Surfactant Effects on Lipase-Catalyzed Hydrolysis of Olive Oil in AOT/ISOOCTANE Reverse Micelles

The effects of surfactant concentration on the hydrolytic activity of Candida rugosa lipase in AOT/isooctane reverse micelles with olive oil as the substrate has been investigated. A noncompetitive inhibition by the surfactant on the enzyme was observed. Strong dependences of the kinetic constants k_cat and k_M, but not k_I on the water-to-surfactant ratio (R value) have been identified. The benefits of carrying out the hydrolysis at higher surfactant and water concentrations were demonstrated from the improvement of the initial rate and time course of conversion.     

Multiplex PCR for detection of the heat-labile toxin gene and shiga-like toxin I and II genes in Escherichia coli isolated from natural waters.

A triplex PCR method was developed to simultaneously amplify a heat-labile toxin sequence (LT) of 258 bp, a shiga-like toxin I sequence (SLT I) of 130 bp, and a shiga-like toxin II sequence (SLT II) of 346 bp from toxigenic strains of Escherichia coli. This method was used to screen 377 environmental E. coli isolates from marine waters or estuaries located in Southern California and North Carolina for enterotoxigenic or enterohemorrhagic E. coli strains. Of the 377 E. coli screened, one isolate was found to belong to the enterotoxigenic group, since it contained a LT homologous sequence, and one isolate was found to belong to the enterohemorrhagic group, since it contained a SLT I homologous sequence. None was found to contain SLT II homologous sequences. The pathogenicity of the positive environmental E. coli isolates was confirmed by standard bioassays with Y-1 adrenal cells and Vero cells to confirm toxin production. Our results suggest that toxigenic E. coli occurs infrequently in environmental waters and that there is a low public health risk from toxigenic E. coli in coastal waters.     

Characterization of a recombinant antibody produced in the course of a high yield fed-batch process

Many mammalian cell fed-batch processes rely on maintaining the cells in a viable and productive state for extended periods of time in order to reach high final concentrations of secreted protein. In the work described herein, a nonamplified NSO cell line was transfected with a vector expressing a recombinant human anti-HIV gp 120 monoclonal antibody (Mab) and a selectable marker, glutamine synthetase. A fed-batch process was developed which improved product yields tenfold over the yields reached in batch culture. In this case, the clone was cultured for a period of 22 days and produced 0.85 g Mab/L. To gauge the effect of extended culture lifetime on product quality, biochemical characteristics of MAb isolated from different time points in the fed-batch culture were determined. The apparent molecular weight of the MAb was constant throughout the course of the culture. Isoelectric focusing revealed four major charged species, with a fifth more acidic species appearing later in the culture. The antigen binding kinetics were constant for MAb isolated throughout the culture period. Glycosylation analysis, on the other hand, revealed that MAb produced later in the culture contained greater percentages of truncated N-acetylglucosamine and highmannose N-glycans. Possible contributions to this underglycosylated material from either cell lysis or synthesis from noviable cells were found to be negligible. Instead, the viable cells appeared to be secreting more truncated and high mannose MAb glycoforms as the culture progressed. (c) 1994 John Wiley & Sons, Inc.     

Passive mechanical behavior of human neutrophils: effect of cytochalasin B.

Actin is a ubiquitous protein in eukaryotic cells. It plays a major role in cell motility and in the maintenance and control of cell shape. In this article, we intend to address the contribution of actin to the passive mechanical properties of human neutrophils. As a framework for assessing this contribution, the neutrophil is modeled as a simple viscous fluid drop with a constant cortical ("surface") tension. The reagent cytochalasin B (CTB) was used to disrupt the F-actin structure, and the neutrophil cortical tension and cytoplasmic viscosity were evaluated by single-cell micropipette aspiration. The cortical tension was calculated by simple force balance, and the viscosity was calculated according to a numerical analysis of the cell entry into the micropipette. CTB reduced the cell cortical tension in a dose-dependent fashion: by 19% at a concentration of 3 microM and by 49% at 30 microM. CTB also reduced the cytoplasmic viscosity by approximately -25% at a concentration of 3 microM and by approximately 65% at a concentration of 30 microM when compared at the same aspiration pressures. All three groups of neutrophils, normal cells, and cells treated with either 3 or 30 microM CTB, exhibited non-Newtonian behavior, in that the apparent viscosity decreased with increasing shear rate. The dependence of the cytoplasmic viscosity on deformation rate can be described empirically by mu = mu c(gamma m/gamma c)-b, where mu is cytoplasmic viscosity, gamma m is mean shear rate, mu c is the characteristic viscosity at the characteristic shear rate gamma c, and b is a material coefficient.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation by angiotensin III of nociception-related and arterial pressure-related neuronal responsiveness in the nucleus reticularis gigantocellularis of the rat

We evaluated possible modulation by angiotensin III (AIII) of the interactive effect of noxious stimuli and elevation in systemic arterial pressure on the responsiveness of neurons in the nucleus reticularis gigantocellularis (NRGC) of the medulla oblongata. Combined extracellular single-neuron recording and microiontophoresis were carried out on male, adult Sprague-Dawley rats anesthetized with pentobarbital sodium. The responsiveness of NRGC neurons to nociception (tail clamp) and/or transient hypertension elicited by phenylephrine (5 μg/kg, i.v.), in the absence or presence of AIII, was used as the experimental index. Microiontophoretic application of the heptapeptide suppressed the responses of spontaneously active NRGC neurons to individually delivered nociception or hypertension. Interestingly, the preferential reduction in responsiveness to tail clamp upon simultaneous elevation in arterial pressure was reversed to one that favored nociception in the presence of AIII. These actions of the heptapeptide appeared to be receptor-specific, since they were discernibly blocked by its selective antagonist, Ile⁷-angiotensin III. Our results reveal that neuropeptides such as AIII may differentially modulate neuronal responsiveness according to the prevailing physiologic input(s) to the central nervous system of the animal.