Expression,purification and substrate specificities of 3-nitrotoluene dioxygenase from Diaphorobacter sp. strain DS2

3-Nitotoluene dioxygenase (3-NTDO) is the first enzyme in the degradation pathway of 3-nitrotoluene (3-NT) by Diaphorobacter sp. strain DS2. The complete gene sequences of 3-NTDO were PCR amplified from genomic DNA of Diaphorobacter sp., cloned, sequenced and expressed. The 3-NTDO gene revealed a multi component structure having a reductase, a ferredoxin and two oxygenase subunits. Clones expressing the different subunits were constructed in pET21a expression vector system and overexpressed in E. coli BL21(DE3) host. Each subunit was individually purified separately to homogeneity. The active recombinant enzyme was reconstituted in vitro by mixing all three purified subunits. The reconstituted recombinant enzyme could catalyse biotransformations on a variety of organic aromatics.     

A simple and serum-free protocol for cryopreservation of human umbilical cord as source of Wharton’s jelly mesenchymal stem cells

Mesenchymal stromal cells (MSCs) show promise in cell-based transplantations and regenerative medicine applications. MSCs from Wharton’s jelly (WJ) of umbilical cord can be easily harvested and exhibit greater proliferative activity than bone marrow MSCs. It is important to develop a practical cryopreservation technique to effectively store umbilical cord for potential future applications. Successful cryopreservation would allow access to umbilical cord from the same donor for repeated WJ MSC-based transplantations. For therapeutic applications, one should be able to obtain clinically-relevant quality and quantity of MSCs from cryopreserved tissues. In this study, we optimised a serum-free formulation of 10% dimethyl sulfoxide (DMSO) and 0.2 M sucrose for cryopreservation of umbilical cord tissue. Slow freezing and rapid thawing were adopted. MSCs harvested from WJ of cryopreserved umbilical cord could undergo robust expansion, differentiate to mesodermal lineages and express MSC-characteristic surface antigens. The cumulative cell yield, however, was less compared to corresponding fresh cord tissue.     

Detection and Partial Characterization of Milk vetch dwarf virus Isolates from Faba Bean (Vicia faba L.) in Yunnan Province, China

A virus disease of faba bean ( Vicia faba L.) in China, characterized by leaf yellowing and rolling and plant stunting, was shown to be caused by a virus of the genus Nanovirus based on serological reactions to nanovirus-specific monoclonal antibodies and the generation of polymerase chain reaction amplicons using nanovirus-specific primers. To identify the faba bean-infecting nanovirus, regions of the DNA components encoding the master replication initiator protein and capsid protein of two nanovirus isolates from China were cloned, sequenced and compared with those of other members of the genus Nanovirus . The two Chinese virus isolates shared nucleotide sequence identities ranging from 95 to 98% with the type isolate of Milk vetch dwarf virus (MDV) from Japan. They were thus identified as isolates of MDV, a virus so far known to cause important diseases of legumes in Japan. This is the first record of MDV-infecting faba bean in China.     

Stress-mediated alteration in V-ATPase and V-PPase of Butea monosperma

The activity and subunit amounts of V-ATPase and V-PPase in various plants of Butea monosperma Taub. (Fabaceae) (ver. Dhak; Palas) growing as a natural inhabitant in varying stress conditions in southeast Rajasthan were studied. Western blot analysis followed by immunological quantification of V-ATPase subunits using specific polyclonal antibodies showed that the subunits A, B, D, E, and c are clearly detectable in all plants, with A, B, and c appearing as intense bands. The other subunits of V-ATPase, viz., C, a, and d, were also detected in majority of the plants. Various subunits exhibited variations in their protein amount in different plants. Besides, a few other clear bands were also detected. Of these, the 30- and 29-kD bands may possibly be Di and Ei. Furthermore, a clear band of V-PPase corresponding to 67–70 kD was also detected. A comparison of the V-ATPase and V-PPase activity revealed that Butea plants in the upper region of the study site showed 70% and 39% higher activity, respectively. Furthermore, the immunological quantification showed that the V-ATPase and V-PPase protein amounts are also higher in the upper Butea plants which have drought stress and, moreover, are also exposed to stronger light intensities for relatively longer duration.     

Functionalization of pine needles by carboxymethylation and network formation for use as supports in the adsorption of Cr

Pine needles and their carboxymethyl forms were functionalized by network formation with 2-acrylamido-2-methylpropanesulphonic acid (AAmPSA) in the presence of N,N-methylene bisacrylamide. N-Tetramethylethylene diamine and ammonium persulfate were used as accelerator-initiator systems to prepare these hydrogels. The hydrogels were characterized by FTIR, SEM, and nitrogen analysis and for water uptake capacities before and after metal ion sorption with a view to evaluating their use in the removal of toxic ionic species from waste water. A detailed study of Cr⁶⁺ adsorption was carried out as a function of time, temperature, pH, and ionic strength. The thermodynamic parameters of adsorption such as ΔH⁰, ΔS⁰, and ΔG⁰ have been evaluated to understand the underlying mechanism of adsorption. In order to understand their reusability in possible technological applications, biodegradability of these hydrogels and their precursors was studied.     

Nuclear transport of Ras-associated tumor suppressor proteins: different transport receptor binding specificities for arginine-rich nuclear targeting signals

Ras proteins regulate a wide range of biological processes by interacting with a variety of effector proteins. In addition to the known role in tumorigensis, the activated form of Ras exhibits growth-inhibitory effects by unknown mechanisms. Several Ras effector proteins identified as mediators of apoptosis and cell-cycle arrest also exhibit properties normally associated with tumor suppressor proteins. Here, we show that Ras effector RASSF5/NORE-1 binds strongly to K-Ras but weakly to both N-Ras and H-Ras. RASSF5 was found to localize both in the nucleus and the nucleolus in contrast to other Ras effector proteins, RASSF1C and RASSF2, which are localized in the nucleus and excluded from nucleolus. A 50 amino acid residue transferable arginine-rich nucleolar localization signal (NoLS) identified in RASSF5 is capable of interacting with importin-beta and transporting the cargo into the nucleolus. Surprisingly, similar arginine-rich signals identified in RASSF1C and RASSF2 interact with importin-alpha and transport the heterologous cytoplasmic proteins to the nucleus. Interestingly, mutation of arginine residues within these nuclear targeting signals prevented interaction of Ras effector proteins with respective transport receptors and abolished their nuclear translocation. These results provide evidence for the first time that arginine-rich signals are able to recognize different nuclear import receptors and transport the RASSF proteins into distinct sub-cellular compartments. In addition, our data suggest that the nuclear localization of RASSF5 is critical for its cell growth control activity. Together, these data suggest that the transport of Ras effector superfamily proteins into the nucleus/nucleolus may play a vital role in modulating Ras-mediated cell proliferation during tumorigenesis.     

Synthesis and evaluation of antifungal properties of a series of the novel 2-amino-5-oxo-4-phenyl-5,6,7,8-tetrahydroquinoline-3-carbonitrile and its analogues

A series of 2-amino-5-oxo-4-phenyl-5,6,7,8-tetrahydroquinoline-3-carbonitrile and various analogues have been synthesized in excellent isolated yields starting from various arylidenemalononitrile and 3-amino-2-cyclohexen-1-one in 1-propanol as solvent at reflux temperature in the absence of any added catalyst. All the synthesized compounds were evaluated for their antifungal activity. The relationship between functional group variation and biological activity of the evaluated compounds is discussed in the article.     

In vivo humoral and cellular reactions, and fate of injected bacteria Aeromonas hydrophila in freshwater prawn Macrobrachium rosenbergii

Live non-opsonized and opsonized Aeromonas hydrophila were injected into juveniles of freshwater prawn Macrobrachium rosenbergii to study the cells involved in phagocytosis, distribution of bacteria, cellular reactions and clearance of both forms of bacteria from the system. The bacteria were rapidly distributed to various tissues viz., gills, heart, hepatopancreas within 1h, and the tissues revealed haemocytic nodule formation after 3 h of injection. There was rapid clearance of both the forms of bacteria from the circulation. However, clearance efficiency was significantly (P < 0.05) faster in the case of opsonized bacteria at 12 h after injection. Similarly, the nodule formation, that was prominent in cardiac musculature, was rapidly eliminated from the tissues of the group injected with opsonized bacteria as compared to non-opsonized bacteria injected group, thus confirming the existence of opsonic factors in haemolymph of this prawn. In another experiment, various dose levels of bacteria were injected intramuscularly into prawns and haemolymph was collected after 1, 6, 24, 72 h and 7 days of injection to study various immune parameters. Although, no major alterations in the total and differential haemocyte counts were observed in bacteria injected prawns compared to control, there was a significant decline in phenoloxidase activity in the highest dose bacteria injected group at the earlier phase and a rise in agglutinin levels at the later phase of the experimental period in the higher dose groups.     

Bacterial synthesis of poly(hydroxybutyrate- co-hydroxyvalerate) using carbohydrate-rich mahua (Madhuca sp.) flowers

AIMS: The objective of the present work was to utilize an unrefined natural substrate namely mahua (Madhuca sp.) flowers, as a carbon source for the production of bacterial polyhydroxyalkanoate (PHA) copolymer by Bacillus sp-256. METHODS AND RESULTS: In the present work, three bacterial strains were tested for PHA production on mahua flower extract (to impart 20 g l(-1) sugar) amongst which, Bacillus sp-256 produced higher concentration of PHA in its biomass (51%) compared with Rhizobium meliloti (31%) or Sphingomonas sp (22%). Biosynthesis of poly(hydroxybutyrate-co-hydroxyvalerate) - P(HB-co-HV)--of 90 : 10 mol% by Bacillus sp-256 was observed by gas chromatographic analysis of the polymer. Major component of the flower is sugars (57% on dry weight basis) and additionally it also contains proteins, vitamins, organic acids and essential oils. The bacterium utilized malic acid present in the substrate as a co-carbon source for the copolymer production. The flowers could be used in the form of aqueous extract or as whole flowers. PHA content of biomass (%) and yield (g l(-1)) in a 3.0-l stirred tank fermentor after 30 h of fermentation under constant pH (7) and dissolved oxygen content (40%) were 54% and 2.7 g l(-1), respectively. Corresponding yields for control fermentation with sucrose as carbon source were 52% and 2.5 g l(-1). The polymer was characterized by proton NMR. CONCLUSIONS: Utilization of mahua flowers, a natural substrate for bacterial fermentation aimed at PHA production, had additional advantage, as the sugars and organic acids present in the flowers were metabolized by Bacillus sp-256 to synthesize P(HB-co-HV) copolymer. SIGNIFICANCE AND IMPACT OF THE STUDY: Literature reports on utilization of suitable cheaper natural substrate for PHA copolymer production is scanty. Mahua flowers used in the present experiment is a cheaper carbon substrate compared with several commercial substrates and it is rich in main carbon as well as co-carbon sources that can be utilized by bacteria for PHA copolymer production.     

Prognostic significance of the histomorphometric features of bone marrow trephine biopsies in patients with chronic myeloid leukemia

OBJECTIVE: To study the correlation of histomorphometric data of bone marrow trephine biopsies at the time of initial diagnosis in chronic myeloid leukemia (CML) patients with the patient prognosis. STUDY DESIGN: A total of 40 CML patients were divided equally in group I (developed accelerated phase or blast crisis within 18 months of initial diagnosis of chronic phase of CML) and group II (developed accelerated phase or blast crisis > 30 months after initial diagnosis of chronic phase of CML). The clinical, hematologic and histomorphometric data were compared in the 2 groups of CML patients. RESULTS: The percentage of bone marrow promyelocytes was significantly increased in group I. On morphometry, the number of blasts per square millimeter, the area of reticulin fibers per square millimeter and the percentage area occupied by reticulin fibers were statistically significant. CONCLUSION: There seems to be grounds for hope for predicting prognosis of CML patients at initial diagnosis based on histomorphometric findings. The percentage area of reticulin fibers and the number of blasts per square millimeter are important prognostic predictors in histomorphometry data.