N-METHYL-N'-NITRO-N-NITROSOGUANIDINE AS A MUTAGEN FOR BLUE-GREEN ALGAE: EVIDENCE FOR REPAIR1

Survival and mutagenesis have been examined in the marine coccoid blue-green alga, Agmenellum quadruplicatum, after treatment with the chemical mutagen, N-methyl-N'-nitro-N-nitrosoguanidine (NTG). When cells were immediately returned to growth conditions following NTG treatment, the survival level was consistently low and essentially independent of the treatment conditions. The types of mutants found paralleled those previously described in the freshwater coccoid, Anacystis nidulans. If, however, cells were kept under very low light intensity, a nongrowth condition, following NTG treatment, viable cell recovery was dramatically increased. This “dim light” repair in A. quadruplicatum has characteristics similar to those reported for the dark repair systems of bacteria and yeast.     

CULTURALLY INDUCED VARIATION IN FOUR MORPHOLOGICALLY DIVERSE BLUEGREEN ALGAE

Characters used in taxonomic placement at levels from family to species appeared, disappeared, or varied widely, while others remained constant, in cultures of four disparate species of blue-green algae (Cyanophyta) under varying conditions of culture. Species of Anabaena and Gloeocapsa displayed fairly stable characteristics. Species of Calothrix and Lyngbya exhibited much greater variability, especially in twisting of filaments, presence or absence of constrictions at the cross walls, abundance of heterocysts, pseudovacuoles, fragmentation of filaments, and change of color. Color change was associated with abrupt pH shift in Lyngbya and could be reversed by adding nitrate. In the putatively nitrogen-fixing Anabaena, similar change in color with age was not associated with sharp pH shift, nor could it be reversed with nitrate. In general, growth on agar in turbulent conditions and with decreased CO₂–O₂ exchange was less than that obtainable under other conditions, and media with acid pH were partially or totally inhibitory to growth.     

The effects of ultraviolet irradiation on a coccoid blue-green alga: survival, photosynthesis, and photoreactivation

The effects of UV irradiation (254 mμ) on a coccoid blue-green alga Agmenellum quadruplicatum, Strain PR-6, have been examined in terms of the survival curve and measurement of short time photosynthetic rates. From study of survival evidence has been found for a strong photoreactivation centered near 430 mμ. Measurements of photosynthetic rate suggest that there is a correlation between decay of photosynthesis and survival after UV exposure. The UV induced decay in photosynthetic activity is reversed by the identical photoreactivation conditions that increase the survival level. The photosynthetic data are interpreted as demonstrating a photoreactivation of photosynthesis in blue-green algae.     

Filament formation in Clostridium acidiurici under conditions of elevated temperatures

Terry, David R. (Brigham Young University, Provo, Utah), Abdul Gaffar, and Richard D. Sagers. Filament formation in Clostridium acidiurici under conditions of elevated temperatures. J. Bacteriol. 91:1625-1634. 1966.-Vegetative cells of Clostridium acidiurici, when grown at temperatures up to 42 C, are straight rods varying from 2.5 to 4 mu in length. When grown at 43 C, the cells show a definite tendency to elongate, and, when grown at 44 C, filaments are formed, often exceeding 500 mu in length. Only an occasional cross wall is apparent in the heat-induced long forms, but as the temperature is lowered they readily form cross walls and fragment into short, single cells. Chromatin material is distributed in evenly spaced clusters throughout the length of the filaments. The filaments grown at 44 C are gram-negative, whereas cells grown at 37 C are gram-positive. However, filament formation and gram-negativity apparently are not due to magnesium deficiency, since the gram-negative filaments are formed in concentrations of magnesium ranging from 10(-6) to 10(-2)m. The rapid transition from filaments to single cells upon lowering the temperature from 44 to 37 C suggests that the temperature-related repression of the cross wall-forming system is a phenotypic response rather than the selection of specific mutants which produce the observed phenomena.     

Isolation of a small-cell mutant in the blue-green bacterium Agmenellum quadruplicatum.

A new type of high-temperature conditional cell division mutant has been isolated in Agmenellum quadruplicatum strain BG1 in which the process of cell division is uncoupled from that of growth at 39 C. This mutant produces abnormally small cells under conditions of nutrient limitation and forms multinucleoid filaments under normal growth conditions.     

Dimorphism of Benjaminiella poitrasii: Isolation and biochemical studies of morphological mutants

The yeast-mycelium dimorphims of the genus Benjaminiella poitrasii has been investigated. To understand the mechanism of dimorphism two stable yeast-phase mutants (Y-1 & Y-2) and one slow growing mycelial mutant (M-1) of B. poitrasii were isolated after NTG treatment of parent strain spores and studied for their biochemical characteristics. Effects of (i) kind and concentration of carbon source, (ii) presence of complex organic nitrogen and (iii) C:N ratio in the growth medium on the morphology of parent and mutant strains were carried out at 28°C under shaking conditions. Ethanol induced morphological change and its reversal were studied in all the strains in order to elucidate the possible mechanism of morphogenesis.     

Mutations of Anacystis nidulans that affect cell division

Summary Following nitrosoguanidine treatment of the blue-green alga Anacystis nidulans strain 6311, mutants which grow predominantly as short or long filaments are commonly produced. Cytological study shows that the filaments are multinucleate, coenocytic structures. Such mutations are therefore best interpreted as ones that impair cell division in a normally unicellular organism.     

Action of mutagenic chemicals on Anacystis nidulans

Summary The tolerance of the unicellular blue-green alga Anacystis nidulans to nitrosoguanidine increased from an initial level of 5 g NTG/ml to 20 g/ml during the course of 10 successive transfers in NTG-supplemented medium.Single treatments of log-phase cells with NTG at pH 5–6 resulted in a significant increase in the frequency of streptomycin-resistant mutants but only a marginal increase in the frequency of ultraviolet-resistant mutants was observed following single or 10 successive treatments with NTG.Attempts to score mutants resistant to penicillin, aminotriazole or NTG were unsuccessful since these agents failed to effectively kill or bleach the sensitive background population on the agar plates.     

Peroxidase isozyme patterns in the skin of maturing tomato fruit

The cessation of tomato fruit growth is thought to be induced by an increase in the activity of enzymes which rigidify cell walls in the fruit skin. Peroxidase could catalyse such wall‐stiffening reactions, and marked rises in peroxidase activity were recently reported in skin cell walls towards fruit maturity. Peroxidase isoforms in the fruit are here analysed using native gel electrophoresis. New isoforms of apparent M_r 44, 48 and 53 kDa are shown to appear in cell walls of the fruit skin at around the time of cessation of growth. It is inferred that these isozymes are present in the cell wall in vivo. Fruit from a range of non‐ripening mutants were also examined. Some of these do not soften or ripen for many weeks after achieving their final size. The new isozymes were found in skin cell walls of mature fruit in each of these mutants, as in the wild‐type and commercial varieties. It is concluded that the late‐appearing isozymes are not associated with fruit ripening or softening, and are probably not ethylene‐induced. They may act to control fruit growth by cross‐linking wall polymers within the fruit skin, thus mechanically stiffening the walls and terminating growth.     

Growth phases of Mycoplasma in liquid media observed with phase-contrast microscope

Razin, Shmuel (University of Connecticut, Storrs), and Benjamin J. Cosenza. Growth phases of Mycoplasma in liquid media observed with phase-contrast microscope. J. Bacteriol. 91:858-869. 1966-Growth of 11 Mycoplasma strains in liquid media was followed by phase-contrast microscopy. A similar pattern of development was common to all strains. Branching filaments, 0.3 to 0.4 mu thick, characterized the early logarithmic phase of growth. The length of the filaments varied according to the strain tested and the growth medium. Addition of oleic acid to the medium induced the formation of very long filaments by M. laidlawii strain B. Upon aging, the filaments were found to break up into chains of coccoid elements. These chains further fragmented to yield shorter chains and single coccoid elements, which characterized the stationary and decline phases of growth. The size of the coccoid elements increased from 0.3 to 0.4 mu, when formed in the filaments, to 0.6 to 0.8 mu after being released from the chains. Further increase in the size of the cells took place at the decline phase of growth, leading to the formation of very large cells reaching a diameter of 10 to 20 mu. However, these large cells had the appearance of empty vesicles and were apparently nonviable as indicated by viable-count experiments.     

Induction of autophagy in hepatocellular carcinoma cells by SB203580 requires activation of AMPK and DAPK but not p38 MAPK

SB203580 is a well-known inhibitor of p38 mitogen-activated protein kinase (MAPK). However, it can suppress cell proliferation in a p38 MAPK independent manner. The inhibitory mechanism remains unknown. Here, we showed that SB203580 induced autophagy in human hepatocellular carcinoma (HCC) cells. SB203580 increased GFP-LC3-positive cells with GFP-LC3 dots, induced accumulation of autophagosomes, and elevated the levels of microtubule-associated protein light chain 3 and Beclin 1. It stimulated the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and p53, but inhibited the phosphorylation of death-associated protein kinase (DAPK). Inhibition of AMPK, p53, or DAPK attenuated SB203580-induced autophagy. AMPK activation appeared to predate the DAPK signal. The activation of both AMPK and DAPK prompted the phosphorylation of p53 and enhanced Beclin 1 expression. Neither the downregulation of p38 MAPK by its siRNA or chemical inhibitor nor the upregulation of p38 MAPK by p38 MAPK DNA transfection affected B203580-induced autophagy. Collectively, the findings demonstrate a novel function of SB203580 to induce autophagy via activating AMPK and DAPK but independent of p38 MAPK. The induction of autophagy can thus account for the antiproliferative effect of SB203580 in HCC cells.     

Relationship between diurnal glucose levels and HbA1c in type 2 diabetes.

AIMS AND METHODS: Study results still conflict on the contribution of diurnal blood glucose (BG) values to Hb (A1c) in type 2 diabetes. We investigated the relationship between Hb (A1c) and diurnal BG obtained under standardized conditions - before breakfast, two hours after breakfast, before lunch, two hours after lunch, before dinner, two hours after dinner, and at 10 PM, 12 midnight and 3 AM in 68 type 2 diabetic patients before and after optimizing glycemic control. The areas under the curve above fasting BG (AUC1) and above 5.6 mmol/l (AUC2) were calculated for further evaluation. Hb (A1c) was measured at baseline and after a mean of 89 (74 to 108) days. RESULTS: Each BG value at baseline and after treatment optimization significantly correlated with baseline and follow-up Hb (A1c), respectively. The pre-breakfast BG showed the closest correlation with Hb (A1c). The relative contribution of postprandial BG concentrations (AUC1) to overall hyperglycemia (AUC2) decreased with poorer glycemic control. However, treatment optimization mainly resulted in improved blood glucose values in patients with the poorest glycemic control at baseline. Multiple regression analysis demonstrated that fasting (AUC2-AUC1) and postprandial (AUC1) hyperglycemia independently determined Hb (A1c) or the change in Hb (A1c) after treatment optimization. CONCLUSIONS: Our findings indicate that intensive blood glucose monitoring during fasting and postprandial states is important for glycemic control, and is therefore an essential part of good clinical practice.     

Conditions for mutagenesis in the cynanobacterium Aphanocapsa 6714

Conditions for induction of mutants have been studied in the unicellular, blue-green alga, Aphanosapsa 6714. Ethylmethanesulfonate, nitrosomethylurea, an acridine (euflavine) and -rays from ⁶⁰Co induced no significant increase in mutant frequency although they produced classical killing effects. Methyl-nitro-nitrosoguanidine (NTG), at similar lethal doses, led to only a small increase in mutant yields. A useful mutagenic action was obtained only with ultraviolet light. This effect proved to be sensitive to photodependent repair processes, but not to dark excision repair systems. Attempts to adapt enrichment procedures by penicillin selective killing were unsuccessful.Abbreviations UV Ultraviolet light - NTG Methyl nitro nitroso guanidine - NMU Nitro methyl urea - EMS Ethyl methane sulfonate     

Oxygen Sensitive Nitrogen-Fixing Mutants of Anabaena

Two Anabaena mutants having heterocysts but incapable of fixing molecular nitrogen in air have been isolated by using ultraviolet radiation or NTG mutagenesis. Their vegetative cells differentiated into heterocysts at a higher frequency than that of the wild type. The phenotype of the mutants is stable and a low frequence of spontaneous reversion was observed. Under microaerobic condition the mutants cells can express the genetic information which encodes nitrogenase synthesis and were capable of utilizing nitrogen for growth with a low acetylene reductiop activity. The level of nitrogenase activity was correlated reciprocally with the content of cell phycocyanin and the light intensity. Both synthesis and activity of the mutant nitrogenase were very sensitive than wild type to the oxygen in vive. Introduction of 1% O2 (v/v) into the gas phase inhibited evidently acetylene reduction. Exposure of the mutant suspension to 20% O2 (v/v) resulted in total and irreversible denaturation of nitrogenase. Withdrawing of O2 in gas phase, the nitrogenase was synthesized de nero; The synthesis process was repressed by chloramphenical or ammonia. The nitrogenase activity of mutant cells increased significantly either by nitrogen- starvating to decrease the phycocyanin content or by lowering the light intensity. Specifically, during the anaerobic induction by treating the mutants filaments with diehloromethylurea which prevents photosynthetic oxygen production, the specific activity of mutant nitrogcnase was equivalent nearly to that of wild type. The ability to reduce 2, 3, 5-triphenyltetrazolium was lower in heterocysts and vegetative cells of mutants than in that of wild type. The results suggest that the oxygen sensitivity of nitrogen fixation by heterocystous bluegreen algal mutants may be duc to the defect of some enzymic systems which might play a role in scavenging oxygen toxity, so that the process of nitrogen fixation is inhibited by the active oxygen produced by vegetative cells. The mechanism of protecting nitrogenase from oxygen damage in blue-green algae is discussed.     

Red light, Phot1 and JAC1 modulate Phot2-dependent reorganization of chloroplast actin filaments and chloroplast avoidance movement

The phototropin (phot)-dependent intracellular relocation of chloroplasts is a ubiquitous phenomenon in plants. We have previously revealed the involvement of a short cp-actin (chloroplast actin) filament-based mechanism in this movement. Here, the reorganization of cp-actin filaments during the avoidance movement of chloroplasts was analyzed in higher time resolution under blue GFP (green fluorescent protein) excitation light in an actin filament-visualized line of Arabidopsis thaliana. Under standard background red light of 89 μmol m(-2) s(-1), cp-actin filaments transiently disappeared at approximately 30 s and reappeared in a biased configuration on chloroplasts approximately 70 s after blue excitation light irradiation. The timing of biased cp-actin reappearance was delayed under the background of strong red light or in the absence of red light. Consistently, chloroplast movement was delayed under these conditions. In phot1 mutants, acceleration of both the disappearance and reappearance of cp-actin filaments occurred, indicating an inhibitory action of phot1 on reorganization of cp-actin filaments. Avoidance movements began sooner in phot1 than in wild-type plants. No reorganization of cp-actin filaments was seen in phot2 or phot1phot2 mutants lacking phot2, which is responsible for avoidance movements. Surprisingly, jac1 (j-domain protein required for chloroplast accumulation response 1) mutants, lacking the accumulation response, showed no avoidance movements under the whole-cell irradiation condition for GFP observation. Cp-actin filaments in jac1 did not show a biased distribution, with a small or almost no transient decrease in the number. These results indicate a close association between the biased distribution of cp-actin filaments and chloroplast movement. Further, JAC1 is suggested to function in the biased cp-actin filament distribution by regulating their appearance and disappearance.     

Protoplasts from the cyanobacterium,Spirulina platensis

Protoplasts were obtained from the filamentous blue-green algaSpirulina platensis by treating the filaments with 0.05% (w/v) lysozyme in 0.03m phosphate buffer. The protoplasts regenerated cell walls and formed colonies when plated on a regeneration medium. The highest percentage of regeneration, 40% was obtained after 21 days.     

纳豆芽孢杆菌产VK2菌株的NTG与N＋注入复合诱变选育

以纳豆芽孢杆菌BN-2-6为出发菌株,利用亚硝基胍（NTG）和N＋注入复合诱变选育产维生素K2的突变株。经过NTG诱变后得到突变株BN-N30—1,其维生素K2的产量提高了53％,继而采用低能N＋注入技术进行处理得到突变株BN-P15—11-1,维生素K2的产量比BN—N30—1提高了96％,比原始菌株提高了166％。结果表明,对纳豆芽孢杆菌BN-2-6进行NTG和低能N＋注入复合诱变的效果明显,突变菌株维生素K2的产量显著提高。     

Oxygen tension regulates reactive oxygen generation and mutation of Helicobacter pylori

Although both bacillary and coccoid forms of Helicobacter pylori reside in human stomach, the pathophysiological significance of the two forms remains obscure. The present work describes the effect of oxygen tension on the transformation and reactive oxygen species (ROS) metabolism of this pathogen. Most H. pylori cultured under an optimum O2 concentration (7%) were the bacillary form, whereas about 80% of cells cultured under aerobic or anaerobic conditions were the coccoid form. The colony-forming unit of H. pylori decreased significantly under both aerobic and anaerobic culture conditions. The bacillary form of H. pylori generated predominantly superoxide radical, whereas the coccoid form generated preferentially hydroxyl radical. Specific activities of cellular respiration, urease, and superoxide dismatase decreased markedly after transformation of the bacillary form to the coccoid form, with concomitant generation of protein carbonyls and 8-hydroxyguanine. The frequency of mutation of cells increased significantly during culture under nonoptimum O2 conditions. These results indicate that ROS generated by H. pylori catalyze the oxidative modification of cellular DNA, thereby enhancing the transformation from the bacillary to the coccoid form. The enhanced generation of mutagenic hydroxyl radicals in the coccoid form might accelerate mutation and increase the genetic diversity of H. pylori.     

Mutation of ndh genes leads to inhibition of CO(2) uptake rather than HCO(3)(-) uptake in Synechocystis sp. strain PCC 6803

Six mutants (B1 to B6) that grew poorly in air on BG11 agar plates buffered at pH 8.0 were rescued after mutations were introduced into ndhB of wild-type (WT) Synechocystis sp. strain PCC 6803. In these mutants and a mutant (M55) lacking ndhB, CO(2) uptake was much more strongly inhibited than HCO(3)(-) uptake, i.e., the activities of CO(2) and HCO(3)(-) uptake in B1 were 9 and 85% of those in the WT, respectively. Most of the mutants grew very slowly or did not grow at all at pH 6.5 or 7.0 in air, and their ability to grow under these conditions was correlated with CO(2) uptake capacity. Detailed studies of B1 and M55 indicated that the mutants grew as fast as the WT in liquid at pH 8.0 under air, although they grew poorly on agar plates. The contribution of CO(2) uptake appears to be larger on solid medium. Five mutants were constructed by inactivating each of the five ndhD genes in Synechocystis sp. strain PCC 6803. The mutant lacking ndhD3 grew much more slowly than the WT at pH 6.5 under 50 ppm CO(2), although other ndhD mutants grew like the WT under these conditions and showed low affinity for CO(2) uptake. These results indicated the presence of multiple NAD(P)H dehydrogenase type I complexes with specific roles.