Characterization of a putative thioredoxin peroxidase prx1 of <Emphasis Type="Italic">Candida albicans</Emphasis>

In this study, we characterized a putative peroxidase Prx1 of Candida albicans by: 1) demonstrating the thioredoxin-linked peroxidase activity with purified proteins, 2) examining the sensitivity to several oxidants and the accumulation of intracellular reactive oxygen species with a null mutant (prx1Δ), a mutant (C69S) with a point mutation at Cys69, and a revertant, and 3) subcelluar localization. Enzymatic assays showed that Prx1 is a thioredoxin-linked peroxidase which reduces both hydrogen peroxide (H₂O₂) and tert-butyl hydroperoxide (t-BOOH). Compared with two other strong H₂O₂ scavenger mutants for TSA1 and CAT1, prx1Δ and C69S were less sensitive to H₂O₂, menadione and diamide at all concentrations tested, but were more sensitive to low concentration of t-BOOH. Intracellular reactive oxygen species accumulated in prx1Δ and C69S cells treated with t-BOOH but not H₂O₂. These results suggest that peroxidase activity of Prx1 is specified to t-BOOH in cells. In both biochemical and physiological cases, the evolutionarily conserved Cys69 was found to be essential for the function. Immunocytochemical staining revealed Prx1 is localized in the cytosol of yeast cells, but is translocated to the nucleus during the hyphal transition, though the significances of this observation are unclear. Our data suggest that PRX1 has a thioredoxin peroxidase activity reducing both t-BOOH and H₂O₂, but its cellular function is specified to t-BOOH.     

Synonymous codon usage in chloroplast genome of Coffea arabica

Synonymous codon usage of 53 protein coding genes in chloroplast genome of Coffea arabica was analyzed for the first time to find out the possible factors contributing codon bias. All preferred synonymous codons were found to use A/T ending codons as chloroplast genomes are rich in AT. No difference in preference for preferred codons was observed in any of the two strands, viz., leading and lagging strands. Complex correlations between total base compositions (A, T, G, C, GC) and silent base contents (A₃, T₃, G₃, C₃, GC₃) revealed that compositional constraints played crucial role in shaping the codon usage pattern of C. arabica chloroplast genome. ENC Vs GC₃ plot grouped majority of the analyzed genes on or just below the left side of the expected GC₃ curve indicating the influence of base compositional constraints in regulating codon usage. But some of the genes lie distantly below the continuous curve confirmed the influence of some other factors on the codon usage across those genes. Influence of compositional constraints was further confirmed by correspondence analysis as axis 1 and 3 had significant correlations with silent base contents. Correlation of ENC with axis 1, 4 and CAI with 1, 2 prognosticated the minor influence of selection in nature but exact separation of highly and lowly expressed genes could not be seen. From the present study, we concluded that mutational pressure combined with weak selection influenced the pattern of synonymous codon usage across the genes in the chloroplast genomes of C. arabica.     

Calcium and cargoes as regulators of myosin 5a activity

Myosin 5a is a two-headed actin-dependent motor that transports various cargoes in cells. Its enzymology and mechanochemistry have been extensively studied in vitro. It is a processive motor that takes multiple 36 nm steps on actin. The enzymatic activity of myosin 5 is regulated by an intramolecular folding mechanism whereby its lever arms fold back against the coiled-coil tail such that the motor domains directly bind the globular tail domains. We show that the structure seen in individual folded molecules is consistent with electron density map of two-dimensional crystals of the molecule. In this compact state, the actin-activated MgATPase activity of the molecule is markedly inhibited and the molecule cannot move processively on surface bound actin filaments. The actin-activated MgATPase activity of myosin 5a is activated by increasing the calcium concentration or by binding of a cargo-receptor molecule, melanophilin, in vitro. However, calcium binding to the calmodulin light chains results in dissociation of some of the calmodulin which disrupts the ability of myosin 5a to move on actin filaments in vitro. Thus we propose that the physiologically relevant activation pathway in vivo involves binding of cargo-receptor proteins.     

Elevated Soluble CD163 in Gestational Diabetes Mellitus: Secretion from Human Placenta and Adipose Tissue

Recently soluble CD163 (sCD163), a cleaved form of the macrophage receptor CD163, was identified as a macrophage-specific risk-predictor for developing Type 2 Diabetes. Here, we investigate circulating levels of sCD163 in gestational diabetes mellitus (GDM). Furthermore, given the role of the placenta in the pathogenesis of GDM, we assessed placental contribution to sCD163 secretion. Paired maternal (venous) and umbilical vein blood samples from GDM (n = 18) and Body Mass Index (BMI) matched control women (n = 20) delivered by caesarean section at 39–40 week gestation were assessed for circulating levels of sCD163, Tumour necrosis factor alpha (TNF-α) and Interleukin 6 (IL-6). Media from explant culture of maternal subcutaneous fat and corresponding placental tissues were assayed for these same molecules. CD163 positive cell numbers were determined in placental and adipose tissues of GDM and control women. We found significantly elevated circulating sCD163 levels in GDM mothers (688.4±46.9 ng/ml vs. 505.6±38.6 ng/ml) and their offspring (418.2±26.6 ng/ml vs. 336.3±24.4 ng/ml [p<0.05 for both]) as compared to controls, together with elevated circulating TNF-α and IL-6 levels. Moreover, both GDM placentae (268.1±10.8 ng/ml/mg vs. 187.6±20.6 ng/ml/mg) and adipose explants (41.1±2.7 ng/ml/mg vs. 26.6±2.4 ng/ml/mg) released significantly more sCD163 than controls. Lastly, significantly more CD163 positive cells were observed in GDM placentae (25.7±1.1 vs. 22.1±1.2) and adipose tissue (19.1±1.1 vs 12.7±0.9) compared to controls. We describe elevated sCD163 levels in GDM and identify human placenta as a novel source of sCD163 suggesting that placental tissues might contribute to the increased levels of circulating sCD163 in GDM pregnancies.     

An oxidative and salinity stress induced peroxisomal ascorbate peroxidase from Avicennia marina: Molecular and functional characterization

APX (EC, 1.11.1.11) has a key role in scavenging ROS and in protecting cells against their toxic effects in algae and higher plants. A cDNA encoding a peroxisomal ascorbate peroxidase, Am-pAPX1, was isolated from salt stressed leaves of Avicennia marina (Forsk.) Vierh. by EST library screening and its expression in the context of various environmental stresses was investigated. Am-pAPX1 contains an ORF of 286 amino acids coding for a 31.4kDa protein. The C-terminal region of the Am-pAPX1 ORF has a putative transmembrane domain and a peroxisomal targeting signal (RKKMK), suggesting peroxisomal localization. The peroxisomal localization of Am-pAPX1 was confirmed by stable transformation of the GFP-(Ala)(10)-Am-pAPX1 fusion in tobacco. RNA blot analysis revealed that Am-pAPX1 is expressed in response to salinity (NaCl) and oxidative stress (high intensity light, hydrogen peroxide application and excess iron). The isolated genomic clone of Am-pAPX1 was found to contain nine exons. A fragment of 1616bp corresponding to the 5' upstream region of Am-pAPX1 was isolated by TAIL-PCR. In silico analysis of this sequence reveals the presence of putative light and abiotic stress regulatory elements.     

Isolation and characterization of marine biofilm forming bacteria from a ship’s hull

Background

Diverse aquatic microorganisms are capable of colonizing living and non-living surfaces leading to the formation of biofilms. Commonly visualized as a slimy layer, these biofilms are filled with hundreds of other microorganisms compared to free living planktonic cells. Microbial surface colonization and surface-associated metabolic activities also exert several macroscale deleterious effects, including biofouling, biocorrosion and the persistence and transmission of harmful or pathogenic microorganisms and virulence determinants. The present study deals with the isolation and screening of marine bacteria for biofilm formation. The screened isolates were characterized and identified as Pychrobacter celer, Pychrobacter alimentarius and Kocuria rhizophila by 16S rRNA sequencing.

Methods

Biofilm forming bacteria were isolated by spread plate technique and subjected to screening by microtiter plate assay. The potent biofilm formers were identified by molecular characterization using 16S rRNA gene sequencing.

Results

Twelve bacterial isolates were obtained by pour plate technique and subjected to biofilm assay. Among the 12 isolates three isolates which showed maximum biofilm formation were subjected to molecular characterizationby 16S rRNA gene sequencing method. The isolates were identified as Pychrobacter celer, Pychrobacter alimentarius and Kocuria rhizophila. The EPS produced by the three biofilm forming bacteria was extracted and the protein and carbohydrate content determined.

Conclusion

Among the isolates screened, isolate 8 (Kocuria rhizophila) produced maximum protein and carbohydrate which was also in accordance with the results of microtiter plate assay.

     

Threat-Sensitive Behavioral Responses to Concentrations of Water-Borne Cues from Predation

Aquatic organisms often detect predators via water-borne chemical cues, and respond by showing reduced activity. Prey responses may be correlated with the concentration of predation cues, which would result in graded antipredator behavioral responses that adjust potentially costly behavioral changes to levels that are commensurate with the risk of predation. Larvae of the predatory mosquito Toxorhynchites rutilus prey upon other container-dwelling insects, including larvae of the mosquito Ochlerotatus triseriatus. Previous work has established that O. triseriatus reduce movement, foraging, and time below the surface, and increase the frequency of resting at the surface, in the presence of water-borne cues from predation by T. rutilus. We tested whether these responses by O. triseriatus are threat sensitive by recording behavior of fourth instar larvae in two runs of an experiment in which we created a series of concentrations (100, 10, 1, 0.1, and 0.01% and 100, 70, 40, 20, and 10%) of water that had held either O. triseriatus larvae alone (control) or a T. rutilus larva feeding on O. triseriatus (predation). We also tested whether associated effects on time spent feeding are threat sensitive by determining whether frequencies of filtering or browsing are also related to concentration of cues. The frequencies of resting and surface declined, whereas frequency of filtering (but not browsing) increased more rapidly with a decrease in concentration of predation cues compared with control cues. Thus, O. triseriatus shows a threat sensitive behavioral response to water-borne cues from this predator, adjusting its degree of behavioral response to the apparent risk of predation.     

Severity of drug resistance and co-existence of <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> in diabetic foot ulcer infections

The genes encoding aminoglycoside resistance in Enterococcus faecalis may promote collateral aminoglycoside resistance in polymicrobial wounds. We studied a total of 100 diabetic foot ulcer samples for infection and found 60 samples to be polymicrobial, 5 to be monomicrobial, and 35 samples to be culture negative. A total of 65 E. faecalis isolates were screened for six genes coding for aminoglycoside resistance, antibiotic resistance patterns, and biofilm production. Infectious Diseases Society of America/International Working Group on the Diabetic Foot system was used to classify the wound ulcers. Majority of the subjects with culture-positive wound were recommended conservative management, while 14 subjects underwent amputation. Enterococcal isolates showed higher resistance for erythromycin, tetracycline, and ciprofloxacin. Isolates from grade 3 ulcer showed higher frequency of aac(6′)-Ie-aph(2″)-Ia, while all the isolates were negative for aph(2″)-Ib, aph(2″)-Ic, and aph(2″)-Id. The isolates from grade 3 ulcers showed higher resistance to aminoglycosides as well as teicoplanin and chloramphenicol. All the 39 biofilm producers were obtained from polymicrobial wound and showed higher resistance when compared to biofilm non-producers. Higher frequency of isolates carrying aac(6′)-Ie-aph(2″)-Ia in polymicrobial community showing resistance to key antibiotics suggests widespread distribution of aminoglycoside-resistant E. faecalis and their role in worsening diabetic foot ulcers.