Protective role of certain chemicals against UV-B-induced damage in the nitrogen-fixing cyanobacterium, Nostoc muscorum.

The protective effects of L-cysteine, ascorbic acid, reduced glutathione, L-tryptophan, and sodium pyruvate against UV-B-induced damages were studied in the nitrogen-fixing cyanobacterium, Nostoc muscorum. When added to the culture suspension during UV-B treatment, these chemicals caused a significant protective effect on survival and growth of the organism. Sodium pyruvate conferred the strongest protection whereas the weakest effect was elicited by tryptophan. A 20 min exposure of a culture suspension to UV-B completely inactivated nitrogenase activity but the inactivation was strongly prevented by exogenous addition of ascorbic acid or reduced glutathione during UV-B exposure, and weakly prevented by pyruvate, cysteine and tryptophan. In vivo nitrate reductase activity was not completely lost even after 80 min of UV-B exposure, and addition of the test chemicals did not confer any significant protection to this enzyme. Whereas (14)CO(2) uptake was drastically inhibited (78% inhibition) by 30 min exposure to UV-B in the absence of any test chemical, about 76% activity remained when the UV-B exposure was given to cultures in the presence of ascorbic acid. These results suggest that the damaging effects of UV-B are substantially minimized by certain reducing agents, the protective effect being particularly strong on the O(2) sensitive enzyme, nitrogenase. Presence of these chemicals in their natural habitat or inside the cells of living organisms may partially protect/repair the damaging effects of UV-B radiation.

The role of UV-B radiation in aquatic and terrestrial ecosystems--an experimental and functional analysis of the evolution of UV-absorbing compounds.

We analysed and compared the functioning of UV-B screening pigments in plants from marine, fresh water and terrestrial ecosystems, along the evolutionary line of cyanobacteria, unicellular algae, primitive multicellular algae, charophycean algae, lichens, mosses and higher plants, including amphibious macrophytes. Lichens were also included in the study. We were interested in the following key aspects: (a) does the water column function effectively as an 'external UV-B filter'?; (b) do aquatic plants need less 'internal UV-B screening' than terrestrial plants?; (c) what role does UV screening play in protecting the various plant groups from UV-B damage, such as the formation of thymine dimers?; and (d) since early land 'plants' (such as the predecessors of present-day cyanobacteria, lichens and mosses) experienced higher UV-B fluxes than higher plants, which evolved later, are primitive aquatic and land organisms (cyanobacteria, algae, lichens, mosses) better adapted to present-day levels of UV-B than higher plants? Furthermore, polychromatic action spectra for the induction of UV screening pigments of aquatic organisms have been determined. This is relevant for translating 'physical' radiation measurements of solar UV-B into 'biological' and 'ecological' effects. From the action spectra, radiation amplification factors (RAFs) have been calculated. These action spectra allow us to determine any mitigating or antagonistic effects in the ecosystems and therefore qualify the damage prediction for the ecosystems under study. We summarize and discuss the main results based on three years of research of four European research groups. The central theme of the work was the investigation of the effectiveness of the various screening compounds from the different species studied in order to gain some perspective of the evolutionary adaptations from lower to higher plant forms. The induction of mycosporine-like amino acids (MAAs) was studied in the marine dinoflagellate Gyrodinium dorsum, the green algal species Prasiola stipitata and in the cyanobacterium Anabaena sp. While visible (400-700 nm) and long wavelength UV-A (315-400 nm) showed only a slight effect, MAAs were effectively induced by UV-B (280-315 nm). The growth of the lower land organisms studied, i.e. the lichens Cladina portentosa, Cladina foliacaea and Cladonia arbuscula, and the club moss Lycopodiumannotinum, was not significantly reduced when grown under elevated UV-B radiation (simulating 15% ozone depletion). The growth in length of the moss Tortula ruralis was reduced under elevated UV-B. Of the aquatic plants investigated the charophytes Chara aspera showed decreased longitudinal growth under elevated UV-B. In the 'aquatic higher plants' studied, Ceratophyllum demersum, Batrachium trichophyllum and Potamogeton alpinus, there was no such depressed growth with enhanced UV-B. In Chara aspera, neither MAAs nor flavonoids could be detected. Of the terrestrial higher plants studied, Fagopyrum esculentum, Deschampsia antarctica, Vicia faba, Calamagrostis epigejos and Carex arenaria, the growth of the first species was depressed with enhanced UV-B, in the second species length growth was decreased, but the shoot number was increased, and in the latter two species of a dune grassland there was no reduced growth with enhanced UV-B. In the dune grassland species studied outdoors, at least five different flavonoids appeared in shoot tissue. Some of the flavonoids in the monocot species, which were identified and quantified with HPLC, included orientin, luteolin, tricin and apigenin. A greenhouse study with Vicia faba showed that two flavonoids (aglycones) respond particularly to enhanced UV-B. Of these, quercetin is UV-B inducible and mainly located in epidermal cells, while kaempferol occurs constitutively. In addition to its UV-screening function, quercetin may also act as an antioxidant. Polychromatic action spectra were determined for induction of the UV-absorbing pigments in three photosynthetic organisms, representing very different taxonomic groups and different habitats. In ultraviolet photobiology, action spectra mainly serve two purposes: (1) identification of the molecular species involved in light absorption; and (2) calculation of radiation amplification factors for assessing the effect of ozone depletion. Radiation amplification factors (RAFs) were calculated from the action spectra. In a somewhat simplified way, RAF can be defined as the percent increase of radiation damage for a 1% depletion of the ozone layer. Central European summer conditions were used in the calculations, but it has been shown that RAF values are not critically dependent on latitude or season. If only the ultraviolet spectral region is considered, the RAF values obtained are 0.7 for the green alga Prasiola stipitata, 0.4 for the dinoflagellate Gyrodinium dorsum, and 1.0 for the cyanobacterium Anabaena sp. In the case of P. stipitata, however, the effect of visible light (PAR, photosynthetically active radiation, 400-700 nm) is sufficient to lower the RAF to about 0.4, while the PAR effect for G. dorsum is negligible. RAFs for some damage processes, such as for DNA damage (RAF=2.1 if protective effects or photorepair are not considered [1]), are higher than those above. Our interpretation of this is that if the ozone layer is depleted, increased damaging radiation could overrule increased synthesis of protective pigments. In addition to investigating the functional effectiveness of the different screening compounds, direct UV effects on a number of key processes were also studied in order to gain further insight into the ability of the organisms to withstand enhanced UV-B radiation. To this end, the temperature-dependent repair of cyclobutane dimers (CPD) and (6-4) photoproducts induced by enhanced UV-B was studied in Nicotiana tabacum, and the UV-B induction of CPD was studied in the lichen Cladonia arbuscula. Also, photosynthesis and motility were monitored and the response related to the potential function of the screening compounds of the specific organism.

Polychromatic action spectrum for the induction of a mycosporine-like amino acid in a rice-field cyanobacterium, Anabaena sp.

A polychromatic action spectrum for the induction of an ultraviolet-absorbing/screening mycosporine-like amino acid (MAA) has been determined in a filamentous and heterocystous nitrogen-fixing rice-field cyanobacterium, Anabaena sp. High-performance liquid chromatographic (HPLC) studies revealed the presence of only one type of MAA, which was identified as shinorine, a bisubstituted MAA containing both glycine and serine groups having a retention time at 2.8 min and an absorption maximum at 334 nm. Exposure of cultures to simulated solar radiation in combination with various cut-off filters (WG 280, 295, 305, 320, 335, 345, GG 400, 420, 455, 475, OG 515, 530, 570, RG 645, 665 and a broad-band filter, UG 11) clearly revealed that the induction of the MAA takes place only in the UV range. Photosynthetic active radiation (PAR) had no significant impact on MAA induction. The ratio of the absorption at 334 nm (shinorine) to 665 nm (chlorophyll a) and the action spectrum also showed the induction of MAA to be UV dependent peaking in the UV-B range at around 290 nm. The results indicate that the studied cyanobacterium, Anabaena sp. may protect itself from deleterious short wavelength solar radiation by its ability to synthesize a mycosporine-like amino acid in response to UV-B radiation and thereby screen the negative effects of UV-B.

Life under solar UV radiation in aquatic organisms.

Aquatic photosynthetic organisms are exposed to solar ultraviolet (UV) radiation while they harvest longer wavelength radiation for energetic reasons. Solar UV-B radiation (280-315 nm) affects motility and orientation in motile organisms and impairs photosynthesis in cyanobacteria, phytoplankton and macroalgae as measured by monitoring oxygen production or pulse amplitude modulated fluorescence analysis. Upon moderate UV stress most organisms respond by photoinhibition which is an active downregulation of the photosynthetic electron transport in photosystem II by degradation of UV-damaged D1 protein. Photoinhibition is readily reversible during recovery in shaded conditions. Excessive UV stress causes photodamage which is not easily reversible. Another major target is the DNA where UV-B mainly induces thymine dimers. Cyanobacteria, phytoplankton and macroalgae produce scytonemin, mycosporine-like amino acids and other UV-absorbing substances to protect themselves from short wavelength solar radiation.

Induction of mycosporine-like amino acids (MAAs) in cyanobacteria by solar ultraviolet-B radiation.

Three filamentous and heterocystous N(2)-fixing cyanobacteria, Anabaena sp., Nostoc commune and Scytonema sp. were tested for the presence of ultraviolet-absorbing mycosporine-like amino acids (MAAs) and their induction by solar ultraviolet-B (UV-B) radiation. High performance liquid chromatographic (HPLC) studies revealed the presence of only one type of MAAs in all three cyanobacteria, that was identified as shinorine, a bisubstituted MAA containing both glycine and serine groups having an absorption maximum at 334 nm and a retention time of around 2.8 min. There was a circadian induction in the synthesis of MAAs when the cultures were exposed to mid-latitude solar radiation (Playa Unión, Rawson, Chubut, Patagonia, Argentina) for 3 days, 4-6th February, 2000. Solar radiation was measured by an ELDONET (European Light Dosimeter Network) filter radiometer permanently installed on the roof of the Estación de Fotobiología Playa Unión (43 degrees 18' S; 65 degrees 03' W). The maximum irradiances were around 450-500, 45-50 and 1.0-1.2 Wm(-2) for PAR (photosynthetic active radiation), UV-A (ultraviolet-A) and UV-B (ultraviolet-B), respectively. PAR and UV-A had no significant impact on MAA induction while UV-B induced the synthesis of shinorine in all three cyanobacteria. Shinorine was found to be induced mostly during the light period. During the dark period the concentration stayed almost constant. In addition to shinorine, another unidentified, water-soluble, brownish compound with an absorption maximum at 315 nm was found to be induced by UV-B only in Scytonema sp. and released into the medium. This substance was neither found in Anabaena sp. nor in Nostoc commune. Judging from the results, the studied cyanobacteria may protect themselves from deleterious short wavelength radiation by their ability to synthesize photoprotective compounds in response to UV-B radiation.

A cyanobacterial recombination study, involving an efficient N2-fixing non-heterocystous partner.

Many filamentous cyanobacteria fix atmospheric nitrogen under natural conditions in specialized anaerobic compartments, heterocysts, interspersed between vegetative cells, which provide protection to the O2-sensitive nitrogenase. A few unicellular cyanobacterial strains are also known to fix nitrogen aerobically at a slower rate. Filamentous cyanobacteria lacking heterocysts are not known so far to fix nitrogen. We describe the isolation and purification of a non-heterocystous filamentous cyanobacterium from the fronds of the water-fern Azolla, fixing nitrogen at 18.7+/-0.2 n moles ethylene microg Chl. a(-1) h(-1) when grown in nitrogen-free medium at a low level of oxygen between two layers of agar. This strain of Anabaena azollae has been designated as het- nif+ (non-heterocystous and nitrogen-fixing), and is found to be easily and effectively preserved in nitrogen-free medium in standard synthetic cyanobacterial nutrient medium (pH 8.5) at a continuous light intensity of 2800 lx at 25+/-1 degrees C. This het- nif+ strain is an effective donor of the nif+ marker to a het+ nif- strain of another cyanobacterium, Nostoc muscorum, when both are grown together in a recombination study.

A cyanobacterial mutant resistant against a bleaching herbicide.

The nitrogen fixing cyanobacterium Nostoc muscorum has been found to be sensitive to the herbicide SAN 6706 [4-chloro-5 (dimethylamino)-2-(a,a,a,-trifluoro-m-tolyl)-3-(2H) pyridazinon] at 30-45 microM within 15 min. The toxicity was more severe in combined nitrogen-free (Ncomb-free) medium than in a combined nitrogen medium; this enhancement was reversible by supplementation of the medium with 3 mM glucose or 5 microM ATP, serving as carbon and/or energy source in this organism. A mutant of this cyanobacterium resistant to 3 mM SAN 6706 has been isolated and characterized to perform nitrogenase activity in exogenous ATP supplemented Ncomb-free medium. However, it exhibited a moderate growth in combined nitrogen media in the absence of external ATP. The resistance factor is higher than 100. Simultaneously, this strain possesses a cross-resistance to methylamine, a well-known inhibitor of photophosphorylation, irrespective of the exogenous ATP supply. The behavior of the mutant suggests a defective phosphorylation in its photosynthetic system.

Photoprotective compounds in cyanobacteria, phytoplankton and macroalgae--a database.

A database on photoprotective compounds in cyanobacteria, phytoplankton and macroalgae has been developed. It contains information on photoprotective compounds such as mycosporine-like amino acids (MAAs), scytonemin and other not yet identified compounds reported in aquatic organisms, their habitat, the collection site and date and the reference. Further information on the absorption maxima and extinction coefficients of different photoprotective compounds as well as experimental procedures are provided (see http:/ /www.biologie.uni-erlangen.de/botanik1/index.html). The database answers the urgent need for a library on these substances and provides scientists in the field with the necessary information to identify and quantify screening pigments in different aquatic organisms from various growing sites and different collection dates.

Effect of dietary calcium and phosphorus concentrations on retention of these nutrients by caged layers.

1. A 5 x 3 factorial experiment was carried out with caged White Leghorn hens with 5 concentrations of calcium (26.0, 29.0, 32.5, 36.0, 39.0 g/kg) and 3 concentrations of phosphorus (4.3, 5.0 and 6.0 g/kg) for 120 d. Variables observed were hen day egg production, food consumption, shell weight, shell weight per unit surface area (SWUSA) and egg content. 2. 36.0 g calcium (Ca) and 4.3 g phosphorus (P) kg were found to be the dietary concentrations that resulted in optimal hen day egg production, shell weight, SWUSA and egg content. 3. Lack of a significant interaction between the effects of calcium and phosphorus on production parameters showed that the dietary Ca:P ratio is not of great importance for the laying hen. 4. Absolute retentions of Ca and P were inversely related to percentage retentions. 5. A balance study of calcium and phosphorus also showed optima at 36.0 g Ca and 4.3 g P per kg. 6. It was inferred that 36.0 g Ca and 5.0 g total P per kg are the optimal concentrations in diets for caged layers in a tropical climate.

Disintegration of phycobilisomes in a rice field cyanobacterium Nostoc sp. following UV irradiation.

The effects of ultraviolet-B (UV-B, 280-315 nm) irradiation on survival, growth and phycobilisome assembly have been studied in the N2-fixing cyanobacterium Nostoc sp. Survival and growth were virtually arrested after 120 min of UV irradiation. The phycobiliprotein fractions showed a decrease in absorption and fluorescence as well as a shift towards shorter wavelengths indicating the impairment of energy transfer from phycobiliproteins to the photosynthetic reaction centers. This was further supported by SDS-PAGE analysis of the fractions which revealed a loss in high molecular mass rod-core and core-membrane linker polypeptides. Also the low molecular mass phycobilin (alpha beta) monomers decreased, showing that the supra-molecular organization of the phycobilisomes disintegrated during UV irradiation.

Effect of combination therapy in Mycobacterium paratuberculosis infected rabbits.

Rabbits, infected with M paratuberculosis of caprine origin, were treated with streptomycin sulphate and rifampicin in combination with levamisole hydrochloride for a period of 2 months. Marked clinical response including significant rise in total serum protein, albumin and globulin were recorded in treated groups. Highly significant higher leucocyte migration inhibition indicating enhanced CMI reaction, occurred in rabbits treated for 4 weeks. Complete elimination of M. paratuberculosis from faeces, intestines and mesenteric lymph nodes was observed. They were also absent from spleen, kidneys, lungs and liver of rabbits treated with rifampicin and levamisole, whereas were present in lungs of the rabbits administered with streptomycin and levamisole. Absence of characteristic lesions of paratuberculosis and evidence of regenerative reaction were observed in visceral organs of rabbits treated with rifampicin and levamisole. Effectiveness of rifampicin is attributed to its effect on lymphocyte, the primary cell involved in cell mediated immunity. Rifampicin-levamisole combination appeared superior to streptomycin-levamisole in eliminating the infection of M. paratuberculosis from infected rabbits. Approximate cost of treatment was calculated to be Rs 8.50 per kg body weight. Cattle, sheep and goats, if treated in early stage of infection may recover from paratuberculosis.

Stability of plasmids in lactococci during extended incubation in growth media.

The stability of plasmids in Lactococcus lactis ssp. lactis strains C2 and ML3, and L. lactis ssp. cremoris strains ML1 and SC607, was investigated by extended incubation of bacterial cells in low nutrient media under acidic conditions. Strains were grown overnight (16-18 h) in skim milk and unbuffered medium (M17-) at 32 degrees C and subsequently held at that temperature for extended periods (greater than or equal to 96 h). Lac- variants were obtained from each strain in milk and (M17-) broth. The plasmid profiles of Lac- variants when compared with their parental Lac+ strains showed loss of one or more plasmid bands. None of the Lac- mutants showed loss of smaller plasmids (less than 5 MDa) indicating that smaller plasmids in lactococci are more stable under these conditions than larger plasmids (greater than 10 MDa). Concomitant loss of the Lac+ phenotype and plasmids by the method used in the present investigation may have application for isolating mutants devoid of one or more plasmids.

Approaches to higher productivity in health care system.

The prosperity of organisations is recognised as being dependent on their comparative productivity. Productivity expresses a relationship between outputs from a system and the inputs which go into this creation. Hospital activities have to be assessed. Results have to be evaluated. The medical and paramedical staff have to quantify procedure and assess them qualitatively in order to improve the standards of care. Assessment techniques already introduced in many hospitals have succeeded in reducing the number of antibiotics (and their cost) and the amount of blood used in certain operations and also cutting the wait for operations. As long as there is unused capability in the individual or the productive system, increases in productivity can be achieved without decline in quality. If one focuses on quality while holding speed constant, quality should improve, waste should be eliminated, and productivity should increase. This can happen as long as the individual, or group of individuals, is willing to exert effort and has the capacity to achieve the quality--productivity levels desired. It is the operations manager's task to provide the facilities, tools and desired (motivation) to do so.

Effect of Carbohydrate on the Viability of Streptococcus thermophilus.

Streptococcus thermophilus strain ATCC 19258 consistently showed low colony counts (<106 CFU/ml) when grown overnight (16-18 h) in M17 at 42°C. There was a rapid decrease in the total viable colony counts after 10 h incubation at 42°C with the maximum decrease after 16 to 18 h incubation. A similar decrease in colony counts was also noticed in M17G and M17S broths containing 0.5% glucose and sucrose, respectively. The decrease in colony counts were more pronounced at 42°C than at 37°C. Such decreases in total viable colony counts was not observed when cells were grown in M17 broth initially supplemented with 2% carbohydrate. The cell survival and turbidity of seven other cultures of S. thermophilus in M17 and M17-2 L (M17 containing 2% lactose) broths were also tested at 42°C. All the cultures showed significantly higher cell survival in M17-2 L broth than that observed in M17 medium. The results demonstrate that both growth and maintenance of S. thermophilus strains can be markedly improved by supplementing M17 medium with 2% carbohydrate as opposed to 0.5% used in regular M17 medium.

Effect of Growth Media and Extended Incubation on the Appearance of Lactose - Nonfermenting Variants in Lactococci.

Growth and storage were investigated for the development of Lac- (lactose-nonfermenting) variants of Lactococcus lactis subsp. lactis C2, ML3 and L. lactis subsp. cremoris ML1, SC607 under different conditions, in an unbuffered medium (M17-), and in media buffered with inorganic or organic phosphates. Strains were grown overnight (16-18 h) at 32°C and subsequently held at 32 and 22°C. The cell survival was much higher at 22°C than at 32°C after storage for 96 h. Most of the survivors in M17- broth were of the Lac- phenotype. Lac- variants were also observed when the cultures were grown in skim milk at 32°C and then held at that temperature for 96 h. These results showed that the lactose-fermenting ability of lactococcus in general is lost when overnight cultures in M17- broth are kept either at room temperature (22°C) or at 32°C for an extended period. However, the cultures in buffered media under similar conditions showed little or no loss of lactose-fermenting ability, suggesting that phosphate in the media had a stabilizing effect on plasmid-encoded lactose-fermenting gene(s). These observations indicate the possibility of utilizing this method as a simple technique for isolating mutants deficient in plasmid-linked genetic traits in lactococci.

A new simple method of curing plasmids in lactic streptococci (Streptococcus cremoris; Streptococcus lactis, plasmids).

A simple method for curing plasmid DNA from lactic streptococci is described. When strains of lactic streptococci are grown overnight at 32 degrees C in an unbuffered medium (M17-) and held at the same temperature for an extended period (96 h), the acid environment induces loss of plasmid DNA of different sizes. If the process of growth in M17- broth followed by extended incubation at 32 degrees C is repeated, most of the plasmids are lost, as revealed by gel electrophoretic profiles of DNA. The method is simple and efficient in curing plasmids of lactic streptococci without use of any mutagenic chemical.

District health and hospitals: a system and concept to achieve health for all.

India has pledged for "health for all" goals and objectives, agreeing to adopt the vehicle of primary health care to this end. However, till the mid eighties, a lot of emphasis was given to the hospitals under national health policies resulting in the relative segregation of these two approaches. This paper discusses the role of district hospitals as the common link between these two systems and gives a model and concept to unify them.