Quantitative determination of the antifungal compound 6-pentyl-α-pyrone (6PAP) using a simple plate bioassay

An assay relating bioactivity to levels of the antifungal compound 6-pentyl-α-pyrone (6PAP) has been developed. Using Cladosporium strain NZ660US as indicator at a spore concentration of 5×10⁶ spores overlay⁻¹ on malt (adjusted to pH 3·5) agar plates, this assay allows quantitative determination of 6PAP down to a detection limit of 0·5% v/v. This will allow rapid screening of Trichoderma culture extracts to compare the bioactivity of isolates grown under different culture conditions, and can be used in combination with analytical chemical analysis to identify the presence of other bioactive metabolites.     

Screening environmental samples for source-specific bacteriophage hosts using a method for the simultaneous pouring of 12 petri plates

A simple apparatus was developed to allow 12 petri plates to be poured simultaneously by hand. It was used when screening bacterial isolates from sewage and dog feces for their ability to detect phages from these sources. This was done to assess the ease with which source-specific phage hosts can be isolated from these sources of fecal pollution. Host bacteria that consistently detected phages from sewage were easily isolated from sewage. These bacterial isolates did not detect phages from dog feces. Host bacteria were not isolated from dog feces even after screening hundreds of colonies from fecal samples from six dogs. Journal of Industrial Microbiology & Biotechnology (2000) 24, 124–126. Received 06 July 1999/ Accepted in revised form 05 November 1999     

Scanning electron microscopic observations of early stages of phagocytosis of E. coli by human neutrophils

Summary Changes in surface morphology, as observed by scanning electron microscopy, appear rapidly when human polymorphonuclear neutrophils (PMN) are challenged with bacteria. Monolayers of PMN adhering to glass were incubated with opsonized E. coli from 5 sec to 10 min, and then fixed and prepared for SEM. As early as 5 sec after phagocytic challenge, E. coli are found in contact with PMN and in the process of engulfment into open cavities formed by lamellipodia. The shape of the mouth of the forming phagocytic vacuole is related to the orientation of bacteria during entry. Bacteria engulfed into early forming phagosomes are surrounded by a large open space between the bacteria and the phagosome wall. As phagocytosis proceeds, the space is reduced and the loose fit around the entering bacteria becomes tight. By 30 sec, bacteria may be completely internalized and by 1 min phagocytized E. coli are packed into bulging PMN. The observations reveal the variability and rapidity of the phagocytic response and confirm the presence of sensitive mechanisms for host defense by PMN.This work was supported by research grants from the University of North Carolina Research Council and the National Institutes of Health (A1 02430)     

A novel tubular bioassay for measuring the production of antagonistic chemicals produced at the fungal/pathogen interface

A novel tubular bioassay system has been developed that allows both concentration profiles and the local production and concentration of antagonistic chemicals at the fungal/pathogen interface to be measured analytically.     

A straightforward radiometric technique for measuring IMP dehydrogenase

[2-3H]Inosinic acid ([2-3H]IMP) has been biosynthesized in good yield from [2-3H]hypoxanthine and PRPP via the action of a partially purified preparation of hypoxanthine/guanine phosphoribosyl transferase from mouse brain. The product was purified in one step by ascending paper chromatography, and used to assess the activity of IMP dehydrogenase. To conduct the assay, tritiated substrate is admixed with enzyme in a final volume of 10 microliters; NAD is present to serve as cofactor for the reaction, and allopurinol to inhibit the oxidation of any hypoxanthine generated as a consequence of side reactions. After an appropriate period of incubation, the 3H2O arising from the oxidation of tritiated IMP via [3H]NAD is isolated by quantitative microdistillation. Performed as described, the assay is facile, sensitive, and accurate, with the capability of detecting the dehydrogenation of as little as 1 pmol of [3H]IMP. Using it, measurements have been made of IMP dehydrogenase in a comprehensive array of mouse organs. Of these, pancreas contained the enzyme at the highest specific activity.     

Activation of AMPK by Bitter Melon Triterpenoids Involves CaMKKβ

We recently showed that bitter melon-derived triterpenoids (BMTs) activate AMPK and increase GLUT4 translocation to the plasma membrane in vitro, and improve glucose disposal in insulin resistant models in vivo. Here we interrogated the mechanism by which these novel compounds activate AMPK, a leading anti-diabetic drug target. BMTs did not activate AMPK directly in an allosteric manner as AMP or the Abbott compound (A-769662) does, nor did they activate AMPK by inhibiting cellular respiration like many commonly used anti-diabetic medications. BMTs increased AMPK activity in both L6 myotubes and LKB1-deficient HeLa cells by 20–35%. Incubation with the CaMKKβ inhibitor, STO-609, completely attenuated this effect suggesting a key role for CaMKKβ in this activation. Incubation of L6 myotubes with the calcium chelator EGTA-AM did not alter this activation suggesting that the BMT-dependent activation was Ca²⁺-independent. We therefore propose that CaMKKβ is a key upstream kinase for BMT-induced activation of AMPK.     

Computer-aided baker's yeast fermentations.

The economics of yeast production depend heavily upon the cellular yield coefficient on the carbon source and the volumetric productivity of the process. The application of an on-line computer to maximize these two terms during the fermentation requires a continuous method of measuring cell density and growth rate. Unfortunately, a direct sensor for biomass concentration suitable for use in industrial fermentations is not available. Material balancing, with the aid of on-line computer monitoring, offers an indirect method of measurement. Laboratory results from baker's yeast production in a 14-liter fermentor (with a PDP-11/10 computer for on-line analyses) show this indirect measurement technique to be a viable alternative. From the oxygen uptake and carbon dioxide production data, gas flow rate, and ammonia addition rate, the cell density during the fermentation has been estimated and found to compare well with actual fermentation data.     

Computer control of bakers' yeast production

The Biotechnology and Bioengineering study presented here was undertaken to demonstrate the usefulness of computer control for the production of yeast from molasses. A flexible control system was developed by using an on-line computer for the monitoring of cell mass and employing anticipatory control to maintain the maximum productivity. Process disturbances were minimized by employing a multivariable feedback control system to prevent ethanol formation. The control strategy acted to keep overall conversion yield at its maximum level, about 0.5 g cell/ g sugar, while maintaining high volumetric productivity between 3 and 5 g/liter-hr. Results are presented to show the effectiveness of simultaneous anticipatory and feedback contol in overcoming problems of oxygen starvation, molasses quality, and variable inoculum size.