Combination of Liposomal CpG Oligodeoxynucleotide 2006 and Miltefosine Induces Strong Cell-Mediated Immunity during Experimental Visceral Leishmaniasis

Immuno-modulators in combination with antileishmanial drug miltefosine is a better therapeutic approach for treatment of Visceral Leishmaniasis (VL) as it not only reduces the dose of miltefosine but also shortens the treatment regimen. However, immunological mechanisms behind the perceived benefits of this combination therapy have not been investigated in detail. In the present study, we hypothesized that potential use of drugs that target the host in addition to the parasite might represent an alternative strategy for combination therapy. We investigated immune responses generated in Leishmania donovani infected animals (hamsters and mice) treated with combination of CpG-ODN-2006 and miltefosine at short dose regimen. Infected animals were administered CpG-ODN-2006 (0.4 mg/kg, single dose), as free and liposomal form, either alone or in combination with miltefosine for 5 consecutive days and parasite clearance was evaluated at day 4 and 7 post treatment. Animals that received liposomal CpG-ODN-2006 (lipo-CpG-ODN-2006) and sub-curative miltefosine (5 mg/kg) showed the best inhibition of parasite multiplication (∼97%) which was associated with a biased Th1 immune response in. Moreover, compared to all the other treated groups, we observed increased mRNA expression levels of pro-inflammatory cytokines (IFN-γ, TNF-α and IL-12) and significantly suppressed levels of Th2 cytokines (IL-10 and TGF-β) on day 4 post treatment in animals that underwent combination therapy with lipo-CpG-ODN-2006 and sub-curative miltefosine. Additionally, same therapy also induced heightened iNOS mRNA levels and NO generation, increased IgG2 antibody level and strong T-cell response in these hamsters compared with all the other treated groups. Collectively, our results suggest that combination of lipo-CpG-ODN-2006 and sub-curative miltefosine generates protective T-cell response in an animal model of visceral leishmaniasis which is characterized by strong Th1 biased immune response thereby underlining our hypothesis that combination therapy, at short dose regimen can be used as a novel way of treating visceral leishmaniasis.     

Cloning of a xylanase gene <Emphasis Type="Italic">xyn2A</Emphasis> from rumen fungus <Emphasis Type="Italic">Neocallimastix</Emphasis> sp. GMLF2 in <Emphasis Type="Italic">Escherichia coli</Emphasis> and its partial characterization

Anaerobic fungi belonging to the family Neocallimastigaceae are native inhabitants in the rumen of the most herbivores, such as cattle, sheep and goats. A member of this unique group, Neocallimastix sp. GMLF2 was isolated from cattle feces and screened for its xylanase encoding gene using polymerase chain reaction. The gene coding for a xylanase (xyn2A) was cloned in Escherichia coli and expression was monitored. To determine the enzyme activity, assays were conducted for both fungal xylanase and cloned xylanase (Xyl2A) for supernatant and cell-associated activities. Optimum pH and temperature of the enzyme were found to be 6.5 and 50°C, respectively. The enzyme was stable at 40°C and 50°C for 20 min but lost most of its activity when temperature reached 60°C for 5-min incubation time. Rumen fungal xylanase was mainly released to the supernatant of culture, while cloned xylanase activity was found as cell-associated. Multiple alignment of the amino acid sequences of Xyl2A with published xylanases from various organisms suggested that Xyl2A belongs to glycoside hydrolase family 11.     

Theoretical mimicry of biomembranes

The study of membrane proteins requires a proper consideration of the specific environment provided by the biomembrane. The compositional complexity of this environment poses great challenges to all experimental and theoretical approaches. In this article a rather simple theoretical concept is discussed for its ability to mimic the biomembrane. The biomembrane is approximated by three mimicry solvents forming individual continuum layers of characteristic physical properties. Several specific structural problems are studied with a focus on the biological significance of such an approach. Our results support the general perception that the biomembrane is crucial for correct positioning and embedding of its constituents. The described model provides a semi-quantitative tool of potential interest to many problems in structural membrane biology.     

Palaeolimnology of the last crater lake in the Eastern Carpathian Mountains: a multiproxy study of Holocene hydrological changes

A multi-proxy investigation (loss-on-ignition, major and trace elements, pollen, plant macrofossil and siliceous algae) was carried out on the sediment of a crater lake (Lake Saint Ana, 950 m a.s.l.) from the Eastern Carpathian Mountains. Diatom-based transfer functions were applied to estimate the lake’s trophic status and pH, while reconstruction of the water-depth changes was based on the plant macrofossil and diatom records. The lowest Holocene water depths were found between 9000 and 7400 calibrated BP years, when the crater was occupied by Sphagnum-bog. Significant increases in water depth were found from 5350(1), 3300(2) and 2700 cal yr BP. Of these, the first two coincided with major terrestrial vegetation changes, namely (1) the establishment of Carpinus betulus on the crater slope and (2) the replacement of the lakeshore Picea abies forest by Fagus sylvatica. The chemical record indicated significant soil changes along with the canopy changes (from coniferous to deciduous) that led to increased in-lake productivity and pH. A further increase in water depth around 2700 cal yr BP resulted in stable thermal stratification and hypolimnetic anoxia that via P-release further increased in-lake productivity and eventually led to phytoplankton blooms with large populations of Scenedesmus. High productivity was depressed by anthropogenic lakeshore forest clearances from ca. 1000 cal yr BP that led to the re-establishment of P. abies on the lakeshore and consequent acidification of the lake water. On the whole, these data suggest that Lake Saint Ana is a vulnerable ecosystem: in-lake productivity is higher under deciduous canopy and litter, and considerably repressed by coniferous canopy and litter. The lake today subsists in a managed environment that is far from its natural state. This would be a dense F. sylvatica forest supplying more nutrients and keeping up a more productive in-lake flora and fauna. Guest editors: K. Buczkó, J. Korponai, J. Padisák & S. W. Starratt Palaeolimnological Proxies as Tools of Environmental Reconstruction in Fresh Water     

Mapping diabetes QTL in an intercross derived from a congenic strain of the Brown Norway and Goto-Kakizaki rats

Genetic studies in experimental crosses derived from the inbred Goto-Kakizaki (GK) rat model of spontaneous diabetes mellitus have identified quantitative trait loci (QTL) for diabetes phenotypes in a large region of rat Chromosome (RNO) 1. To test the impact of GK variants on QTL statistical and biological features, we combined genetic and physiologic studies in a cohort of F₂ hybrids derived from a QTL substitution congenic strain (QTLSCS) carrying a 110-cM GK haplotype of RNO1 introgressed onto the genetic background of the Brown Norway (BN) strain. Glucose intolerance and altered insulin secretion in QTLSCS rats when compared with BN controls were consistent with original QTL features in a GK × BN F₂ cross. Segregating GK alleles in the QTLSCS F₂ cross account for most of these phenotypic differences between QTLSCS and BN rats. However, significant QTL for diabetes traits in both the QTLSCS and GK × BN F₂ cohorts account for a similar small proportion of their variance. Comparing results from these experimental systems provides indirect estimates of the contribution of genetic interactions and environmental factors to QTL architecture as well as locus and biological targets for future post-QTL mapping studies in congenic substrains. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. Stephan C. Collins and Robert H. Wallis contributed equally to this work.     

Antimicrobial activity of marine bacteria associated with sponges from the waters off the coast of South East India

Seventy-five marine bacterial strains associated with four species of sponges (Echinodictyum sp., Spongia sp., Sigmadocia fibulatus and Mycale mannarensis) were isolated from the Tuticorin coast, Gulf of Mannar region. The agar-overlay method was used to screen for antibiotic production by these strains against four bacteria, viz., Bacillus subtilis, Escherichia coli, Vibrio parahaemolyticus, and Vibrio harveyi and one fungal pathogen, viz., Candida albicans. Twenty-one per cent of the bacterial strains were found to be antibiotic producers and their activities ranged from broad spectral to species specific. A strain coded SC3 was found to be highly potent and was mass cultured. The ethyl acetate extract of the culture broth was further fractionated by reverse phase HPLC and the active fraction identified. In addition, SC3 was subjected to morphological and physiological characterization. The results of the tests showed SC3 to be a Gram-positive rod, sporulating, motile, catalase and oxidase positive. Phylogenetic analysis based on comparative analysis of sequenced 16s rRNA of the active strains indicated a preponderance of bacteria belonging to Vibrio and Bacillus genera with 95-99% sequence similarities. To our knowledge this is the first report on phylogenetic identification of antibiotic producing bacteria associated with sponges from Indian waters.     

Protein-protein interactions of tandem affinity purification-tagged protein kinases in rice

Forty-one rice cDNAs encoding protein kinases were fused to the tandem affinity purification (TAP) tag and expressed in transgenic rice plants. The TAP-tagged kinases and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by mass spectrometry. Ninety-five percent of the TAP-tagged kinases were recovered. Fifty-six percent of the TAP-tagged kinases were found to interact with other rice proteins. A number of these interactions were consistent with known protein complexes found in other species, validating the TAP-tag method in rice plants. Phosphorylation sites were identified on four of the kinases that interacted with either 14-3-3 proteins or cyclins.