Use of a synthetic polymer (Pluronic F-127) as an agar substitute in culture medium to detect and measure microbial enzyme production

Summary A simple and rapid detection of proteolytic activity of microbial sources on a thermally reversible gel (Pluronic F-127) is described.     

Aminopeptidase-A. I. CDNA cloning and expression and localization in rat tissues

Aminopeptidase-A (APA) is an ectoenzyme that selectively hydrolyzes acidic residues from the amino terminus of oligopeptides, including biologically active [Asp(1)]ANG II and [Asp(1)]CCK-8. We sought to characterize rat APA by cDNA cloning and expression and to determine its tissue distribution by in situ hybridization and immunohistochemistry. Sequence analysis of overlapping cDNA clones isolated from rat kidney cDNA libraries indicated that the full-length cDNA encoded a 945-amino acid protein with a predicted molecular mass of 108 kDa; the size was confirmed by in vitro translation of a full-length cDNA construct. Transient transfection of the full-length cDNA construct in mammalian cells yielded a protein approximately 140 kDa in size, a size that agrees with the immunoblots of APA from rat tissue and is consistent with APA being known as a glycosylated protein. Tissue APA activity and mRNA expression were highest in the kidney and ileum. Localization of APA by in situ hybridization and immunohistochemistry indicated that, with the exception of the kidney and ileum, where APA was localized to the luminal brush border of proximal tubules and enterocytes, respectively, APA was associated with either capillaries or the lining of sinusoids. Areas known to be physiological targets for ANG II, including glomeruli, the zona glomerulosa, and anterior pituitary, had high levels of APA. The localization pattern suggests that APA may subserve multiple functions, i.e., a generalized role in peptide scavenging and perhaps a more specific role in metabolism of circulating or locally produced ANG II or CCK-8.     

A Simple ERP Method for Quantitative Analysis of Cognitive Workload in Myoelectric Prosthesis Control and Human-Machine Interaction

Common goals in the development of human-machine interface (HMI) technology are to reduce cognitive workload and increase function. However, objective and quantitative outcome measures assessing cognitive workload have not been standardized for HMI research. The present study examines the efficacy of a simple event-related potential (ERP) measure of cortical effort during myoelectric control of a virtual limb for use as an outcome tool. Participants trained and tested on two methods of control, direct control (DC) and pattern recognition control (PRC), while electroencephalographic (EEG) activity was recorded. Eighteen healthy participants with intact limbs were tested using DC and PRC under three conditions: passive viewing, easy, and hard. Novel auditory probes were presented at random intervals during testing, and significant task-difficulty effects were observed in the P200, P300, and a late positive potential (LPP), supporting the efficacy of ERPs as a cognitive workload measure in HMI tasks. LPP amplitude distinguished DC from PRC in the hard condition with higher amplitude in PRC, consistent with lower cognitive workload in PRC relative to DC for complex movements. Participants completed trials faster in the easy condition using DC relative to PRC, but completed trials more slowly using DC relative to PRC in the hard condition. The results provide promising support for ERPs as an outcome measure for cognitive workload in HMI research such as prosthetics, exoskeletons, and other assistive devices, and can be used to evaluate and guide new technologies for more intuitive HMI control.     

Molecular cloning,characterization and expression of a nitrofuran reductase gene ofescherichia coli

Mini-mu derivatives carrying plasmid replicons can be used to clone genesin vivo. This method was adopted to generate phasmid clones which were later screened for their ability of restore nitrofurantoin sensitivity of a nitrofuran-resistant host by eliciting nitroreductase activity. One phasmid-derived clone (pAJ101) resulted in considerable increase in nitroreductase activity when introduced into a nitrofurantoin-resistant mutant ofEscherichia coli with reduced nitroreductase activity. Subsequently, a 1.8 kb fragment obtained from pAJ101 by partial digestion with 5au3A, was subcloned into pUC18 to yield pAJ102. The nitroreductase activity attributable to pAJ102 was capable of reducing both nitrofurantoin and nitrofurazone. The polypeptides encoded by pAJ102 were identified by the minicell method. A large, well-defined band corresponding to 37 kDa and a smaller, less-defined band corresponding to 35 kDa were detected. Tnl000 mutagenesis was used to delineate the coding segment of the 1.8 kb insert of pAJ102. A 0.8 kb stretch of DNA was shown to be part of the nitroreductase gene. The gene was mapped at 19 min on theEscherichia coli linkage map.     

A theoretical model for water flux through the artery wall

A two layer model for water flux through the artery is studied using a mathematical model based on the theory for the consolidation of water saturated soils. The matrix is considered to be constituted by two layers with different permeabilities and different elastic constants and the two systems of equations are coupled with the condition of continuity of pressure, total stress, solid displacement and fluid seepage velocity at the interface. The luminal pressure is considered to be harmonic in time. Exact solutions are obtained for displacements and pressures in both the layers. For large consolidation times, large pressure gradients are found to exist near the boundaries and at the interface. The heterogeneous model may not only be useful to understand the mechanics of transport in the physiological system but it will also help the bioengineers to choose proper implant materials to design artificial vascular organs for the purpose of prosthesis.     

Citric acid production

Techniques for possible higher and rapid production of citric acid from the well known industrial medium i.e. molasses has been reported using Aspergillus niger. This includes optimization of the total reducing sugar (TRS) and nutrients like nitrogen and phosphorous. The long and unproductive lag periods normally associated with this type of fermentation has been reduced. These strategies are discussed in detail. Dr. M. Chellapandian is thankful to the Council of Scientific and Industrial Research, New Delhi, for the award of research associateship.     

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.Download video file.^{(143M, mp4)}     

Binding of nucleotide triphosphates to cardiotoxin analogue II from the Taiwan cobra venom (Naja naja atra). Elucidation of the structural interactions in the dATP-cardiotoxin analogue ii complex.

Snake venom cardiotoxins have been recently shown to block the enzymatic activity of phospholipid protein kinase and Na+,K+-ATPase. To understand the molecular basis for the inhibitory effects of cardiotoxin on the action of these enzymes, the nucleotide triphosphate binding ability of cardiotoxin analogue II (CTX II) from the Taiwan cobra (Naja naja atra) venom is investigated using a variety of spectroscopic techniques such as fluorescence, circular dichroism, and two-dimensional NMR. CTX II is found to bind to all the four nucleotide triphosphates (ATP, UTP, GTP, and CTP) with similar affinity. Detailed studies of the binding of dATP to CTX II indicated that the toxin molecule is significantly stabilized in the presence of the nucleotide. Molecular modeling, based on the NOEs observed for the dATP.CTX II complex, reveals that dATP binds to the CTX II molecule at the groove enclosed between the N- and C-terminal ends of the toxin molecule. Based on the results obtained in the present study, a molecular mechanism to account for the inhibition of the enzymatic activity of the phospholipid-sensitive protein kinase and Na+,K+-ATPase is also proposed.