Seasonal changes in fungal production and biomass on standing dead Scirpus lacustris litter in a northern prairie wetland

Decaying macrophytes are an important source of carbon and nutrients in fungal and bacterial communities of northern prairie wetlands. Dead macrophytes do not collapse into the water column immediately after death, and decomposition by fungi and bacteria begins while the plants are standing. The seasonal variations in fungal biomass and production on Scirpus lacustris stems, both above and below water, were measured to assess which environmental factors were dominant in affecting these variations in a typical prairie wetland. Fungal biomass and production were measured from early May to November, just prior to freeze-up. Fungal decomposition began and was greatest in the spring despite low water temperatures. The fungal production, as measured by the incorporation of [1-(14)C]acetate into ergosterol, ranged from 1.8 to 376 microg of C g of ash-free dry mass (AFDM)(-1) day(-1), and the biomass, as estimated by using ergosterol, ranged from nondetectable to 5.8 mg of C g of AFDM(-1). There was no significant difference in biomass or production between aerial and submerged portions of Scirpus stems. The water temperature was correlated with fungal production (r = 0.7, P < 0.005) for aerial stem pieces but not for submerged pieces. However, in laboratory experiments water temperature had a measurable effect on both biomass and production in submerged stem pieces. Changes in fungal biomass and productivity on freshly cut green Scirpus stems decaying in the water either exposed to natural solar radiation or protected from UV radiation were monitored over the summer. There was no significant difference in either fungal biomass (P = 0.76) or production (P = 0.96) between the two light treatments. The fungal biomass and rates of production were within the lower range of the values reported elsewhere, probably as a result of the colder climate and perhaps the lower lability of Scirpus stems compared to the labilities of the leaves and different macrophytes examined in other studies performed at lower latitudes.     

A unified nomenclature for Arabidopsis dynamin-related large GTPases based on homology and possible functions

Plant Molecular Biology -     

Development of a new antigen detection dot-ELISA for diagnosis of tubercular lymphadenitis in fine needle aspirates

A sandwich dot enzyme-linked immunosorbent assay (ELISA) was standardized to detect mycobacterial antigen in fine needle aspirates of patients with tubercular lymphadenitis (TBLN). The assay was performed on nitrocellulose paper by using antibodies raised in mice and rabbits against crude soluble protein (CSP) of Mycobacterium tuberculosis. The test was able to detect as low as 5 ng protein/ml. A total of 225 suspected cases of tubercular lymphadenopathy were screened, out of which 96 were cytomorphologically confirmed as cases of tubercular lymphadenitis (50 acid-fast bacilli (AFB)-positive and 46 AFB-negative). These were considered as positive controls. Only 28 cases were proven to be of nontubercular etiology and were considered as negative controls. In the remaining 101 (39 scanty) aspirates, tubercular etiology could neither be ruled out nor confirmed. Out of 50 AFB-positive confirmed cases of tubercular lymphadenitis, 46 were ELISA-positive. Out of 46 AFB-negative but cytomorphologically confirmed aspirates, antigen could be demonstrated in only 42 aspirates. Four samples from patients with nontubercular etiology were also found to be ELISA-positive. Antigen was picked up in a total of 90.3% of aspirates with suspicion of tuberculosis and 79.5% of scanty aspirates. The assay was found to be 91.6% sensitive and 85.7% specific. The assay was found to be simple and rapid, and hence, could be performed in areas where health facilities are rudimentary.     

Starvation-induced impairment of metabolism in a freshwater catfish

Starvation induced changes in citrate synthase (CS), glucose-6-phosphate dehydrogenase (G6-PDH), lactate dehydrogenase (LDH), DNA, RNA, RNA/DNA ratio and protein were studied in the freshwater catfish Clarias batrachus. Starvation gradually decreased the activity of CS, G6-PDH and LDH in brain, liver and skeletal muscle of the freshwater catfish. The maximum reduction in these enzyme activities upto 35-45% was observed after 35 days of fasting. This shows substantial decline in aerobic and biosynthetic capacity during starvation period. DNA, RNA, RNA/DNA ratio and protein contents were also reduced from 40-67% which reflects reduction in an overall capacity of the protein synthesis. Starvation-induced macromolecular changes indicate impairment of metabolism in fish.     

Decolourization of synthetic dyes by a newly isolated strain of Serratia marcescens

A novel dye-decolourizing strain of the bacterium Serratia marcescens efficiently decolourized two chemically different dyes Ranocid Fast Blue (RFB) and Procion Brilliant Blue-H-GR (PBB-HGR) belonging respectively to the azo and anthraquinone groups. Extracellular laccase and manganese peroxidase (MnP) activity were detected during dye decolourization. The involvement of MnP activity was found in the decolourization of both dyes. More than 90% decolourization of PBB-HGR and RFB was obtained on days 8 and 5, respectively at 26 °C under static conditions at pH 7.0. MnP activity was increased by the addition of Mn²⁺ · At 50 M Mn²⁺, high MnP (55.3 U/ml) but low laccase activity (8.3 U/ml) was observed. Influence of oxalic acid on MnP activity was also observed.     

Melittin as model system for probing interactions between proteins and cyclodextrins

Cylcodextrin sugars are cyclic sugars that have a hydrophilic exterior and a hydrophobic center. This enables cyclodextrins to solubilize hydrophobic molecules in aqueous media. Cyclodextrins may inhibit aggregation by intercalating surface aromatic residues and competing with interprotein aromatic clusters (pi-pi interactions). In order to investigate this concept, the interaction of hydroxypropyl-beta-cyclodextrin (HPBCD) with melittin is studied with steady-state and time-resolved fluorescence, fluorescence polarization, circular dichroism, and IR spectroscopy. HPBCD inhibits the aggregation of melittin. This inhibition and the spectroscopic results are consistent with the lone aromatic tryptophan of the peptide being intercalated within HPBCD.     

Specificity in lipases: a computational study of transesterification of sucrose

Computational conformational searches of putative transition states of the reaction of sucrose with vinyl laurate catalyzed by lipases from Candida antarctica B and Thermomyces lanuginosus have been carried out. The dielectric of the media have been varied to understand the role of protein plasticity in modulating the observed regioselective transesterification. The binding pocket of lipase from Candida adapts to the conformational variability of the various substates of the substrates by small, local adjustments within the binding pocket. In contrast, the more constrained pocket of the lipase from Thermomyces adapts by adjusting through concerted global motions between subdomains. This leads to the identification of one large pocket in Candida that accommodates both the sucrose and the lauroyl moieties of the transition state, whereas in Thermomyces the binding pocket is smaller, leading to the localization of the two moieties in two distinct pockets; this partly rationalizes the broader specificity of the former relative to the latter. Mutations have been suggested to exploit the differences towards changing the observed selectivities.     

Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome system

Recruitment of ubiquitinated proteins to the 26S proteasome lies at the heart of the ubiquitin-proteasome system (UPS). Genetic studies suggest a role for the multiubiquitin chain binding proteins (MCBPs) Rad23 and Rpn10 in recruitment, but biochemical studies implicate the Rpt5 ATPase. We addressed this issue by analyzing degradation of the ubiquitinated Cdk inhibitor Sic1 (UbSic1) in vitro. Mutant rpn10Delta and rad23Delta proteasomes failed to bind or degrade UbSic1. Although Rpn10 or Rad23 restored UbSic1 recruitment to either mutant, rescue of degradation by Rad23 uncovered a requirement for the VWA domain of Rpn10. In vivo analyses confirmed that Rad23 and the multiubiquitin binding domain of Rpn10 contribute to Sic1 degradation. Turnover studies of multiple UPS substrates uncovered an unexpected degree of specificity in their requirements for MCBPs. We propose that recruitment of substrates to the proteasome by MCBPs provides an additional layer of substrate selectivity in the UPS.     

The biosynthesis of D-Galacturonate in plants. functional cloning and characterization of a membrane-anchored UDP-D-Glucuronate 4-epimerase from Arabidopsis

Pectic cell wall polysaccharides owe their high negative charge to the presence of D-galacturonate, a monosaccharide that appears to be present only in plants and some prokaryotes. UDP-D-galacturonate, the activated form of this sugar, is known to be formed by the 4-epimerization of UDP-D-glucuronate; however, no coding regions for the epimerase catalyzing this reaction have previously been described in plants. To better understand the mechanisms by which precursors for pectin synthesis are produced, we used a bioinformatics approach to identify and functionally express a UDP-D-glucuronate 4-epimerase (GAE1) from Arabidopsis. GAE1 is predicted to be a type II membrane protein that belongs to the family of short-chain dehydrogenases/reductases. The recombinant enzyme expressed in Pichia pastoris established a 1.3:1 equilibrium between UDP-D-galacturonate and UDP-D-glucuronate but did not epimerize UDP-D-Glc or UDP-D-Xyl. Enzyme assays on cell extracts localized total UDP-D-glucuronate 4-epimerase and recombinant GAE1 activity exclusively to the microsomal fractions of Arabidopsis and Pichia, respectively. GAE1 had a pH optimum of 7.6 and an apparent Km of 0.19 mm. The recombinant enzyme was strongly inhibited by UDP-D-Xyl but not by UDP, UDP-D-Glc, or UDP-D-Gal. Analysis of Arabidopsis plants transformed with a GAE1:GUS construct showed expression in all tissues. The Arabidopsis genome contains five GAE1 paralogs, all of which are transcribed and predicted to contain a membrane anchor. This suggests that all of these enzymes are targeted to an endomembrane system such as the Golgi where they may provide UDP-D-galacturonate to glycosyltransferases in pectin synthesis.