependence of on P was similar, although the pressure dependence could not be measured on single cells. The Lp-values are compared with literature data obtained for Elodea by a nuclear magnetic resonance (NMR)-technique. The dependence of Lp on P is discussed in terms of pressure dependent structural changes of the cell membranes and interactions between solute and water transport.Abbreviations P cell turgor pressure - Lp hydraulic conductivity - volumetric elastic modulus - T _1/2 half-time of water exchange of individual cell     

Export of β-1,3-glucanase from mutant rice cells rechallenged and stressed with lysine plus threonine

Mutant rice cells (Oryza sativa L.) grown in liquid suspension cultures exported greater quantities of protein and -glucanases than controls. These mutants were isolated from anther calli resistant to 1 mM lysine plus threonine (LT), regenerated and reestablished as cell suspension cultures from seeds. Cellular protein levels are genetically conditioned, and the levels of extracellular proteins and enzyme activities are inversely related to that of the cellular portions. The rechallenge of cells with 1 mM LT inhibited the expression of both -1,3-glucanases and -1,4-glucosidases but had no significant effect upon the levels of chitinase activity. Mutant cells were more sensitive than controls to stress caused by exogenous LT. In general, under exogenous LT stress the mutant/control ratio for extracellular glucanases increased as the assay conditions were changed from a basic to an acidic pH. The specific activity of glucanases was highest in media and lowest in cells. Both the mutant and control cells exported -glucanases into the suspension medium, but the level of activity in media was greater in that in which the mutant was suspended. The export was probably modulated by the internal protein levels which were highest in mutant cells without LT. Seedlings from mutants with enhanced lysine also had enhanced acidic -glucanase activity.     

Selection for kanamycin resistance in transformed petunia cells leads to the co-amplification of a linked gene

A cell suspension culture was established from a transgenic petunia (Petunia hybrida L.) plant which carried genes encoding neomycin phosphotransferase II (nptII) and -glucuronidase (uidA, GUS). Two selection experiments were performed to obtain cell lines with increased resistance to kanamycin. In the first, two independently selected cell lines grown in the presence of 350 g/ml kanamycin were eight to ten-fold more resistant to kanamycin than unselected cells. Increased resistance was correlated with amplification of the nptII gene and an increase in nptII mRNA levels. Selection for kanamycin resistance also produced amplification of the linked GUS gene, resulting in increased GUS mRNA levels and enzyme activity. Selected cells grown in the absence of kanamycin for twelve growth cycles maintained increased copy numbers of both genes, and GUS enzyme activity was also stably overexpressed. In a second selection experiment, a cell line grown continuously in medium containing 100 g/ml kanamycin exhibited higher nptII and GUS gene copy numbers and an increase in GUS enzyme activity after eleven growth cycles. In this cell line, amplification of the two genes was accompanied by DNA rearrangement.     

5′-AMP hydrolysis by suspensions and homogenates of pancreatic islet cells from normal and cortisone-treated rats

Summary Suspensions of endocrine pancreas cells were prepared by shaking collagenase-isolated rat islets of Langerhans in calcium-free buffer. When incubated with 1.0 mM substrate at pH 7.4, the cells split,P _i from 5-AMP at a rate of 87 nmol/h per g DNA, and from-glycerophosphate at a rate of 25 nmol/h per g DNAK _m for 5 AMP was about 54 M. Adenosine or theophylline inhibited the 5-AMP hydrolysis. Homogenization of the cells increased the activity toward 5-AMP by 23% and that toward-glycerophosphate by 115%. Injecting rats with cortisone had no effect on the 5-AMP hydrolysis by whole cells but significantly increased the activity in cell homogenates; the intracellular activity toward 5-AMP was more than doubled by the cortisone treatment. Staining whole islet cells for 5-AMP-splitting activity resulted in a demarcation of the cell periphery in control rats. Cells from cortisone-treated rats showed heavier deposits of reaction product, and their cell periphery did not stand out as clearly. It is suggested that 5-nucleotidase is largely an ectoenzyme in normal rat islet cells. The cells also contain an as yet unidentified intracellular phosphatase that seems to be solely responsible for the increased hydrolysis of 5-AMP in cortisone-treated rats.     

Interrelation between HeLa-S3 cell transfection and hemolysis in red blood cell suspension using pulsed ultrasound of various duty cycles

We have studied the in vitro transfection of a plasmid DNA with the lacZ gene to HeLa-S3 cells and hemolysis in a red blood cell (RBC) suspension under pulsed ultrasound with duty cycles of 10, 20 and 30% using a digital sonifier at a frequency of 20 kHz and an intensity of 6.2 W/cm² on the surface of a horn tip. Cultured HeLa-S3 cells in suspension were exposed to pulsed ultrasound for an apparent exposure time t from 0 to 60 s. HeLa-S3 viability decreased as a single exponential function of the total exposure time t=t with a common time constant =3.8 s for three duty cycles. Transfection was evaluated by counting the number of -galactosidase(-Gal)-positive cells relative to the total number of cells. Pulsed ultrasound provided an enhanced transfer of the -Gal plasmid to HeLa-S3 cells, 3.4-fold as compared with that in the case of the control. The optimal transfection efficiencies were 0.75, 0.80 and 0.74% near t= with =10, 20 and 30%, respectively. The number ratio of -Gal-positive cells to the surviving cells after exposure increased with t according to a modified logistic equation. The degree of hemolysis also increased exponentially with t at a time constant =₀/ for the RBC suspension in physiological saline at a hematocrit concentration of 0.5% with ₀=0.9 s. Thus the total exposure time for the optimal transfection efficiency was , that is, nearly four times of ₀. Hemolysis in the RBC suspension may be a useful model for determining optimal transfection by pulsed ultrasound of various duty cycles.     

The significance of hydrogenase activity for the energy metabolism of green algae: anaerobiosis favours ATP synthesis in cells of Chlorella with active hydrogenase

In cells of the green alga Chlorella fusca, which contain active hydrogenase(s), the concentration of ATP, NADH and NADPH were measured during a 5 h period of anaerobiosis in the dark and upon subsequent illumination with high light intensities (770 W/m²), conditions which favour optimal hydrogen photoproduction.ATP concentrations were also determined in cells of Chlorella fusca, whose hydrogenase was inactivated prior to illumination, and in cells of Chlorella vulgaris which do not contain hydrogenase. In the dark, the ATP concentration increased slightly during anaerobiosis in cells with active hydrogenase. This increase in ATP concentration was accompanied by an increase of NADH and a decrease of NADPH content.Upon illumination, the ATP content increased in cells with an active hydrogenase, whereas the NADH content decreased. The rate of phosphorylation was twice that observed in cells without active hydrogenase.This ATP synthesis in the light was not inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) (10 mol/l) nor by carbonylcyanide-3-chlorophenyl-hydrazone (CCCP) (1 mol/l) but was diminished by 500 mol/l dibromothymoquinone (DBMIB) and 6 mol/l carbonylcyanide-3-chlorophenyl-hydrazone (CCCP).It was concluded that an active hydrogenase can support ATP production under anaerobic conditions in the dark as well as in the light. NADH might serve in vivo as electron donor for a fermentative production of hydrogen in the light.Possible mechanisms underlying ATP production under anaerobiosis and hydrogen productive conditions are discussed.Abbreviations CCCP Carbonylcyanide-3-chlorophenyl-hydrazone - DBMIB dibromothymoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FCCP carbonylcyanide-p-trifluormethoxyphenyl-hydrazone - HEPES N-2-hydroxyethylpiperazin-N-2-ethan-sulfonic acid - PSI II, photosystem I, II respectively - PQ plastoquinone     

Histology and histochemistry of the testis and ovary of the platyfish,Xiphophorus maculatus,from birth to sexual maturity

Summary The gonads of 3-day- to 7-month-old male and female platyfish (Xiphophorus maculatus) were examined for the presence of 5-3-hydroxysteroid dehydrogenase (3-HSD) and glucose-6-phosphate dehydrogenase (G6PD) by histochemical means. In 3-day-old males a positive response for both enzymes is localized in the Leydig cells. With subsequent testicular development, these cells increase in number and display greater activity at the periphery of the testis and around the efferent ducts. In 3-day-old females 3-HSD and G6PD are localized in the stromal cells of the ovary. These cells increase in number and activity as the animals become sexually mature. Sertoli cells, efferent duct epithelium, and ovarian granulosa cells are negative at all stages of development examined. Our findings suggest that the gonads of neonatal fish possess the potential for steroidogenesis. The role played by sexsteroid hormones in the maturation of the brain-pituitary-gonad axis is discussed.     

Repair of double-strand breaks in plasmid DNA in the yeast Saccharomyces cerevisiae

Summary We studied the repair of double-strand breaks (DSB) in plasmid DNA introduced into haploid cells of the yeast Saccharomyces cerevisiae. The efficiency of repair was estimated from the frequency of transformation of the cells by an autonomously replicated linearized plasmid. The frequency of lithium transformation of Rad⁺ cells was increased greatly (by 1 order of magnitude and more) compared with that for circular DNA if the plasmid was initially linearized at the XhoI site within the LYS2 gene. This effect is due to recombinational repair of the plasmid DNA. Mutations rad52, rad53, rad54 and rad57 suppress the repair of DSB in plasmid DNA. The kinetics of DSB repair in plasmid DNA are biphasic: the first phase is completed within 1 h and the second within 14–18 h of incubating cells on selective medium.     

Characterization of the photosynthetic electron transport chain in normal and photobleachedAnabaena cylindrica by flash spectroscopy

Electron transport of normal and photobleachedAnabaena cylindrica was studied using spectral and kinetic analyses of absorbance transients induced by single turnover flashes. Between 500 and 600 nm two positive bands (540 and 566 nm) and two negative bands (515 and 554 nm) were found. Absorbance changes at 515 and 540 nm were partly characterized. None of these absorbance changes represent an electrochromic shift. Absorbance changes at 554 and 566 nm correspond to the oxidation of cytochromef and the reduction of cytochromeb ₅₆₃, respectively. We found a very slight 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) sensitivity of cytochromef in normal cells, while DCMU was completely ineffective for cytochromef reduction in photobleached cells. The absorbance change of cytochromeb ₅₆₃ increased, while the absorbance change of cytochromef was smaller than in normal cells. The increased O₂ evolution in photobleached cells and the negligible electron transport via cytochromef suggest the participation of other electron acceptor(s) in the electron-transport chain of photobleachedAnabaena cylindrica.     

Response histogram shapes and tuning curves: The predicted responses of several cortical cell types to drifting gratings stimuli

The responses to visual stimuli of simple cortical cells show linear spatial summation within and between their receptive field subunits. Complex cortical cells do not show this linearity. We analyzed the simulated responses to drifting sinusoidal grating stimuli of simple and of several types of complex cells. The complex cells, whose responses are seen to be half-wave rectified before pooling, have receptive fields consisting of two or more DOG (difference-of-Gaussians) shaped subunits. In both cases of stimulation by contrast-reversal gratings or drifting gratings, the cells' response as a function of spatial frequency is affected by the subunit distances 2 and the stimulation frequency . Furthermore, an increased number of subunits (a larger receptive field) yields a narrower peak tuning curve with decreased modulation depth for many of the spatial frequencies. The average and the peak response tuning curves are compared for the different receptive field types.     

High concentrations of PEG as a possible uncoupler of the proton motive force: α-Amylase production with Bacillus amyloliquefaciens in aqueous two-phase systems and PEG solutions

Summary -Amylase production with Bacillus amyloliquefaciens was investigated in two different aqueous two-phase systems and in polyethylene glycol (PEG) 600 solutions of different concentrations. The cells did not partition totally to the bottom phases of the aqueous two-phase systems, and the enzyme production was repressed in both systems as well as in PEG 600 solutions. Concomitantly, the cultivation time was prolonged, indicating an increased maintenance metabolism. The surface properties of cells grown in 200 g/kg PEG 600 were investigated by phase partitioning and compared to the surface properties of Bacillus subtilis, which under these conditions showed increased -amylase production. The cells of B. amyloliquefaciens partitioned to the top phase in a PEG-dextran system, whereas the cells of B. subtilis partitioned to the bottom phase. The results are discussed in relation to water activity, oxygen transfer rate and PEG-induced changes of the surface properties of the cells. The possible role of PEG as an uncoupler of the proton motive force at high concentrations is also discussed.     

Expression of a cloned beta-glucanase gene from Bacillus amyloliquefaciens in an Escherichia coli relA strain after plasmid amplification

Summary Amino acid starvation of cells of the Escherichia coli relA strain, CP79, which cannot accumulate guanosine tetraphosphate (ppGpp) in response to amino acid limitation, increased the pEG1 plasmid content about 5- to 7-fold in comparison with exponentially growing cells (pEG1: pBR322 with an insertion of Bacillus amyloliquefaciens DNA coding for -glucanase). In contrast, no pEG1 amplification occurred in E. coli CP78, the stringently controlled counterpart, after amino acid starvation. In order to verify these results, the plasmid DNA content was monitored by measuring the expression of pEG1-encoded -glucanase from B. amyloliquefaciens both before and after plasmid amplification. When amino acid starved CP79 cells were given an additional dose of amino acids, a more than 10-fold increase in pEG1-encoded -glucanase activity (per cell mass) was measured. This increase in enzyme activity correlates with pEG1 amplification during amino acid limitation. Under comparable conditions the activity of -glucanase was not increased in strain CP78, which did not amplify the plasmid. We suggest that the replication of pEG1 in amino acid starved E. coli cells is somehow under negative control by ppGpp. Moreover, we found the Bacillus -glucanase in E. coli relA cells to be excreted into the growth medium after starvation and overexpression.     

Immunocytological localization of LH,FSH, TSH and their subunits in the pituitary of normal and anencephalic human fetuses

Summary Immunostaining with antisera to oLH, hCG, hLH, pLH, hFSH, hFSH, hTSH and bTSH was used to delineate the gonadotropic and thyrotropic cells of the human fetal anterior pituitary. Hypophyses from 29 normal fetuses, 3 newborn infants, and 5 totally ancencephalic fetuses were used. Several controls to check for the specificity of the immunocytological reaction were made.In normal fetuses, observations showed that: 1) the subunit was detected from the eighth week and throughout gestation without sex differences; 2) intact LH was detected during the third month, however, age and sex differences were observed during the fourth and fifth months; 3) intact FSH was detected in female fetuses from the beginning of the fourth month, a sex difference was observed; 4) LH and FSH were detected in the same cells; 5) the thyrotropic cells were detectable from 15 weeks of gestation and their number increased during gestation without sex difference; 6) at birth the gonadotropic cells were scarce and were located in the ventromedian zone of the anterior pituitary, while the thyrotropic cells remained numerous and were located in the dorsomedian zone.In anencephalic fetuses: 1) the subunit existed at each stage studied; 2) the reaction induced by anti-pLH and anti-hFSH sera was always very weak regardless of sex or age; 3) the thyrotropic cells were more numerous in comparison to the gonadotropic cells. These data are discussed in terms of the relationship of the hypophysiotropic hypothalamic factors to the appearance and evolution of the glycoprotein hormones and their subunits.     

Kidney-Specific Activity of the Bovine Uromodulin Promoter

A 10-kilobase (kb) bacteriophage bovine genomic clone containing 5.4 kb of the 5-flanking region, exons, and introns of bovine uromodulin gene was isolated. Transgenic mice containing 3.9 kb of the bovine uromodulin promoter and a lacZ reporter gene were generated by pronuclear microinjection. RT-PCR and northern blot analyses of transgene expression in various tissues of founder and F1 mice showed that the transgene was expressed exclusively in the kidney. In situ hybridization and histochemistry for lacZ demonstrated that transgene expression was restricted to tubule epithelial cells of the loop of Henle in the kidney. Stepwise 5 deletion analysis revealed that transfection of luciferase reporter constructs fused to various proximal 5-flanking regions of the bovine uromodulin gene markedly increased luciferase activity in mouse renal epithelial cells but not in mesenchymal cells and that the most critical cis elements of the uromodulin gene are located within the 600 bp upstream region.     

Role of light and photosynthesis on the acclimation process of the cyanobacteriumSpirulina platensis to salinity stress

The response ofSpirulina platensis cells to salinity stress was studied. Once adapted to the higher osmoticum, photosynthetic parameters such as the maximum rate of photosynthesis under saturating irradiance (P_max) and the initial slope of the P-I curve () are reduced by 15% and 25% in 0.5 M NaCl grown cells, respectively. Salt-adapted cells have a modified biochemical composition; reduced protein and chlorophyll content, and an increased level of carbohydrates. The reduction in the photosynthetic capacity of the salt-adaptedSpirulina cells reflects a lower ability to utilize light energy and results in an increase in the susceptibility of the stressed cells to photoinhibition. This conclusion is supported by the finding that cultures exposed to salt stress show not only a decrease in growth rate (), but lose the ability to respond to increased irradiance with an increase in growth. The use of variable fluorescence as a fast and reliable measurement to follow the changes in PSII of salt-stressesSpirulina cells enables following the early events of salinity shock. It indicates that as soon as the cells are exposed to salt, a protection mechanism is induced. This mechanism does not require any protein synthesis and may take place even in the dark, though at somewhat reduced effectiveness. The significance of the result in providing a better understanding of the interaction between two environmental stresses — light and salinity — and their application in the outdoor mass cultivation ofSpirulina are discussed.Author for correspondence     

Isolation and characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by the mating hormone a-factor

Summary Nine independent mutants which are supersensitive (ssl ^–) to G1 arrest by the mating hormone a-factor were isolated by screening mutagenized Saccharomyces cerevisiae MAT cells on solid medium for increased growth inhibition with a-factor. These mutants carried lesions in two complementation groups, ssl1 and ssl2. Mutations at the ssl1 locus were mating type specific: MAT ssl1 ^– cells were supersensitive to -factor but MAT ssl1 ^– were not supersensitive to -factor. In contrast, mutations at the ssl2. locus conferred supersensitivity to the mating hormone of the opposite mating type on both MAT, and MATa cells. The -cell specific capacity to inactivate externally added a-factor was shown to be lacking in MAT ssl1 ^– mutants whereas MAT ssl2. cells were able to inactivate a-factor. Complementation analysis showed that ssl2 and sst2, a mutation originally isolated as conferring supersensitivity to -factor to MATa cells, are lesions in the same gene. The ssl1 gene was mapped 30.5 centi-Morgans distal to ilv5 on chromosome XII.