Evaluation of Medium Components by Plackett-Burman Statistical Design for Lipase Production by Candida rugosa and Kinetic Modeling

Lipase production by Candida rugosa was carried out in submerged fermentation. Plackett-Burman statistical experimental design was applied to evaluate the fermentation medium components. The effect of twelve medium components was studied in sixteen experimental trials. Glucose, olive oil, peptone and FeCl3?6H2O were found to have more significance on lipase production by Candida rugosa. Maximum lipase activity of 3.8 u mL-1 was obtained at 50 h of fermentation period. The fermentation was carried out at optimized temperature of 30oC, initial pH of 6.8 and shaking speed of 120 r/min. Unstructured kinetic models were used to simulate the experimental data. Logistic model, Luedeking-Piret model and modified Luedeking-Piret model were found suitable to efficiently predict the cell mass, lipase production and glucose consumption respectively with high determination coefficient(R2). From the estimated values of the Luedeking-Piret kinetic model parameters, α and β, it was found that the lipase production by Candida rugosa is growth associated.     

Could adrenal insufficiency serve as a predictor of immune reconstitution inflammatory syndrome (IRIS) in HIV disease?

Immune reconstitution inflammatory syndrome (IRIS) is an inflammatory manifestation that occurs subsequent to initiation of highly active antiretroviral therapy in terminal (HAART) HIV infection, mainly due to the restoration of robust immune responses directed against latent microbial antigens. IRIS is believed to be multifactorial and less studied. Herein, we postulate that hypothalamo-pituitary-adrenal (HPA) dysregulation, a well-documented manifestation in HIV/AIDS, could possibly disturb the balance between pro-inflammatory and anti-inflammatory cytokines leading to clinical IRIS. Drugs, opportunistic infections, stress and numerous intrinsic and extrinsic factors have been described to be the possible causes of IRIS in HIV illness.     

Hesperidin, a flavanone glycoside, on lipid peroxidation and antioxidant status in experimental myocardial ischemic rats

Myocardial infarction continues to be a leading cause of mortality world-wide. Novel therapies are needed to treat the myocardial ischemia. This study was undertaken to evaluate the cardioprotective role of hesperidin on isoproterenol-induced myocardial ischemia in rats. Myocardial ischemia was induced by subcutaneous injection of isoproterenol hydrochloride (85 mg/kg body weight), for two consecutive days. Isoproterenol-administered rats showed elevated levels of cardiac markers (aspartate transaminase, alanine transaminase, lactate dehydrogenase, creatine kinase, creatine kinase-MB, cardiac troponins T and I) when compared with control and hesperidin treatment groups (100, 200 and 400 mg/kg body weight). The serum levels of cardiac markers were significantly reduced at the doses of 200 mg and 400 mg. All further experiments were carried out at the 200 mg dose. Lipid peroxidation markers (thiobarbituric acid reactive substances, lipid hydroperoxides and conjugated dienes) were elevated significantly in the plasma and heart whereas non-enzymic antioxidants (vitamin C, vitamin E and reduced glutathione) were decreased significantly. Activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase and glutathione reductase declined significantly in the heart of ischemic rats. However, after hesperidin treatment, all the above parameters reverted to normal levels. This study demonstrated that the cardioprotective effect of hesperidin on ischemic rats could be due to its anti-lipid peroxidative and antioxidant properties.     

Non-nodulating pink-pigmented facultative Methylobacterium sp. with a functional nifH gene

Among the 250 Methylobacterium isolates studied, 11 were able to grow in Nitrogen-free methanol mineral salts medium. Out of the eleven isolates, except MV10, 10 isolates had the nodA gene. ARA and presence of nifH gene confirmed the ability of isolate MV10 to fix biological nitrogen. Plant infection tests conducted with Crotalaria sp. confirmed the inability of isolate MV10 to nodulate Crotalaria sp. The presence of a functional nifH gene and absence of a nodA gene differentiate this isolate from the other 15 species so far described in the genus Methylobacterium and suggest that it is a new species.     

Synthesis and evaluation of affinity adsorbents for glycoproteins: an artificial lectin

A combination of rational design based on mimicking natural protein–carbohydrate interactions and solid-phase combinatorial chemistry has led to the identification of an affinity ligand which displays selectivity for the mannose moiety of glycoproteins. The ligand, denoted 18/18 and comprising a triazine scaffold bis-substituted with 5-aminoindan, has been synthesised in solution, characterised by TLC, ¹H-NMR and MS. When immobilised to amine-derivatised agarose at concentrations >24 μmol/g moist weight gel, ligand 18/18 selectively binds glucose oxidase. The adsorbed enzyme was quantitatively eluted with 0.5 M α- -methyl-mannoside and to a lesser extent with the equivalent glucoside. An investigation of the comparative retention times of saccharidic solutes showed that significant retardation was observed for α- -mannose, mannobiose and mannan, with little or no evidence for selective retention of other saccharides, with the exception of α- -fucose. Interestingly, α- -fucose and α- -mannose share an identical configuration of the hydroxyl groups on C-2, C-3 and C-4. Analysis of Scatchard plots from partition equilibrium studies on the interaction of glucose oxidase and the p-nitrophenyl-glycosides of -mannose, -glucose, -fucose and -galactose with immobilised 18/18 establish that the affinity constants (K_AX) for the enzyme, the glycosides of mannose, glucose and fucose, and the p-nitrophenyl-galactoside are 4.3×10⁵ M⁻¹, 1.9×10⁴ M⁻¹ and 1.2×10⁴ M⁻¹ respectively. ¹H-NMR studies on the interaction of α- -methyl-mannoside with ligand 18/18 in solution confirm the involvement of the hydroxyl group in the C-2 position. Molecular modelling suggests the formation of four hydrogen bonds between the hydroxyl groups at positions C-2, C-3 and C-4 of α- -methyl-mannoside and the bridging and ring nitrogen atoms of the triazine scaffold, with aromatic stacking of a second ligand against the carbohydrate face. The greater specificity of ligand 18/18 for mannose and glucose than for galactose parallels that exhibited by concanavalin A.     

Design,synthesis and characterisation of affinity ligands for glycoproteins

The concepts of rational design and solid phase combinatorial chemistry were used to develop affinity adsorbents for glycoproteins. A detailed assessment of protein–carbohydrate interactions was used to identify key residues that determine monosaccharide specificity, which were subsequently exploited as the basis for the synthesis of a library of glycoprotein binding ligands. The ligands were synthesised using solid phase combinatorial chemistry and were assessed for their sugar‐binding ability with the glycoenzymes, glucose oxidase and RNase B. Partial and completely deglycosylated enzymes were used as controls. The triazine‐based ligand, histamine/tryptamine (8/10) was identified as a putative glycoprotein binding ligand, since it displayed particular affinity for glucose oxidase and other mannosylated glycoproteins. Experiments with deglycosylated control proteins, specific eluants and retardation in the presence of competing sugars strongly suggest that the ligand binds the carbohydrate moiety of glucose oxidase rather than the protein itself. Copyright © 1999 John Wiley & Sons, Ltd.     

A Review on Saponin Biosynthesis and its Transcriptomic Resources in Medicinal Plants

Plant Molecular Biology Reporter - Saponins are naturally occurring glycosides which are produced by various plant species with diverse biological properties. The surface-active properties of...     

An electrically-controlled programmable microfluidic concentration waveform generator

Background

Biological systems have complicated environmental conditions that vary both spatially and temporally. It becomes necessary to impose time-varying soluble factor concentrations to study such systems, including cellular responses to pharmaceuticals, inflammation with waxing and waning cytokine concentrations, as well as circadian rhythms and their metabolic manifestations. There is therefore a need for platforms that can achieve time-varying concentrations with arbitrary waveforms.

Results

To address this need, we developed a microfluidic system that can deliver concentration waveforms in a fast and accurate manner by adopting concepts and tools from electrical engineering and fluid mechanics. Specifically, we employed pulse width modulation (PWM), a commonly used method for generating analog signals from digital sources. We implement this technique using three microfluidic components via laser ablation prototyping: low-pass filter (lower frequency signals permitted, high frequency signals blocked), resistor, and mixer. Each microfluidic component was individually studied and iteratively tuned to generate desired concentration waveforms with high accuracy. Using fluorescein as a small-molecule soluble factor surrogate, we demonstrated a series of concentration waveforms, including square, sawtooth, sinusoidal, and triangle waves with frequencies ranging from 100 mHz to 400 mHz.

Conclusion

We reported the fabrication and characterization of microfluidic platform that can generate time-varying concentrations of fluorescein with arbitrary waveforms. We envision that this platform will enable a wide range of biological studies, where time-varying soluble factor concentrations play a critical role. In addition, the technology is expected to assist in the development of biomedical devices that allow precise dosing of pharmaceuticals for enhanced therapeutic efficacy and reduced toxicity.

     

Comparison of various maturation treatments on in vitro maturation of goat oocytes and their early embryonic development and cell numbers

In the present study, comparison of 2 different culture media (Ham's F-12 and M-199) for supporting in vitro maturation of goat oocytes, and their subsequent embryonic development was evaluated in the presence or absence of sera (estrous goat serum, EGS and fetal calf serum, FCS) and hormones (FSH, 0.5 ug/ml, LH, 5 ug/ml and estradiol, 1 ug/ml). Neither medium (Ham's F-12 or M-199) when supplemented with EGS and hormones showed any notable changes in the maturation rate nor in cleavage and blastocyst development. The mean cell number for blastocysts was also significantly low (P < 0.05). However, Ham's F-12 medium supplemented with FCS and hormones showed a considerable increase in the maturation rate, but subsequent embryonic development was not appreciably increased. However, maturation, cleavage and blastocyst development rates of oocytes matured in M-199 medium in combination with 10% FCS and hormones were significantly higher (P < 0.05). Mean cell number per blastocyst was also significantly increased in this latter treatment compared with that of the other groups (P < 0.05). The results thus indicated that both the culture medium and serum have a marked effect on maturation and subsequent embryonic development. Further, the results also showed that the combination of M-199 with FSH, LH and E2 supplemented with 10% FCS was the most efficacious medium for in vitro maturation and subsequent embryonic development of the media, sera and hormone combinations studied.     

Variability of Indian Isolates of Tilletia indica Assessed by Pathogenicity and Molecular Markers

Twenty isolates of Tilletia indica collected from sites in North and North‐western India showed pathogenic variation on 18 host differentials. Sixteen aggressive pathotypes were identified on the basis of percent coefficient of infection (PCI). Two major clusters were apparent in the dendrogram; cluster 1 comprised 13 isolates and cluster two consisted of seven isolates. One of the isolate Kashipur had a high PCI on most of the host differentials compared to other isolates. Polymerase chain reaction‐based random amplified polymorphic DNA (PCR – RAPD) analysis also divided isolates into two major clusters, one comprising of 5 isolates collected from hill and foot‐hill sites and another group comprising of 15 isolates collected from plain sites. Thus, the clusters identified based on PCI did not match closely with those identified by molecular analysis based on RAPD. Although diversity among the isolates of T. indica was absent in the rDNA‐ITS region, our study based on pathogenicity and molecular markers confirms the existence of great diversity in the pathogen, also shifting of ‘hot spot’ areas from one place to another within Karnal bunt prevailing areas.