Purification and partial characterization of novel penicillin V acylase from Acinetobacter sp. AP24 isolated from Loktak Lake,an Indo-Burma biodiversity hotspot

Members of the bacterial genus Acinetobacter have attracted great attention over the past few decades, on account of their various biotechnological applications and clinical implications. In this study, we are reporting the first experimental penicillin V acylase (PVA) activity from this genus. Penicillin acylases are pharmaceutically important enzymes widely used in the synthesis of semisynthetic beta-lactam antibiotics. The bacterium, identified as Acinetobacter sp. AP24, was isolated from the water of Loktak Lake (Manipur, India), an Indo-Burma biodiversity hotspot. PVA production was increased threefold in an optimized medium with 0.2% sodium glutamate and 1% glucose as nitrogen and carbon sources respectively, after 24 hr of fermentation at 28°C and pH 7.0 with shaking at 180 rpm. The enzyme was purified to homogeneity by cation-exchange chromatography using SP-sepharose resin. The PVA is a homotetramer with subunit molecular mass of 34 kD. The enzyme was highly specific toward penicillin V with optimal hydrolytic activity at 40°C and pH 7.5. The enzyme was stable from pH 5.0 to 9.0 at 25 °C for 2 hr. The enzyme retained 75% activity after 1 hr of incubation at 40°C at pH 7.5.     

Structural and functional diversities in lepidopteran serine proteases

Primary protein-digestion in Lepidopteran larvae relies on serine proteases like trypsin and chymotrypsin. Efforts toward the classification and characterization of digestive proteases have unraveled a considerable diversity in the specificity and mechanistic classes of gut proteases. Though the evolutionary significance of mutations that lead to structural diversity in serine proteases has been well characterized, detailing the resultant functional diversity has continually posed a challenge to researchers. Functional diversity can be correlated to the adaptation of insects to various host-plants as well as to exposure of insects to naturally occurring antagonistic biomolecules such as plant-derived protease inhibitors (PIs) and lectins. Current research is focused on deciphering the changes in protease specificities and activities arising from altered amino acids at the active site, specificity-determining pockets and other regions, which influence activity. Some insight has been gained through in silico modeling and simulation experiments, aided by the limited availability of characterized proteases. We examine the structurally and functionally diverse Lepidopteran serine proteases, and assess their influence on larval digestive processes and on overall insect physiology. Invited paper     

3D QSAR studies of N-4-arylacryloylpiperazin-1-yl-phenyl-oxazolidinones: a novel class of antibacterial agents

Three-dimensional QSAR studies for N-4-arylacryloylpiperazin-1-yl-phenyl-oxazolidinones were conducted using TSAR 3.3. The in vitro activities (MICs) of the compounds against Staphylococcus aureus ATCC 25923 exhibited a strong correlation with the prediction made by the model developed in the present study.     

Novel 4-N-substituted aryl pent-2-ene-1,4-dione derivatives of piperazinyloxazolidinones as antibacterials

A few substituted piperazinylphenyloxazolidinone compounds 6-13 having substitution on the distant nitrogen atom of piperazine ring scaffold have been synthesized and evaluated for their antibacterial activity in Gram-positive bacteria. A few compounds showed superior in vitro antibacterial activity against Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Streptococcus pyogenes than linezolid and eperezolid.     

A hill-climbing approach for automatic gridding of cDNA microarray images

Image and statistical analysis are two important stages of cDNA microarrays. Of these, gridding is necessary to accurately identify the location of each spot while extracting spot intensities from the microarray images and automating this procedure permits high-throughput analysis. Due to the deficiencies of the equipment used to print the arrays, rotations, misalignments, high contamination with noise and artifacts, and the enormous amount of data generated, solving the gridding problem by means of an automatic system is not trivial. Existing techniques to solve the automatic grid segmentation problem cover only limited aspects of this challenging problem and require the user to specify the size of the spots, the number of rows and columns in the grid, and boundary conditions. In this paper, a hill-climbing automatic gridding and spot quantification technique is proposed which takes a microarray image (or a subgrid) as input and makes no assumptions about the size of the spots, rows, and columns in the grid. The proposed method is based on a hill-climbing approach that utilizes different objective functions. The method has been found to effectively detect the grids on microarray images drawn from databases from GEO and the Stanford genomic laboratories.     

Dynamics of vitellogenin synthesis in juvenile giant freshwater prawn Macrobrachium rosenbergii

The dynamics of vitellogenin (Vg) mRNA expression and patterns of Vg and vitellin distribution in the hepatopancreas and ovary of juvenile Macrobrachium rosenbergii were examined using real-time RT-PCR and immunohistochemical methods. Eyestalk ablation was seen to induce rapid development of the gonads and Vg synthesis in females. In the female hepatopancreas, Vg mRNA expression was observed several days following ablation, after which levels increased gradually with increasing gonadosomatic index (GSI). Vitellin accumulation in the oocytes also increased with increasing Vg mRNA synthesis; expression was however negligible in the ovary. Hemolymph Vg levels in females ranged from 0.04 to 2.2 mg/ml. SDS PAGE/Western blotting analysis of hemolymph samples revealed that juvenile Vg was composed of 199 and 90 kDa subunits; the 102 kDa subunit present in adult female Vg (Okuno et al., 2002. J Exp Zool 292:417-429) could not be detected at any stage of vitellogenesis in juveniles. Vg was not detectable in non-ablated juveniles. The results of this study confirmed that the mode of involvement of eyestalk factors in regulating vitellogenesis is intrinsic to both juveniles and adults, and that a basic pattern of Vg synthesis and processing is conserved. However, the fact that juveniles are not able to produce the same Vg levels observed in adult females, and do not reach high GSI levels culminating in spawning suggests that other factors and physiological conditions specific to adult females are necessary to demonstrate full reproductive ability.     

Ral Overactivation in Malignant Peripheral Nerve Sheath Tumors

Ras leads an important signaling pathway that is deregulated in neurofibromatosis type 1 and malignant peripheral nerve sheath tumor (MPNST). In this study, we show that overactivation of Ras and many of its downstream effectors occurred in only a fraction of MPNST cell lines. RalA, however, was overactivated in all MPNST cells and tumor samples compared to nontransformed Schwann cells. Silencing Ral or inhibiting it with a dominant-negative Ral (Ral S28N) caused a significant reduction in proliferation, invasiveness, and in vivo tumorigenicity of MPNST cells. Silencing Ral also reduced the expression of epithelial mesenchymal transition markers. Expression of the NF1-GTPase-related domain (NF1-GRD) diminished the levels of Ral activation, implicating a role for neurofibromin in regulating RalA activation. NF1-GRD treatment caused a significant decrease in proliferation, invasiveness, and cell cycle progression, but cell death increased. We propose Ral overactivation as a novel cell signaling abnormality in MPNST that leads to important biological outcomes with translational ramifications.The Ras family of guanine-nucleotide bound proteins exerts a fundamental role in cell biology and constitutes an important area of cancer research due to its significant involvement in the development and progression of malignancies (8, 10, 17, 18, 32). Ras-like (Ral) proteins are crucial members of this family and have been shown to play a pivotal role in human tumors (7, 28, 41, 66, 70). Because Ral guanine nucleotide exchange factors (Ral-GEFs) are direct effectors of Ras, the Ral signaling pathway has been traditionally considered a Ras-effector pathway. Activation of Ras (in resemblance to Ral) is regulated by two classes of proteins: Ras-GEFs (e.g., SOS) and Ras- GTPase activating proteins (Ras-GAPs such as neurofibromin). The latter induces hydrolysis of Ras from the active (GTP) form to the inactive (GDP) form (13). Ral-GEFs include two main groups: the proteins that are stimulated by Ras because of their carboxy-terminal Ras binding domain (RalGDS, RGL1, and RGL2) and the proteins that are activated by substrates of PI3K through a pleckstrin homology domain on their C-terminal (RALGPS1 and RALGPS2) (19). Although highly similar to Ras, Ral proteins (RalA and RalB) involve a series of distinctly different effectors that influence gene expression and translation through interaction with ZO-1-associated nucleic acid binding protein (ZONAB) and RalA binding protein 1 (RalBP1) (11, 23, 33). RalB directly interacts with the SEC5 subunit of exocyst to facilitate the host defense response (48, 58).In addition to overactivation of GEFs, inactivation of GAPs is another mechanism for overactivation of GTP-bound proteins. The lack of neurofibromin (encoded by NF1 on human chromosome 17q11.2), a Ras-GAP protein, is the main molecular event in neurofibromatosis type 1 (NF-1), an autosomal-dominant human genetic disease occurring in approximately 1 in 2,500 to 3,500 births (22, 27, 42). One of the main tumor-causing effects of inactivating mutations in the tumor suppressor NF1 gene is postulated to be the subsequent activation of Ras (3, 29, 53, 57, 69). With two main functional domains, SEC14 and Ras-GAP, neurofibromin is best known for its Ras-GAP function. Although the yeast SEC14p is shown to be involved in regulating intracellular proteins and lipid trafficking, the function of its homologous domain in neurofibromin is unknown (49, 62). Although neurofibromas are the most common tumors in NF-1, 10% of patients with plexiform develop malignant peripheral nerve sheath tumors (MPNSTs), which are typically high grade and often fatal (21, 34, 65).The molecular events involved in the malignant transformation of benign neurofibromas to MPNST are poorly defined. Usually arising in the third through sixth decades of life, these tumors are composed of tightly packed hyperchromatic spindle-shaped cells with frequent mitotic figures. Inactivation of both copies of the NF1 gene has been demonstrated in benign human neurofibromas and shown to cause tumors in murine models (56). Loss of heterozygosity of NF1 and p53 has frequently been observed in human MPNST (35, 47, 54). Recombinant mouse strains (NP mice), which harbor inactivated Nf1 and p53 alleles (cis-Nf1^+/−:p53^+/−), demonstrate the cumulative effects of loss of both Nf1 and p53 genes in the etiology of MPNST (14, 68).In the present study, we show that while both Ras activation and activation of a series of its downstream effector pathways are observed in a fraction of MPNST cells, RalA is activated globally in all studied mouse and human MPNST cells and tumor samples. Our results also explain the involvement of this signaling molecule in a series of key biological functions of MPNST cells, as shown in a variety of in vitro assays and an in vivo model of MPNST. Such information may play a role in designing novel therapies for treatment of MPNST or other tumors with overactivation of the Ral pathway.