Naphthalene Degradation and Incorporation of Naphthalene-Derived Carbon into Biomass by the Thermophile Bacillus thermoleovorans

The thermophilic aerobic bacterium Bacillus thermoleovorans Hamburg 2 grows at 60°C on naphthalene as the sole source of carbon and energy. In batch cultures, an effective substrate degradation was observed. The carbon balance, including naphthalene, metabolites, biomass, and CO₂, was determined by the application of [1-¹³C]naphthalene. The incorporation of naphthalene-derived carbon into the bulk biomass as well as into specified biomass fractions such as fatty acids and amino acids was confirmed by coupled gas chromatography-mass spectrometry (GC-MS) and isotope analyses. Metabolites were characterized by GC-MS; the established structures allow tracing the degradation pathway under thermophilic conditions. Apart from typical metabolites of naphthalene degradation known from mesophiles, intermediates such as 2,3-dihydroxynaphthalene, 2-carboxycinnamic acid, and phthalic and benzoic acid were identified for the pathway of this bacterium. These compounds indicate that naphthalene degradation by the thermophilic B. thermoleovorans differs from the known pathways found for mesophilic bacteria.     

Biochemical Characterization of Two Thermostable Xylanolytic Enzymes Encoded by a Gene Cluster of Caldicellulosiruptor owensensis

The xylanolytic extremely thermophilic bacterium Caldicellulosiruptor owensensis provides a promising platform for xylan utilization. In the present study, two novel xylanolytic enzymes, GH10 endo-β-1,4-xylanase (Coxyn A) and GH39 β-1,4-xylosidase (Coxyl A) encoded in one gene cluster of C.owensensis were heterogeneously expressed and biochemically characterized. The optimum temperature of the two xylanlytic enzymes was 75°C, and the respective optimum pH for Coxyn A and Coxyl A was 7.0 and 5.0. The difference of Coxyn A and Coxyl A in solution was existing as monomer and homodimer respectively, it was also observed in predicted secondary structure. Under optimum condition, the catalytic efficiency (k _cat/K _m) of Coxyn A was 366 mg ml⁻¹ s⁻¹ on beechwood xylan, and the catalytic efficiency (k _cat/K _m) of Coxyl A was 2253 mM⁻¹ s⁻¹ on pNP-β-D-xylopyranoside. Coxyn A degraded xylan to oligosaccharides, which were converted to monomer by Coxyl A. The two intracellular enzymes might be responsible for xylooligosaccharides utilization in C.owensensis, also provide a potential way for xylan degradation in vitro.     

Cadmium Ion Biosorption by the Thermophilic Bacteria Geobacillus stearothermophilus and G. thermocatenulatus

This study reports surface complexation models (SCMs) for quantifying metal ion adsorption by thermophilic microorganisms. In initial cadmium ion toxicity tests, members of the genus Geobacillus displayed the highest tolerance to CdCl₂ (as high as 400 to 3,200 μM). The thermophilic, gram-positive bacteria Geobacillus stearothermophilus and G. thermocatenulatus were selected for further electrophoretic mobility, potentiometric titration, and Cd²⁺ adsorption experiments to characterize Cd²⁺ complexation by functional groups within and on the cell wall. Distinct one-site SCMs described the extent of cadmium ion adsorption by both studied Geobacillus sp. strains over a range of pH values and metal/bacteria concentration ratios. The results indicate that a functional group with a deprotonation constant pK value of approximately 3.8 accounts for 66% and 80% of all titratable sites for G. thermocatenulatus and G. stearothermophilus, respectively, and is dominant in Cd²⁺ adsorption reactions. The results suggest a different type of functional group may be involved in cadmium biosorption for both thermophilic strains investigated here, compared to previous reports for mesophilic bacteria.     

A genome-guided analysis of energy conservation in the thermophilic,cytochrome-free acetogenic bacterium Thermoanaerobacter?kivui

Background

Acetogenic bacteria are able to use CO₂ as terminal electron acceptor of an anaerobic respiration, thereby producing acetate with electrons coming from H₂. Due to this feature, acetogens came into focus as platforms to produce biocommodities from waste gases such as H₂ + CO₂ and/or CO. A prerequisite for metabolic engineering is a detailed understanding of the mechanisms of ATP synthesis and electron-transfer reactions to ensure redox homeostasis. Acetogenesis involves the reduction of CO₂ to acetate via soluble enzymes and is coupled to energy conservation by a chemiosmotic mechanism. The membrane-bound module, acting as an ion pump, was of special interest for decades and recently, an Rnf complex was shown to couple electron flow from reduced ferredoxin to NAD⁺ with the export of Na⁺ in Acetobacterium woodii. However, not all acetogens have rnf genes in their genome. In order to gain further insights into energy conservation of non-Rnf-containing, thermophilic acetogens, we sequenced the genome of Thermoanaerobacter kivui.

Results

The genome of Thermoanaerobacter kivui comprises 2.9 Mbp with a G + C content of 35% and 2,378 protein encoding orfs. Neither autotrophic growth nor acetate formation from H₂ + CO₂ was dependent on Na⁺ and acetate formation was inhibited by a protonophore, indicating that H⁺ is used as coupling ion for primary bioenergetics. This is consistent with the finding that the c subunit of the F₁F_O ATP synthase does not have the conserved Na⁺ binding motif. A search for potential H⁺-translocating, membrane-bound protein complexes revealed genes potentially encoding two different proton-reducing, energy-conserving hydrogenases (Ech).

Conclusions

The thermophilic acetogen T. kivui does not use Na⁺ but H⁺ for chemiosmotic ATP synthesis. It does not contain cytochromes and the electrochemical proton gradient is most likely established by an energy-conserving hydrogenase (Ech). Its thermophilic nature and the efficient conversion of H₂ + CO₂ make T. kivui an interesting acetogen to be used for the production of biocommodities in industrial micobiology. Furthermore, our experimental data as well as the increasing number of sequenced genomes of acetogenic bacteria supported the new classification of acetogens into two groups: Rnf- and Ech-containing acetogens.     

Streptomyces thermoautotrophicus sp. nov., a Thermophilic CO- and H2-Oxidizing Obligate Chemolithoautotroph

The novel thermophilic CO- and H₂-oxidizing bacterium UBT1 has been isolated from the covering soil of a burning charcoal pile. The isolate is gram positive and obligately chemolithoautotrophic and has been named Streptomyces thermoautotrophicus on the basis of G+C content (70.6 ± 0.19 mol%), a phospholipid pattern of type II, MK-9(H₄) as the major quinone, and other chemotaxonomic and morphological properties. S. thermoautotrophicus could grow with CO (t_d = 8 h), H₂ plus CO₂ (t_d = 6 h), car exhaust, or gas produced by the incomplete combustion of wood. Complex media or heterotrophic substrates such as sugars, organic acids, amino acids, and alcohols did not support growth. Molybdenum was required for CO-autotrophic growth. For growth with H₂, nickel was not necessary. The optimum growth temperature was 65°C; no growth was observed below 40°C. However, CO-grown cells were able to oxidize CO at temperatures of 10 to 70°C. Temperature profiles of burning charcoal piles revealed that, up to a depth of about 10 to 25 cm, the entire covering soil provides a suitable habitat for S. thermoautotrophicus. The K_m was 88 μl of CO liter⁻¹ and V_max was 20.2 μl of CO h⁻¹ mg of protein⁻¹. The threshold value of S. thermoautotrophicus of 0.2 μl of CO liter⁻¹ was similar to those of various soils. The specific CO-oxidizing activity in extracts with phenazinemethosulfate plus 2,6-dichlorophenolindophenol as electron acceptors was 246 μmol min⁻¹ mg of protein⁻¹. In exception to other carboxydotrophic bacteria, S. thermoautotrophicus CO dehydrogenase was able to reduce low potential electron acceptors such as methyl and benzyl viologens.     

Effect of Tungstate on Nitrate Reduction by the Hyperthermophilic Archaeon Pyrobaculum aerophilum

Pyrobaculum aerophilum, a hyperthermophilic archaeon, can respire either with low amounts of oxygen or anaerobically with nitrate as the electron acceptor. Under anaerobic growth conditions, nitrate is reduced via the denitrification pathway to molecular nitrogen. This study demonstrates that P. aerophilum requires the metal oxyanion WO₄²⁻ for its anaerobic growth on yeast extract, peptone, and nitrate as carbon and energy sources. The addition of 1 μM MoO₄²⁻ did not replace WO₄²⁻ for the growth of P. aerophilum. However, cell growth was completely inhibited by the addition of 100 μM MoO₄²⁻ to the culture medium. At lower tungstate concentrations (0.3 μM and less), nitrite was accumulated in the culture medium. The accumulation of nitrite was abolished at higher WO₄²⁻ concentrations (<0.7 μM). High-temperature enzyme assays for the nitrate, nitrite, and nitric oxide reductases were performed. The majority of all three denitrification pathway enzyme activities was localized to the cytoplasmic membrane, suggesting their involvement in the energy metabolism of the cell. While nitrite and nitric oxide specific activities were relatively constant at different tungstate concentrations, the activity of nitrate reductase was decreased fourfold at WO₄²⁻ levels of 0.7 μM or higher. The high specific activity of the nitrate reductase enzyme observed at low WO₄²⁻ levels (0.3 μM or less) coincided with the accumulation of nitrite in the culture medium. This study documents the first example of the effect of tungstate on the denitrification process of an extremely thermophilic archaeon. We demonstrate here that nitrate reductase synthesis in P. aerophilum occurs in the presence of high concentrations of tungstate.     

Production and Purification of Extracellular D-Xylose Isomerase from an Alkaliphilic, Thermophilic Bacillus sp.

An alkaliphilic, thermophilic Bacillus sp. (NCIM 59) produced extracellular xylose isomerase at pH 10 and 50°C by using xylose or wheat bran as the carbon source. The distribution of xylose isomerase as a function of growth in comparison with distributions of extra- and intracellular marker enzymes such as xylanase and β-galactosidase revealed that xylose isomerase was truly secreted as an extracellular enzyme and was not released because of sporulation or lysis. The enzyme was purified to homogeneity by ammonium sulfate precipitation followed by gel filtration, preparative polyacrylamide gel electrophoresis, and ion-exchange chromatography. The molecular weight of xylose isomerase was estimated to be 160,000 by gel filtration and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of three subunits. The enzyme is most active at pH 8.0 and with incubation at 85°C for 20 min. Divalent metal ions Mg²⁺, Co²⁺, and Mn²⁺ were required for maximum activity of the enzyme. The K_m values for D-xylose and D-glucose at 80°C and pH 7.5 were 6.66 and 142 mM, respectively, while K_cat values were 2.3 × 10² s^-1 and 0.5 × 10² s^-1, respectively.     

Anaerobic digestion of cattle waste at mesophilic and thermophilic temperatures

Methanogenesis was studied using stirred, bench-top fermentors of 3-1 working volume fed on a semi-continuous basis with waste obtained from cattle fed a high grain, finishing diet. Digestion was carried out at 40 and 60°C. CH₄ production was 11.8, 18.3, 61.9 and 84.5% higher in the thermophilic than the mesophilic digestor at the 3, 6, 9 and 12 g volatile solids (VS) l^–1 reactor volume loading rates, respectively. When compared on an energetic basis CH₄ production was 7.4, 18.3, 72.9 and 107.3 kJ day higher in the thermophilic than the mesophilic digestor. CH₄ production decreased more rapidly with each increase in VS loading rate and decrease in retention time (RT) in the mesophilic than the thermophilic digestor. When expressed as l g^–1 VS fed or as kJ kJ^–1 fed, the amount of CH₄ was 49% less at the highest compared to the lowest loading rate in the mesophilic digestor. In the thermophilic digestor the decrease was only 16%. Propionate accumulated in the mesophilic digestor at the two highest loading rates, reaching concentrations of about 50 mM, but were only about 13 mM in the thermophilic digestor. Isobutyrate, isovalerate plus 2-methylbutyrate, and valerate also accumulated at the higher loading rates.     

Inorganic composition and microbial characteristics of methanogenic granular sludge grown in a thermophilic upflow anaerobic sludge blanket reactor

An upflow anaerobic sludge blanket reactor was operated under thermophilic conditions (55° C) for 160 days by feeding a wastewater containing sucrose as the major carbon source. The reactor exhibited a satisfactory performance due to the formation of well-settling granulated sludge, achieving a total organic carbon (TOC) removal of above 80% at an organic loading rate of 30 kg total organic C m^–3 day^–1. Structural and microbial properties of the methanogenic granular sludge were examined using scanning electron microscope X-ray analyses and serum vial activity tests. All the thermophilic granules developed showed a double-layered structure, comprised of a black core portion and a yellowish exterior portion. The interior cope portion contained abundant crystalline precipitates of calcium carbonate. Calcium-bound phosphorus was also present more prominently in the core portion than in the exterior portion. Methanogenic activities of the thermophilic granules both from acetate and from H₂ increased with increasing vial-test temperature in the range of 55–65° C [from 1.43 to 2.36 kg CH₄ chemical oxygen demand (COD) kg volatile suspended solids (VSS)^–1 day^–1 for acetate and from 0.85 to 1.11 kg CH₄ COD kg VSS^–1 day^–1 for H₂]. On the other hand, propionate-utilizing methanogenic activity was independent of vial-test temperature, and was much lower (0.1–0.12 kg CH₄ COD kg VSS^–1 day^–1) than that from either acetate or H₂. Acetate consumption during vial tests was considerably inhibited by the presence of H₂ in the headspace, indicating that a syntrophic association between acetate oxidizers and H₂-utilizing methane-producing bacteria was responsible for some portion of the overall acetate elimination by the theromophilically grown sludge.     

Biochemical and Genetic Characterization of an FK506-Sensitive Peptidyl Prolyl cis-trans Isomerase from a Thermophilic Archaeon, Methanococcus thermolithotrophicus

A peptidyl prolyl cis-trans isomerase (PPIase) was purified from a thermophilic methanogen, Methanococcus thermolithotrophicus. The PPIase activity was inhibited by FK506 but not by cyclosporine. The molecular mass of the purified enzyme was estimated to be 16 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 42 kDa by gel filtration. The enzyme was thermostable, with the half-lives of its activity at 90 and 100°C being 90 and 30 min, respectively. The catalytic efficiencies (k_cat/K_m) measured at 15°C for the peptidyl substrates, N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, were 0.35 and 0.20 μM⁻¹ s⁻¹, respectively, in chymotrypsin-coupled assays. The purified enzyme was sensitive to FK506 and therefore was called MTFK (M. thermolithotrophicus FK506-binding protein). The MTFK gene (462 bp) was cloned from an M. thermolithotrophicus genomic library. The comparison of the amino acid sequence of MTFK with those of other FK506-binding PPIases revealed that MTFK has a 13-amino-acid insertion in the N-terminal region that is unique to thermophilic archaea. The relationship between the thermostable nature of MTFK and its structure is discussed.     

Hyperthermophilic DNA Methyltransferase M.PabI from the Archaeon Pyrococcus abyssi

Genome sequence comparisons among multiple species of Pyrococcus, a hyperthermophilic archaeon, revealed a linkage between a putative restriction-modification gene complex and several large genome polymorphisms/rearrangements. From a region apparently inserted into the Pyrococcus abyssi genome, a hyperthermoresistant restriction enzyme [PabI; 5′-(GTA/C)] with a novel structure was discovered. In the present work, the neighboring methyltransferase homologue, M.PabI, was characterized. Its N-terminal half showed high similarities to the M subunit of type I systems and a modification enzyme of an atypical type II system, M.AhdI, while its C-terminal half showed high similarity to the S subunit of type I systems. M.PabI expressed within Escherichia coli protected PabI sites from RsaI, a PabI isoschizomer. M.PabI, purified following overexpression, was shown to generate 5′-GTm6AC, which provides protection against PabI digestion. M.PabI was found to be highly thermophilic; it showed methylation at 95°C and retained at least half the activity after 9 min at 95°C. This hyperthermophilicity allowed us to obtain activation energy and other thermodynamic parameters for the first time for any DNA methyltransferases. We also determined the kinetic parameters of k_cat, K_{m, DNA}, and K_{m, AdoMet}. The activity of M.PabI was optimal at a slightly acidic pH and at an NaCl concentration of 200 to 500 mM and was inhibited by Zn²⁺ but not by Mg²⁺, Ca²⁺, or Mn²⁺. These and previous results suggest that this unique methyltransferase and PabI constitute a type II restriction-modification gene complex that inserted into the P. abyssi genome relatively recently. As the most thermophilic of all the characterized DNA methyltransferases, M.PabI may help in the analysis of DNA methylation and its application to DNA engineering.     

Change in Microbial Numbers during Thermophilic Composting of Sewage Sludge with Reference to CO2 Evolution Rate

Dewatered sewage sludge was composted in a laboratory-scale autothermal reactor in which a constant temperature of 60°C was kept as long as possible by regulating the air feed rate. The change in CO₂ evolution rate was measured continuously from the start up through the cessation of compositing. The succession of mesophilic bacteria, thermophilic bacteria, and thermophilic actinomycetes was also observed during the composting. Specific CO₂ evolution rates of thermophilic bacteria and actinomycetes in the constant-temperature region of 60°C were assessed quantitatively. It was found that the CO₂ evolution rate was attributed to thermophilic bacteria at the initial stage of 60°C and to thermophilic actinomycetes at the later stage of 60°C.     

Microbial Diversity in Ultra-High-Pressure Rocks and Fluids from the Chinese Continental Scientific Drilling Project in China

Microbial communities in ultra-high-pressure (UHP) rocks and drilling fluids from the Chinese Continental Scientific Drilling Project were characterized. The rocks had a porosity of 1 to 3.5% and a permeability of ~0.5 mDarcy. Abundant fluid and gas inclusions were present in the minerals. The rocks contained significant amounts of Fe₂O₃, FeO, P₂O₅, and nitrate (3 to 16 ppm). Acridine orange direct counting and phospholipid fatty acid analysis indicated that the total counts in the rocks and the fluids were 5.2 × 10³ to 2.4 × 10⁴ cells/g and 3.5 × 10⁸ to 4.2 × 10⁹ cells/g, respectively. Enrichment assays resulted in successful growth of thermophilic and alkaliphilic bacteria from the fluids, and some of these bacteria reduced Fe(III) to magnetite. 16S rRNA gene analyses indicated that the rocks were dominated by sequences similar to sequences of Proteobacteria and that most organisms were related to nitrate reducers from a saline, alkaline, cold habitat; however, some phylotypes were either members of a novel lineage or closely related to uncultured clones. The bacterial communities in the fluids were more diverse and included Proteobacteria, Bacteroidetes, gram-positive bacteria, Planctomycetes, and Candidatus taxa. The archaeal diversity was lower, and most sequences were not related to any known cultivated species. Some archaeal sequences were 90 to 95% similar to sequences recovered from ocean sediments or other subsurface environments. Some archaeal sequences from the drilling fluids were >93% similar to sequences of Sulfolobus solfataricus, and the thermophilic nature was consistent with the in situ temperature. We inferred that the microbes in the UHP rocks reside in fluid and gas inclusions, whereas those in the drilling fluids may be derived from subsurface fluids.     

Thermophilic Sulfate Reduction in Hydrothermal Sediment of Lake Tanganyika, East Africa

In environments with temperatures above 60°C, thermophilic prokaryotes are the only metabolically active life-forms. By using the ³⁵SO₄^2- tracer technique, we studied the activity of sulfate-reducing microorganisms (SRM) in hot sediment from a hydrothermal vent site in the northern part of freshwater Lake Tanganyika (East Africa). Incubation of slurry samples at 8 to 90°C demonstrated meso- and thermophilic sulfate reduction with optimum temperatures of 34 to 45°C and 56 to 65°C, respectively, and with an upper temperature limit of 80°C. Sulfate reduction was stimulated at all temperatures by the addition of short-chain fatty acids and benzoate or complex substrates (yeast extract and peptone). A time course experiment showed that linear thermophilic sulfate consumption occurred after a lag phase (12 h) and indicated the presence of a large population of SRM in the hydrothermal sediment. Thermophilic sulfate reduction had a pH optimum of about 7 and was completely inhibited at pH 8.8 to 9.2. SRM could be enriched from hydrothermal chimney and sediment samples at 60 and 75°C. In lactate-grown enrichments, sulfide production occurred at up to 70 and 75°C, with optima at 63 and 71°C, respectively. Several sporulating thermophilic enrichments were morphologically similar to Desulfotomaculum spp. Dissimilatory sulfate reduction in the studied hydrothermal area of Lake Tanganyika apparently has an upper temperature limit of 80°C.     

Product Inhibition of Butyrate Metabolism by Acetate and Hydrogen in a Thermophilic Coculture

Studies on product inhibition of a thermophilic butyrate-degrading bacterium in syntrophic association with Methanobacterium thermoautotrophicum showed that a gas phase containing more than 2 × 10⁻² atm (2.03 kPa) of hydrogen prevented growth and butyrate consumption, while a lower hydrogen partial pressure of 1 × 10⁻³ to 2 × 10⁻² atm (0.1 to 2.03 kPa) gradually inhibited the butyrate consumption of the coculture. No inhibition of butyrate consumption was found on the addition of 0.75 × 10⁻³ atm (76 Pa) of hydrogen to the gas phase. A slight inhibition of butyrate consumption by the coculture occurred at an acetate concentration of 16.4 mM. Inhibition gradually increased with increasing acetate concentration up to 81.4 mM, when complete inhibition of butyrate consumption occurred. When the culture contained an acetate-utilizing methanogen in addition to M. thermoautotrophicum, the inhibition of the triculture by acetate was gradually reversed as the acetate concentration was lowered by the aceticlastic methanogen. The results show that optimal growth conditions for the thermophilic butyrate-degrading bacterium depend on both hydrogen and acetate removal.     

Characterization of an ATP-dependent DNA ligase from the thermophilic archaeon Methanobacterium thermoautotrophicum

We report the production, purification and characterization of a DNA ligase encoded by the thermophilic archaeon Methanobacterium thermoautotrophicum. The 561 amino acid Mth ligase catalyzed strand-joining on a singly nicked DNA in the presence of a divalent cation (magnesium, manganese or cobalt) and ATP (K_m 1.1 µM). dATP can substitute for ATP, but CTP, GTP, UTP and NAD⁺ cannot. Mth ligase activity is thermophilic in vitro, with optimal nick-joining at 60°C. Mutational analysis of the conserved active site motif I (KxDG) illuminated essential roles for Lys251 and Asp253 at different steps of the ligation reaction. Mutant K251A is unable to form the covalent ligase–adenylate intermediate (step 1) and hence cannot seal a 3′-OH/5′-PO₄ nick. Yet, K251A catalyzes phosphodiester bond formation at a pre-adenylated nick (step 3). Mutant D253A is active in ligase–adenylate formation, but defective in activating the nick via formation of the DNA–adenylate intermediate (step 2). D253A is also impaired in phosphodiester bond formation at a pre-adenylated nick. A profound step 3 arrest, with accumulation of high levels of DNA–adenylate, could be elicited for the wild-type Mth ligase by inclusion of calcium as the divalent cation cofactor. Mth ligase sediments as a monomer in a glycerol gradient. Structure probing by limited proteolysis suggested that Mth ligase is a tightly folded protein punctuated by a surface-accessible loop between nucleotidyl transferase motifs III and IIIa.     

Biochemical characterization of Santalum album (Chandan) leaf peroxidase

The Santalum peroxidase was extracted from the leaves and precipitated with double volume of chilled acetone. The optimum percent relative activity for the Santalum peroxidase was observed at pH 5.0 and 50 °C temperature. The Santalum peroxidase per cent relative activity was stimulated in the presence of phenolic compounds like ferrulic acid and caffeic acids; however, indole-3-acetic acid (IAA) and protocatechuic acid act as inhibitors. All divalent cations Fe²⁺, Mn²⁺, Mg²⁺, Cu²⁺ and Zn²⁺ stimulate the relative activity of the Santalum peroxidase at concentration of 2.0 μM. Amino acids like L-alanine and L-valine activate the per cent relative activity, while L-proline and DL-methionine showed moderate inhibition for the Santalum peroxidase. However, a very low a concentration of cysteine acts as a strong inhibitor of Santalum peroxidase at the concentration of 0.4 mM. Native polyacrylamide gel electrophoresis (Native-PAGE) was performed for isoenzyme determination and two bands were observed. K_m and V_max values were calculated from Lineweaver-Burk graph. The apparent V_max/K_m value for O-dianisidine and H₂O₂ were 400 and 5.0 × 105 Units/min/mL respectively.     

Group V Secretory Phospholipase A2 Is Involved in Tubular Integrity and Sodium Handling in the Kidney

Group V (GV) phospholipase A₂ (PLA₂) is a member of the family of secreted PLA₂ (sPLA2) enzymes_. This enzyme has been identified in several organs, including the kidney. However, the physiologic role of GV sPLA₂ in the maintenance of renal function remains unclear. We used mice lacking the gene encoding GV sPLA₂ (Pla2g5^−/−) and wild-type breeding pairs in the experiments. Mice were individually housed in metabolic cages and 48-h urine was collected for biochemical assays. Kidney samples were evaluated for glomerular morphology, renal fibrosis, and expression/activity of the (Na⁺ + K⁺)-ATPase α1 subunit. We observed that plasma creatinine levels were increased in Pla2g5^−/− mice following by a decrease in creatinine clearance. The levels of urinary protein were higher in Pla2g5^−/− mice than in the control group. Markers of tubular integrity and function such as γ-glutamyl transpeptidase, lactate dehydrogenase, and sodium excretion fraction (FE_Na⁺) were also increased in Pla2g5^−/− mice. The increased FE_Na⁺ observed in Pla2g5^−/− mice was correlated to alterations in cortical (Na⁺ + K⁺) ATPase activity/ expression. In addition, the kidney from Pla2g5^−/− mice showed accumulation of matrix in corticomedullary glomeruli and tubulointerstitial fibrosis. These data suggest GV sPLA₂ is involved in the maintenance of tubular cell function and integrity, promoting sodium retention through increased cortical (Na⁺ + K⁺)-ATPase expression and activity.     

Intra- specific variation in response of Jatropha (Jatropha curcas L.) to elevated CO2 conditions

Twenty genotypes of Jatropha collected from diverse eco-geographic regions from the states of Chhattisgarh (3), Andhra Pradesh (12), Rajasthan (4) and Uttarakhand (1) of India were subjected to elevated CO₂ conditions. All the genotypes showed significant difference (p < 0.05 and 0.01) in the phenotypic traits in both the environments (elevated and ambient) and genotype x environment interaction. Among the physiological traits recorded, maximum photosynthetic rate was observed in IC565048 (48.8 μmol m⁻² s⁻¹) under ambient controlled conditions while under elevated conditions maximum photosynthetic rate was observed in IC544678 (41.3 μmol m⁻² s⁻¹), and there was no significant difference in the genotype x environment interaction. Stomatal conductance (Gs) emerged as the key factor as it recorded significant difference among the genotypes, between the environments and also genotype x environment interaction. The Gs and transpiration (E) recorded a significant decline in the genotypes under the elevated CO₂ condition over the ambient control. Under elevated CO₂ conditions, the minimum values recorded for Gs and E were 0.03 mmol m⁻² s⁻¹ and 0.59 mmol m⁻² s⁻¹ respectively in accession IC565039, while the maximum values for Gs and E were 1.8 mmol m⁻² s⁻¹ and 11.5 mmol m⁻² s⁻¹ as recorded in accession IC544678. The study resulted in the identification of potential climate ready genotypes viz. IC471314, IC544654, IC541634, IC544313, and IC471333 for future use.     

Thermal Characterization,Crystal Field Analysis and In-Band Pumped Laser Performance of Er Doped NaY(WO4)2 Disordered Laser Crystals

Undoped and Er-doped NaY(WO₄)₂ disordered single crystals have been grown by the Czochralski technique. The specific heat and thermal conductivity (κ) of these crystals have been characterized from T = 4 K to 700 K and 360 K, respectively. It is shown that κ exhibits anisotropy characteristic of single crystals as well as a κ(T) behavior observed in glasses, with a saturation mean free phonon path of 3.6 Å and 4.5 Å for propagation along a and c crystal axes, respectively. The relative energy positions and irreducible representations of Stark Er³⁺ levels up to ⁴G_7/2 multiplet have been determined by the combination of experimental low (<10 K) temperature optical absorption and photoluminescence measurements and simulations with a single-electron Hamiltonian including both free-ion and crystal field interactions. Absorption, emission and gain cross sections of the ⁴I_13/2↔⁴I_15/2 laser related transition have been determined at 77 K. The ⁴I_13/2 Er³⁺ lifetime (τ) was measured in the temperature range of 77–300 K, and was found to change from τ (77K) ≈ 4.5 ms to τ (300K) ≈ 3.5 ms. Laser operation is demonstrated at 77 K and 300 K by resonantly pumping the ⁴I_13/2 multiplet at λ≈1500 nm with a broadband (FWHM≈20 nm) diode laser source perfectly matching the 77 K crystal ⁴I_15/2 → ⁴I_13/2 absorption profile. At 77 K as much as 5.5 W of output power were obtained in π-polarized configuration with a slope efficiency versus absorbed pump power of 57%, the free running laser wavelength in air was λ≈1611 nm with the laser output bandwidth of 3.5 nm. The laser emission was tunable over 30.7 nm, from 1590.7 nm to 1621.4 nm, for the same π-polarized configuration.