Pichia pastoris fermentation optimization: energy state and testing a growth-associated model

A growth-associated model was applied to the production of recombinant ovine interferon-τ (rOvIFN-τ) with Pichia pastoris for the purpose of manufacturing preclinical and clinical active material. This model predicts that product yields will be the greatest when the specific growth of the culture is maintained at a steady and optimal rate. However, rOvIFN-τ yields did not meet the expected linear model but most closely corresponded to a polynomial relationship. After transitioning from glycerol to methanol, product accumulated for 31–45 h, and then the yield decreased. This production shift, which has been termed decoupling, was clearly related to time on methanol and not culture density. It was determined that a correlation exists between the decoupling point and a drop in energy state of the cell when expressing β-galactosidase. By assigning decoupling as a constraint that limits productivity and by reformulating the growth medium, the time prior to decoupling increased to 46.8±2.4 h, product yield improved for rOvIFN-τ from 203 to 337 mg l⁻¹, and the coefficient of variation for yield decreased from 67.9 to 23.3%. A robust and stable fermentation process was realized, resulting in a 210% improvement in total yield from 557±357 to 1,172±388 mg.     

Comparative biodegradation of HDPE and LDPE using an indigenously developed microbial consortium

A variety of bacterial strains were isolated from waste disposal sites of Uttaranchal, India, and some from artificially developed soil beds containing maleic anhydride, glucose, and small pieces of polyethylene. Primary screening of isolates was done based on their ability to utilize high- and low-density polyethylenes (HDPE/LDPE) as a primary carbon source. Thereafter, a consortium was developed using potential strains. Furthermore, a biodegradation assay was carried out in 500-ml flasks containing minimal broth (250 ml) and HDPE/ LDPE at 5 mg/ml concentration. After incubation for two weeks, degraded samples were recovered through filtration and subsequent evaporation. Fourier transform infrared spectroscopy (FTIR) and simultaneous thermogravimetric-differential thermogravimetry-differential thermal analysis TG-DTG-DTA) were used to analyze these samples. Results showed that consortium-treated HDPE (considered to be more inert relative to LDPE) was degraded to a greater extent 22.41% weight loss) in comparison with LDPE (21.70% weight loss), whereas, in the case of untreated samples, weight loss was more for LDPE than HDPE (4.5% and 2.5%, respectively) at 400 degrees . Therefore, this study suggests that polyethylene could be degraded by utilizing microbial consortia in an eco-friendly manner.     

Diurnal and Seasonal Modulation of Sucrose Phosphate Synthase Activity in Leaves of Prosopis juliflora

Diurnal changes of sucrose-phosphate synthase (SPS) activity in different seasons were measured in Prosopis juliflora (Swartz) DC. leaves. SPS activity showed large variations with two distinct peaks, one around 09:00 and another at 21:00. The diurnal pattern was apparently not due to circadian rhythm since the activities were directly related to the changes in environmental parameters (irradiance, temperature, and leaf to air vapour pressure deficit) in different seasons. During the day, the enzyme showed changes in kinetic properties, differential sensitivity to allosteric modulators, differential response to ATP concentration, to concentration of endogenous sucrose, and to protein phosphatase inhibitors. These results taken together indicate the modulation of SPS in synchrony with photosynthesis and suggest the existence of multiple levels of modulation, presumably as an adaptive response to changing environmental extremes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Serotonin synthesis inhibition by pre-treatment of p-CPA alters sleep-electrophysiology in an animal model of acute and chronic heat stress

The effects of pre-treatment of para-chlorophenylalanine (p-CPA) on sleep–wake electroencephalograms (EEG) have been demonstrated in three age groups of rats subjected to heat stress. Each age group for both p-CPA pre-treated and untreated subjects was sub-divided into three groups: (i) acute heat stress—subjected to a single heat exposure for 4 h at 38 °C; (ii) chronic heat stress—exposed for 21 days daily for 1 h in the incubator at 38 °C; and (iii) handling control groups. Digital polygraphic sleep recordings were performed just after the heat exposure from acute stressed rats and on the 22nd day from chronic stressed rats. The analyses of results demonstrated that many changes associated with sleep-EEG (either in sleep–wake parameter or in EEG frequencies) due to acute and chronic heat stress were reversed (changes were analyzed; P<0.05 or better) in p-CPA pre-treated groups of rats. However, differential observations between acute and chronic heat stress groups of subjects were recorded, which are thought to have happened due to acclimatization of subjects to the hot environment. The results of present study supported the previous hypothesis about the significant involvement of serotonin in sleep–wake parameters and also demonstrated its participation in brain electrophysiological alterations in stressed conditions.     

Phosphorylation of p130Cas initiates Rac activation and membrane ruffling

Background  

Non-receptor tyrosine kinases (NTKs) regulate physiological processes such as cell migration, differentiation, proliferation, and survival by interacting with and phosphorylating a large number of substrates simultaneously. This makes it difficult to attribute a particular biological effect to the phosphorylation of a particular substrate. We developed the Functional Interaction Trap (FIT) method to phosphorylate specifically a single substrate of choice in living cells, thereby allowing the biological effect(s) of that phosphorylation to be assessed. In this study we have used FIT to investigate the effects of specific phosphorylation of p130Cas, a protein implicated in cell migration. We have also used this approach to address a controversy regarding whether it is Src family kinases or focal adhesion kinase (FAK) that phosphorylates p130Cas in the trimolecular Src-FAK-p130Cas complex.     

Randomized controlled experiments in health and social sciences: Some conceptual issues

This brief article outlines some difficulties as well as benefits in conducting randomized controlled trials in social science settings especially in developing countries. Some of the historical developments are summarized and certain applications in health sciences are discussed from methodological and policy standpoints.     

Apoptotic resistance exhibited by dexamethasone-resistant murine 7TD1 cells is controlled independently of interleukin-6 triggered signaling

Interleukin-6 (IL6)-mediated signaling is known to play a role in pathogenesis and resistance in several cancers like multiple myeloma (MM). In this report we used the IL6-dependent 7TD1 murine B-cell hybridoma as an in vitro model to study the interactions between IL6-signaling pathways and the development of dexamethasone resistance. Though in initial stages, 7TD1 cells grew IL6-dependent and were sensitive to dexamethasone-induced apoptosis, chronic exposure to dexamethasone led to a dexamethasone-resistant phenotype (7TD1-Dxm) that grew independent of exogenous IL6. While IL6-mediated JAK/STAT3 and PI3K/AKT signaling was important for proliferation of both cell lines, as shown in proliferation assays using the respective pathway inhibitors, AG490 and LY294002, the resistant cells were insensitive to induction of apoptosis using the same. STAT3 was constitutively phosphorylated in resistant cells and inhibition of its dimerization induced apoptosis but did not alter their insensitivity to dexamethasone. Our results suggest a role of entities downstream of IL6-mediated JAK/STAT3 signaling in development of dexamethasone resistance by 7TD1-Dxm cells.     

Enhanced biotransformation of sitosterol to androstenedione by <Emphasis Type="Italic">Mycobacterium</Emphasis> sp. using cell wall permeabilizing antibiotics

Mycobacterial cell wall is rigid and offers a high resistance to the transport of sitosterol into cytosol. The effect of ethambutol, penicillin, polymixin and bacitracin on biotransformation of sitosterol to androstenedione by modification of cell wall permeability was examined. Drug sensitivity assay results established that bacitracin increased the permeability of the cell wall to hydrophobic compounds. Growth inhibitory study of bacitracin and rifamycin, individually as well as in combination showed that these two antibiotics act synergistically to reduce cell growth. A comparison of transmission electron micrograph results of the bacitracin-treated cells with untreated cells, revealed deformities caused in the cell wall structure by bacitracin treatment. These deformities increased the cell wall permeability and transport of sitosterol inside the cell, and thus enhanced androstenedione (AD) production. A maximum of 1.37, 1.44, 1.65 and 1.76 g AD per gram dry cell weight of mycobacterial cells was produced in the presence of ethambutol, penicillin, polymixin and bacitracin, respectively. Below the minimum inhibitory concentration, bacitracin can be used as potent enhancer of permeability of hydrophobic substances across the mycobacterial cell wall.     

Nutrient broth/PEG200/TritonX114/Tween80/Chloroform microemulsion as a reservoir of solubilized sitosterol for biotransformation to androstenedione

Microemulsions (ME) can act as a reservoir of solubilized hydrophobic substrates. The biotransformation of hydrophobic sitosterol to androstenedione (AD) with MEs prepared from nutrient broth and PEG 200 (1:1) as aqueous phase, 40 g/l sitosterol dissolved in chloroform as organic phase, Triton X114 and Tween 80 (1:1) as surfactant phase, was investigated. The phase behavior of this system was studied for ten different ratios(w/w), 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9 and 0:10 of the organic phase and surfactant at 30 °C. A pseudoternary phase diagram was constructed to demarcate the region giving stable MEs. The maximum solubility of sitosterol in ME medium was observed to be 8 g/l, which is 3 orders of magnitude higher than the reported sitosterol solubility of 2–4 mg/l in aqueous medium. The ME medium was used for biotransformation studies and a comparative result has been reported. Transmission electron microscopy of cells grown in ME having oil, surfactant and aqueous phase in the ratio of 6:14:80 showed a weakened cell wall structure that permitted production of 465.86 mg/l AD.