Production of podophyllotoxin from Podophyllum hexandrum: a potential natural product for clinically useful anticancer drugs

Podophyllum hexandrum Royle of family Berberidaceae is an endangered medicinal plant. Rhizome ofP.hexandrum contains several lignans which posses antitumor activity. Podphyllotoxin is the most active cytotoxic natural product. It is used as starting compound for the synthesis of anticancer drug etoposide and teniposide. Podophyllotoxin acts as an inhibitor of microtubule assembly. These drugs are used for lung cancer, testicular cancer, neuroblastoma, hepatoma and other tumors. Besides this, it also shows antiviral activities by interfering with some critical viral processes. Availabilityof podophyllotoxin from plants has its limitations because of its intense collection from nature and lack of organized cultivation. The chemical synthesis of podophyllotoxin is considered to be very complicated as yet. The use of biotechnological approaches for the production of podophyllotoxin using cell cultures, organ cultures, and biotransformation route or by manipulating biosynthetic pathway proves to be an attractive alternative for production of podophyllotoxin. The present paper discusses the current status of research, limitations and future prospects for theproduction of podophyllotoxinin vitro.     

A report on <Emphasis Type="Italic">Penicillium</Emphasis> in the intramural and extramural air of residential areas of Nagpur city (India)

Prevalence of different species of Penicillium and their concentrations per cubic meter of air were evaluated with the use of Hi-Air sampler system Mark II (Hi-Media Laboratories Ltd., India) in the air of homes (bed-rooms) at four different sites in Nagpur. At each of these sites, air sampling was done fortnightly in triplicate for 2 years duration from June 2000 to May 2002. The sampling was also done in triplicate for the outdoor air in the vicinity of each home on the same day immediately after the indoor sampling was over. The mean concentration of Penicillium colony forming units at four different sites in the indoor air was 32, 46.9, 35 and 35.4 CFU/m³, respectively, whereas in the outdoor air at these same four sites, the mean concentration was 24, 28, 25 and 25.8 CFU/m³ respectively. The Penicillium concentration in the indoor air was found to be higher in winter than in other seasons (ANOVA, p < 0.05). Concentration of Penicillium spp. in intramural environment was always higher than that in extramural environment. Statistically significant difference existed between intramural and extramural environments at all the sites, with maximum difference at a site, which is old crowded area of the city. During the 2-years investigations, 11 species of Penicillium were isolated from the indoor air while nine species were isolated from the air outside the homes. The dominant species of Penicillium in indoor as well as outdoor air were P. citrinum (33.78 and 32.81), P. oxalicum (19.70 and 22.60), and P. chrysogenum (17.64 and 14.50). The percentage of the Penicillium in the indoor air was 10.70 while it was 8.36 in outdoor air. Indoor air showed the presence of P. glaber and P. sclerotiorum, which were absent in the outdoor air.     

Reductive dehalogenation mediated initiation of aerobic degradation of 2-chloro-4-nitrophenol (2C4NP) by Burkholderia sp. strain SJ98

Burkholderia sp. strain SJ98 (DSM 23195) was previously isolated and characterized for degradation and co-metabolic transformation of a number nitroaromatic compounds. In the present study, we evaluated its metabolic activity on chlorinated nitroaromatic compounds (CNACs). Results obtained during this study revealed that strain SJ98 can degrade 2-chloro-4-nitrophenol (2C4NP) and utilize it as sole source of carbon, nitrogen, and energy under aerobic conditions. The cells of strain SJ98 removed 2C4NP from the growth medium with sequential release of nearly stoichiometric amounts of chloride and nitrite in culture supernatant. Under aerobic degradation conditions, 2C4NP was transformed into the first intermediate that was identified as p-nitrophenol by high-performance liquid chromatography, LCMS-TOF, and GC-MS analyses. This transformation clearly establishes that the degradation of 2C4NP by strain SJ98 is initiated by "reductive dehalogenation"; an initiation mechanism that has not been previously reported for microbial degradation of CNAC under aerobic conditions.     

Src kinase activity is essential for osteoclast function

Deletion of the c-src gene impairs osteoclast bone resorbing activity, causing osteopetrosis. Although it has been concluded that restoring only the Src adaptor function at least partly rescues the cell attachment and skeletal phenotypes, the contribution of Src kinase activity remains controversial. Src forms a complex with Pyk2 and Cbl after adhesion-induced stimulation of alpha(V)beta(3) integrin. To demonstrate the importance of the Pyk2-Src association in osteoclasts and to distinguish the contributions of the Src adaptor and kinase activities in cytoskeletal organization and osteoclast function, we expressed mutants of Src and Pyk2 in osteoclasts using adenovirus vectors. Eliminating the Src-binding site on Pyk2 (Pyk2(Y402F)) markedly inhibited bone resorption by osteoclast-like cells, whereas kinase-dead Pyk2 had little effect. Kinase-dead Src, unlike kinase-dead Pyk2, markedly inhibited the bone-resorbing activity of wild type osteoclasts and failed to significantly restore bone-resorbing activity to Src(-/-) osteoclast-like cells. Activation of Src kinase by overexpressing kinase-dead Csk failed to reverse the inhibitory effect of Pyk2(Y402F), suggesting that osteoclastic bone resorption requires both c-Src kinase activity and the targeting of Src kinase by Pyk2. Src-catalyzed phosphorylation of Cbl on Tyr-731 is reported to induce the activation and recruitment of phosphatidylinositol 3-kinase to the cell membrane in a signaling pathway that is critical for osteoclast function. Expressing the Cbl(Y731F) mutant in osteoclasts markedly reduced their bone resorbing activity, suggesting that phosphorylation of Cbl(Y731) and the subsequent recruitment and activation of phosphatidylinositol 3-kinase may be critical signaling events downstream of Src in osteoclasts.     

Adenovirus Internalization and Infection Require Dynamin

The cell receptors that facilitate adenovirus internalization into cells have been identified; however, the infectious pathway of virus entry has not been established. Adenovirus entry and infection were examined in HeLa cells lacking or overexpressing mutant dynamin, a protein that specifically regulates clathrin-mediated endocytosis. Expression of mutant dynamin significantly reduced adenovirus internalization and gene delivery, indicating a functional requirement for this molecule. These findings are consistent with virus entry via the clathrin-coated pit pathway.     

Effect of Multimeric Structure of CaMKII in the GluN2B-Mediated Modulation of Kinetic Parameters of ATP

Interaction of GluN2B subunit of N-methyl-D-aspartate receptor with calcium/calmodulin dependent protein kinase II (CaMKII) is critical for the induction of long term potentiation at hippocampal CA3-CA1 synapses. We have previously reported that CaMKII binding to GluN2B increases its affinity but abolishes the cooperativity for ATP. In the present study, we demonstrate that the reduction in S_0.5 for ATP of an individual CaMKII subunit seems to be directly induced by the binding of GluN2B to the same subunit, while any GluN2B induced effects on the cooperativity and maximal velocity would additionally require the CaMKII holoenzyme structure. We measured the apparent kinetic parameters for ATP using an association domain truncated monomeric CaMKII and a heteromultimeric CaMKII (having subunits that are either GluN2B binding defective or ATP binding defective), in the presence of GluN2A or GluN2B substrates. The S_0.5 value for ATP of monomeric CaMKII is reduced ∼ 3 fold by the presence of GluN2B suggesting that the induced change in affinity for ATP is independent of the holoenzyme structure. The heteromultimeric mutant of CaMKII, did not exhibit cooperativity of ATP binding probably because of the interspersing of ATP binding defective subunits in the holoenzyme. In contrast to the wild type holoenzyme, presence of GluN2B increased the V_max of monomeric CaMKII which resulted in an approximately 4.0 fold increase in the apparent catalytic constant (V_max/S_0.5) as compared to GluN2A. The kinetic parameter values of the heteromultimeric CaMKII for ATP, on the other hand, did not show any significant difference between the phosphorylation of GluN2B and GluN2A suggesting that modulation requires binding of GluN2B to the same subunit. Overall, our present study provides insights into the role of multimeric structure of CaMKII in GluN2B-mediated regulation.     

Airborne Penicillium in the grain shops of Nagpur (India)

The airborne Penicillium spp. and total airborne fungal spore concentration was investigated in the grain shops of Nagpur city, India, using a volumetric Hi‐Air sampler system Mark II (Hi Media Laboratories Ltd., India). The mycotoxins were analysed from the Penicillium isolates obtained from the seeds by thin layer chromatography.

The mean concentration of the total fungi isolated from different grain shops ranged from 7.8×10² to 1.1×10³ CFU/m³. The mean concentration of Penicillium isolated from the air of grain shops ranged from 8.6×10¹ CFU/m³ (10.8%) to 1.7×10² CFU/m³ (19.9%). Among the 13 species of Penicillium which were isolated, P. citrinum Thom was the most prevalent species (24.2%), followed by P. oxalicum Currie & Thom (16.5), P. digitatum Saccardo (8.9%), P. janthinellum Biourge (8.7%), P. funiculosum Thom (8.3%), P. chrysogenum Thom (6.4%), P. purpurogenum Stoll (6.2%), P. brevicompactum Dierckx (4.8%), P. frequentans Westling (4.2%), P. italicum Wehmer (3.8%), P. rubrum Stoll (3.4%), P. expansum Link (2.9%) and P. cyclopium Westling (1.6%).

Penicillium species were also isolated from seeds such as wheat, maize, soybean, and groundnut. The mycotoxins roquefortin C, citrinin, rubratoxin B, cyclopiazonic acid, verrucosidin, mitorubrinic acid and two unknown metabolites were isolated from Penicillium isolates.