Modeling of esterase production from Saccharomyces cerevisiae

A suitable simple model tested by experiments is required to address complex biological reactions like esterase synthesis by Saccharomyces cerevisiae. Such an approach might be the answer to a proper bioprocessing strategy. In this regard, a logistic model for esterase production from Saccharomyces cerevisiae has been developed, which predicts well the cell mass, the carbon source (glucose) consumption, and the esterase activity. The accuracy of the model has been statistically examined by using the Student's t-test. The parameter sensitivity analysis showed that all five parameters (microm, Ks, Xm, Yx/s, and Yp/x) have significant influence on the predicted values of esterase activity.     

Toward in situ biochemistry: combining chemical kinetics approaches with biomolecular imaging in living cells

Our knowledge of protein-protein interactions comes primarily from experimentation with reconstituted proteins in dilute solutions. However, dilute solutions are poor approximations of the intracellular microenvironment, which contains exquisite and dynamic structure that is impossible to recreate inside test tubes. New approaches are needed that will allow the in situ characterization of protein-protein interactions inside living, intact cells. In this paper, we discuss recent efforts to measure the kinetics of protein binding within complexes inside living cells. While the experimental effort in these studies requires the confluence of techniques ranging from molecular imaging to cell and molecular biology, the experimental design and analysis requires a strong background in chemical kinetics and transport phenomena. Thus, we argue that chemical engineers can play a central role in furthering in situ approaches to cellular analysis. Such efforts may aid significantly in advancing quantitative knowledge of cellular signaling and physiology.     

Use of response surface optimization for the production of biosurfactant from Rhodococcus spp. MTCC 2574

The production of biosurfactant from Rhodococcus spp. MTCC 2574 was effectively enhanced by response surface methodology (RSM). Rhodococcus spp. MTCC 2574 was selected through screening of seven different Rhodococcus strains. The preliminary screening experiments (one-factor at a time) suggested that carbon source: mannitol, nitrogen source: yeast extract and meat peptone and inducer: n-hexadecane are the critical medium components. The concentrations of these four media components were optimized by using central composite rotatable design (CCRD) of RSM. The adequately high R2 value (0.947) and F score 19.11 indicated the statistical significance of the model. The optimum medium composition for biosurfactant production was found to contain mannitol (1.6 g/L), yeast extract (6.92 g/L), meat peptone (19.65 g/L), n-hexadecane (63.8 g/L). The crude biosurfactant was obtained from methyl tert-butyl ether extraction. The yield of biosurfactant before and after optimization was 3.2 g/L of and 10.9 g/L, respectively. Thus, RSM has increased the yield of biosurfactant to 3.4-fold. The crude biosurfactant decreased the surface tension of water from 72 mN/m to 30.8 mN/m (at 120 mg L(-1)) and achieved a critical micelle concentration (CMC) value of 120 mg L(-1).     

Acute pharmacogenetic activation of medial prefrontal cortex excitatory neurons regulates anxiety-like behaviour

The medial prefrontal cortex (mPFC) is implicated in anxiety-like behaviour. In rodent models, perturbations of mPFC neuronal activity through pharmacological manipulations, optogenetic activation of mPFC neurons or cell-type specific pharmacogenetic inhibition of somatostatin interneurons indicate conflicting effects on anxiety-like behaviour. In the present study we examined the effects of pharmacogenetic activation of Ca²⁺/calmodulin-dependent protein kinase α (CamKIIα)-positive excitatory neurons on anxiety-like behaviour. We used clozapine-N-oxide (CNO) to pharmacogenetically activate virally delivered CamKIIα-hM3Dq-DREADD in mPFC excitatory neurons. The effects of acute CNO or vehicle treatment on anxiety-like behaviour in the open field and elevated plus maze tests were examined in rats virally infected with either CamKIIα-hM3Dq-DREADD or CamKIIα-GFP. In addition, the effects of acute CNO treatment on the expression of the neuronal activity marker c-Fos were examined in the mPFC as well as downstream target neuronal circuits using immunohistochemistry. Acute pharmacogenetic activation of mPFC excitatory neurons evoked a significant decrease in anxiety-like behaviour selectively on the elevated plus maze task, but not the open field test. Acute CNO treatment resulted in enhanced c-Fos-immunopositive cell number in the infralimbic, prelimbic and cingulate subdivisions of the mPFC. This was also accompanied by enhanced c-Fos-immunopositive cell number in multiple downstream circuits of the mPFC in CNO-treated hM3Dq animals. Acute pharmacogenetic activation of mPFC excitatory neurons reduces anxiety-like behaviour in a task-specific fashion accompanied by enhanced c-Fos expression in the mPFC and multiple target circuits implicated in the regulation of anxiety-like behaviour.     

Genetic Characterization of <Emphasis Type="Italic">Barilius barna</Emphasis> (Hamilton, 1822) in the Teesta River of Sub-Himalayan West Bengal,India, Through RAPD and ISSR Fingerprinting

Genetic characterization of Barilius barna, an economically important freshwater fish in the Indian scenario, is unexplored in the sub-Himalayan Dooars region of West Bengal, India. This study is the first attempt to characterize the genetic architecture of Barilius barna from the Teesta river of this region. We have studied loci polymorphism, genetic diversity, Shannon’s information index and the measure of evenness in the two populations of this river through ten RAPD and seven ISSR primer-based PCR amplifications. The result showed 89.52 and 82.21% polymorphisms in RAPD and ISSR amplification respectively. The Nei’s genetic diversity and Shannon’s information index varied from 0.172 ± 0.189SD to 0.293 ± 0.164SD and 0.265 ± 0.268SD to 0.445 ± 0.220SD respectively, which indicated low level of genetic variation. AMOVA revealed significant level of variance within the population and gene flow between the populations. Low levels of genetic variation and moderate to high levels of genetic relatedness were found in the studied populations. Expectedly, the populations were genetically not very distant from each other, as evident from the Nei’s unbiased measure of genetic distance and identity. As the species is commercially important and the region is located in the sub-Himalayan region, the management and proper rehabilitation of this ichthyofauna in the wild is urgently required. Our results may serve as a guideline for adopting such management decisions.     

Stress enhanced calcium kinetics in a neuron

Accurate modeling of the mechanobiological response of a Traumatic Brain Injury is beneficial toward its effective clinical examination, treatment and prevention. Here, we present a stress history-dependent non-spatial kinetic model to predict the microscale phenomena of secondary insults due to accumulation of excess calcium ions (Ca\(^{2+}\)) induced by the macroscale primary injuries. The model is able to capture the experimentally observed increase and subsequent partial recovery of intracellular Ca\(^{2+}\) concentration in response to various types of mechanical impulses. We further establish the accuracy of the model by comparing our predictions with key experimental observations.     

Enhancing ligand–protein binding in affinity thermoprecipitation: Elucidation of spacer effects

Copolymers of N‐isopropylacrylamide and N‐acryloyl amino acid spacers of varying chain length were synthesized. p‐Aminobenzamidine (PABA) was chemically linked to the pendant carboxyl groups of these polymers to obtain thermoprecipitating affinity polymers. The inhibition constant (K_i) of these polymers for trypsin decreased, i.e., the efficiency of PABA–trypsin binding increased with increase in the spacer chain length. The polymer to which PABA was linked through a spacer of five methylene groups exhibited eleven times lower K_i than that of the polymer containing PABA without a spacer. Investigations on model inhibitors N‐acyl‐p‐aminobenzamidines showed that this enhancement in trypsin binding by the polymers was due to the spacer as well as to microenvironmental effects. Recovery and specific activity of the trypsin recovered increased with the spacer chain length. Separation of trypsin from a mixture of trypsin and chymotrypsin was also enhanced with the spacer chain length. The inhibition constants of these affinity polymers were not adversely affected by the crowding effect. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 418–425, 1999.