Molecular identification of <Emphasis Type="Italic">Staphylococcus xylosus</Emphasis> MAK2, a new α-<Emphasis Type="SmallCaps">l</Emphasis>-rhamnosidase producer

A bacterial strain, MAK-2, was isolated as a producer of α-l-rhamnosidase from a soil sample of Dehradoon, India. The strain was identified based on morphology, physiological tests and 16S rDNA analysis. The phylogenetic analysis based on the 16S rDNA sequence, identified the isolate as Staphylococcus xylosus, a non-pathogenic member of CNS (coagulase-negative staphylococci) family. The strain was capable of producing α-l-rhamnosidase by hydrolysing flavonoids thus confirming potential application in the citrus-processing industry.     

Coupled mathematical model of tumorigenesis and angiogenesis in vascular tumours

Objectives: Mathematical models are useful for studying vascular and avascular tumours, because these allow for more logical experimental design and provide valuable insights into the underlying mechanisms of their growth and development. The processes of avascular tumour growth and the development of capillary networks through tumour‐induced angiogenesis have already been extensively investigated, albeit separately. Despite the clinical significance of vascular tumours, few studies have combined these approaches to develop a single comprehensive growth and development model. Materials and methods: We develop a continuum‐based mathematical model of vascular tumour growth. In the model, angiogenesis is initiated through the release of angiogenic growth factors (AGFs) by cells in the hypoxic regions of the tumour. The nutrient concentration within the tumour reflects the influence of capillary growth and invasion induced by AGF. Results and conclusions: Parametric and sensitivity studies were performed to evaluate the influence of different model parameters on tumour growth and to identify the parameters with the most influence, which include the rates of proliferation, apoptosis and necrosis, as well as the diffusion of sprout tips and the size of the region affected by angiogenesis. An optimization was performed for values of the model parameters that resulted in the best agreement with published experimental data. The resulting model solution matched the experimental data with a high degree of correlation (r = 0.85).     

Activation of Mutant Enzyme Function In Vivo by Proteasome Inhibitors and Treatments that Induce Hsp70

Missense mutant proteins, such as those produced in individuals with genetic diseases, are often misfolded and subject to processing by intracellular quality control systems. Previously, we have shown using a yeast system that enzymatic function could be restored to I278T cystathionine β-synthase (CBS), a cause of homocystinuria, by treatments that affect the intracellular chaperone environment. Here, we extend these studies and show that it is possible to restore significant levels of enzyme activity to 17 of 18 (94%) disease causing missense mutations in human cystathionine β-synthase (CBS) expressed in Saccharomyces cerevisiae by exposure to ethanol, proteasome inhibitors, or deletion of the Hsp26 small heat shock protein. All three of these treatments induce Hsp70, which is necessary but not sufficient for rescue. In addition to CBS, these same treatments can rescue disease-causing mutations in human p53 and the methylene tetrahydrofolate reductase gene. These findings do not appear restricted to S. cerevisiae, as proteasome inhibitors can restore significant CBS enzymatic activity to CBS alleles expressed in fibroblasts derived from homocystinuric patients and in a mouse model for homocystinuria that expresses human I278T CBS. These findings suggest that proteasome inhibitors and other Hsp70 inducing agents may be useful in the treatment of a variety of genetic diseases caused by missense mutations.     

Therapeutic Efficacy of Cintredekin Besudotox (IL13-PE38QQR) in Murine Lung Fibrosis Is Unaffected by Immunity to Pseudomonas aeruginosa Exotoxin A

Background

We have previously explored a therapeutic strategy for specifically targeting the profibrotic activity of IL-13 during experimental pulmonary fibrosis using a fusion protein comprised of human IL-13 and a mutated form of Pseudomonas aeruginosa exotoxin A (IL13-PE) and observed that the intranasal delivery of IL13-PE reduced bleomycin-induced pulmonary fibrosis through its elimination of IL-13-responsive cells in the lung. The aim of the present study was to determine whether the presence of an immune response to P. aeruginosa and/or its exotoxin A (PE) would diminish the anti-fibrotic properties of IL13-PE.

Methodology/Principal Findings

Fourteen days after P. aeruginosa infection, C57BL/6 mice were injected with bleomycin via the intratracheal route. Other groups of mice received 4 doses of saline or IL13-PE by either intranasal or intraperitoneal application, and were challenged i.t. with bleomycin 28 days later. At day 21 after bleomycin, all mice received either saline vehicle or IL13-PE by the intranasal route and histopatological analyses of whole lung samples were performed at day 28 after bleomycin. Intrapulmonary P. aeruginosa infection promoted a neutralizing IgG2A and IgA antibody response in BALF and serum. Surprisingly, histological analysis showed that a prior P. aeruginosa infection attenuated the development of bleomycin-induced pulmonary fibrosis, which was modestly further attenuated by the intranasal administration of IL13-PE. Although prior intranasal administration of IL13-PE failed to elicit an antibody response, the systemic administration of IL13-PE induced a strong neutralizing antibody response. However, the prior systemic sensitization of mice with IL13-PE did not inhibit the anti-fibrotic effect of IL13-PE in fibrotic mice.

Conclusions

Thus, IL13-PE therapy in pulmonary fibrosis works regardless of the presence of a humoral immune response to Pseudomonas exotoxin A. Interestingly, a prior infection with P. aeruginosa markedly attenuated the pulmonary fibrotic response suggesting that the immune elicitation by this pathogen exerts anti-fibrotic effects.     

Dodecameric structure and ATPase activity of the human TIP48/TIP49 complex

TIP48 and TIP49 are two related and highly conserved eukaryotic AAA(+) proteins with an essential biological function and a critical role in major pathways that are closely linked to cancer. They are found together as components of several highly conserved chromatin-modifying complexes. Both proteins show sequence homology to bacterial RuvB but the nature and mechanism of their biochemical role remain unknown. Recombinant human TIP48 and TIP49 were assembled into a stable high molecular mass equimolar complex and tested for activity in vitro. TIP48/TIP49 complex formation resulted in synergistic increase in ATPase activity but ATP hydrolysis was not stimulated in the presence of single-stranded, double-stranded or four-way junction DNA and no DNA helicase or branch migration activity could be detected. Complexes with catalytic defects in either TIP48 or TIP49 had no ATPase activity showing that both proteins within the TIP48/TIP49 complex are required for ATP hydrolysis. The structure of the TIP48/TIP49 complex was examined by negative stain electron microscopy. Three-dimensional reconstruction at 20 A resolution revealed that the TIP48/TIP49 complex consisted of two stacked hexameric rings with C6 symmetry. The top and bottom rings showed substantial structural differences. Interestingly, TIP48 formed oligomers in the presence of adenine nucleotides, whilst TIP49 did not. The results point to biochemical differences between TIP48 and TIP49, which may explain the structural differences between the two hexameric rings and could be significant for specialised functions that the proteins perform individually.     

Biochemical and immunological characterization of E. coli expressed 42 kDa fragment of Plasmodium vivax and P. cynomolgi bastianelli merozoite surface protein-1

Plasmodium vivax is one of the most widely distributed human malaria parasites and due to drug-resistant strains, its incidence and prevalence has increased, thus an effective vaccine against the parasites is urgently needed. One of the major constraints in developing P. vivax vaccine is the lack of suitable in vivo models for testing the protective efficacy of the vaccine. P. vivax and P. cynomolgi bastianelli are the two closely related malaria parasites and share a similar clinical course of infection in their respective hosts. The merozoite surface protein-1 (MSP-1) of these parasites has found to be protective in a wide range of host-parasite systems. P. vivax MSP-1 is synthesized as 200 kDa polypeptide and processed just prior to merozoite release from the erythrocytes into smaller fragments. The C- terminal 42 kDa cleavage product of MSP-1 (MSP-1(42)) is present on the surface of merozoites and a major candidate for blood stage malaria vaccine. In the present study, we have biochemically and immunologically characterized the soluble and refolded 42 kDa fragment of MSP-1 of P. vivax (PvMSP-1(42)) and P. cynomolgi B (PcMSP-1(42)). SDS-PAGE analysis showed that both soluble and refolded E. coli expressed P. vivax and P. cynomolgi B MSP-1(42) proteins were homogenous in nature. The soluble and refolded MSP-1(42) antigens of both parasites showed high reactivity with protective monkey sera and conformation-specific monoclonal antibodies against P. cynomolgi B and P. vivax MSP-1(42) antigens. Immunization of BALB/c mice with these antigens resulted in the production of high titres of cross-reactive antibodies primarily against the conformational epitopes of MSP-1(42) protein. The immune sera from rhesus monkeys. immunized with soluble and refolded MSP-1(42) antigens of both parasites also showed high titered cross-reactive antibodies against MSP-1(42) conformational epitopes. These results suggested that the soluble and refolded forms of E. coli expressed P. vivax MSP-1(42) antigens were highly immunogenic and thus a viable candidate for vaccine studies.     

Immunomodulatory activity of an extract of the novel fungal endophyte Entrophospora infrequens isolated from Nothapodytes foetida (Wight) Sleumer

A novel camptothecin-producing endophytic fungus viz., Entrophospora infrequens was isolated from an important Indian medicinal plant Nothapodytes foetida. The present study reports evaluation ofbioactivities of two novel extracts viz., chloroform (CEEI) and methanolic (MEEI) extracts of Entrophospora infrequens with respect to their immunomodulatory potential in vitro and in vivo (in Balb/c mice). The endophyte E. infrequens was found to synthesize camptothecin, which tested positive in CEEI. The immunomodulatory potential of CEEI and MEEI was compared with standard camptothecin (CPT). Doses of the chloroform extract (CEEI) ranging from 12.5-100 mg/kg body weight, significantly (p < 0.05) stimulated the humoral and cell-mediated immune responses in a dose-dependent manner. MEEI on the other hand significantly (p < 0.05) stimulated the delayed type hypersensitivity (DTH) reaction (by nearly 80%), plaque forming cell (PFC) assay (33%), phagocytic response (38%) and haemagglutination antibody (HA) titre [IgM by 79.07% and IgG by 62.05%] at a dose of 12.5 mg/kg body weight. The present study is the first report of the immunomodulatory potential of this neoteric camptothecin-producing endophyte from Nothapodytes foetida.     

Production of high fructose syrup from <Emphasis Type="Italic">Asparagus</Emphasis> inulin using immobilized exoinulinase from <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> YS-1

Extracellular exoinulinase from Kluyveromyces marxianus YS-1, which hydrolyzes inulin into fructose, was immobilized on Duolite A568 after partial purification by ethanol precipitation and gel exclusion chromatography on Sephadex G-100. Optimum temperature of immobilized enzyme was 55 °C, which was 5 °C higher than the free enzyme and optimal pH was 5.5. Immobilized biocatalyst retained more than 90% of its original activity after incubation at 60 °C for 3 h, whereas in free form its activity was reduced to 10% under same conditions, showing a significant improvement in the thermal stability of the biocatalyst after immobilization. Apparent K _m values for inulin, raffinose and sucrose were found to be 3.75, 28.5 and 30.7 mM, respectively. Activation energy (E _a) of the immobilized biocatalyst was found to be 46.8 kJ/mol. Metal ions like Co²⁺ and Mn²⁺ enhanced the activity, whereas Hg²⁺ and Ag²⁺ were found to be potent inhibitors even at lower concentrations of 1 mM. Immobilized biocatalyst was effectively used in batch preparation of high fructose syrup from Asparagus racemosus raw inulin and pure inulin, which yielded 39.2 and 40.2 g/L of fructose in 4 h; it was 85.5 and 92.6% of total reducing sugars produced, respectively.