Bioengineered nisin derivatives with enhanced activity in complex matrices

Nisin A is the best known and most extensively characterized lantibiotic. As it is ribosomally synthesized, bioengineering‐based strategies can be used to generate variants. We have previously demonstrated that bioengineering of the hinge region of nisin A can result in the generation of variants with enhanced anti‐microbial activity against Gram‐positive pathogens. Here we created a larger bank of hinge variant producers and screened for producers that exhibit enhanced bioactivity as assessed by agar‐based assays against a selection of target strains. Further analysis of 12 ‘lead’ variants reveals that in many cases enhanced bioactivity is not attributable to enhanced specific activity but is instead as a consequence of an enhanced ability to diffuse through complex polymers. In the case of two variants, which contain the residues SVA and NAK, respectively, within the hinge region, we demonstrate that this enhanced trait enables the peptides to dramatically outperform nisin A with respect to controlling Listeria monocytogenes in commercially produced chocolate milk that contains carrageenan as a stabilizer.     

Single cellular origin of somatostatin and calcitonin in the rat thyroid gland

Summary Immunostaining of thin serial paraffin sections has shown that somatostatin is present in the same parafollicular cells as calcitonin in the adult rat thyroid gland. The number of cells containing both peptides is much smaller than the number containing calcitonin but not somatostatin.     

Synchronous cell growth occurs upon synchronizing the two regulatory steps of the Saccharomyces cerevisiae cell cycle

There are two known asynchronous steps in the budding yeast Saccharomyces cerevisiae cell cycle, where an asynchronous step is one which is completed in different lengths of time by different cells in an isogenic population. It is shown here that elimination of the asynchrony due to cell size by preincubation of cells with the mating pheromone alpha-factor, and decreasing the asynchrony in the cdc28 'start' step by lowering the pH, yields highly synchronous cell growth measured as the time period between the emergence of buds. In one experiment, cell budding for 92% of cells occurred within a 12-min period for at least two generations. Under identical conditions, cell number increase is not as synchronous as bud emergence indicating that there is a third asynchronous step, which is concluded to be at cell separation. These results are consistent with there being two--and only two--asynchronous steps in the cell cycle, measured from bud emergence to bud emergence. Surprisingly, these two steps are also the two major regulatory steps of the cell cycle. It is concluded that asynchrony may be a general feature of cell cycle regulatory steps. The asynchrony in the completion of the cdc28 'start' step which occurs in the first cell cycle after alpha-factor washout is shown here to be almost or entirely eliminated for the second passage through this step after alpha-factor washout. The 'true' time between the onset of budding and the point where 50% of cells have budded (called t50BE) is 17 and less than or equal to 2 min for the first and second budding, respectively, after alpha-factor washout. The cell cycle models requiring a transition probability, or asynchrony, at 'start' for every cell cycle are therefore incorrect.     

Morphological, molecular and functional differences of adult bone marrow- and adipose-derived stem cells isolated from rats of different ages

Adult mesenchymal stem cells have self-renewal and multiple differentiation potentials, and play important roles in regenerative medicine. However, their use may be limited by senescence or age of the donor, leading to changes in stem cell functionality. We investigated morphological, molecular and functional differences between bone marrow-derived (MSC) and adipose-derived (ASC) stem cells isolated from neonatal, young and old rats compared to Schwann cells from the same animals. Immunocytochemistry, RT-PCR, proliferation assays, western blotting and transmission electron microscopy were used to investigate expression of senescence markers. Undifferentiated and differentiated ASC and MSC from animals of different ages expressed Notch-2 at similar levels; protein-38 and protein-53 were present in all groups of cells with a trend towards increased levels in cells from older animals compared to those from neonatal and young rats. Following co-culture with adult neuronal cells, dMSC and dASC from animals of all ages elicited robust neurite outgrowth. Mitotracker^® staining was consistent with ultrastructural changes seen in the mitochondria of cells from old rats, indicative of senescence. In conclusion, this study showed that although the cells from aged animals expressed markers of senescence, aged MSC and ASC differentiated into SC-like cells still retain potential to support axon regeneration.     

Does the Future Affect the Present? The Effects of Future Weather on the Current Collection of Planted Crops and Wildlife in a Native Amazonian Society of Bolivia

Unlike neighboring disciplines, anthropology rarely studies how actual future events affect current behavior. Such studies could lay the groundwork for studies of ethno-forecasting. Psychologists argue that people forecast poorly, but some empirical work in cultural anthropology suggests that at least with weather, rural people might make reasonably accurate forecasts. Using data from a small-scale, pre-industrial rural society in the Bolivian Amazon, this study estimates the effects of future weather on the current collection of planted crops and wildlife. If actual future events affect current behavior, then this would suggest that people must forecast accurately. Longitudinal data covering 11 consecutive months (10/2002–8/2003, inclusive) from 311 women and 326 men ≥age 14 in 13 villages of a contemporary society of forager-farmers in Bolivia’s Amazon (Tsimane’) are used. Individual fixed-effect panel linear regressions are used to estimate the effect of future weather (mean hourly temperature and total daily rain) over the next 1–7 days from today on the probability of collecting wildlife (game, fish, and feral plants excluding firewood) and planted farm crops (annuals and perennials) today. Daily weather records come from a town next to the Tsimane’ territory and data on foraging and farming come from scans (behavioral spot observations) and surveys of study participants done during scans. Short-term future weather (≤3 days) affected the probability of collecting planted crops and wildlife today, although the effect was greater on the amount of planted crops harvested today than on the amount of wildlife collected today. Future weather beyond 3 days bore no significant association with the amount of planted crops harvested today nor with the amount of wildlife collected today. After controlling for future and past weather, today’s weather (mean hourly temperature, but not rain) affected the probability of collecting wildlife today, but today’s weather (temperature or rain) did not affect the probability of collecting planted crops today. The study supports prior work by anthropologists suggesting that rural people forecast accurately. If future weather affects the probability of harvesting planted crops and collecting wildlife today, then this suggests that Tsimane’ must forecast accurately. We discuss possible reasons for the finding. The study also supports growing evidence from rural areas of low-income nations that rural people tend to protect their food production and food consumption well against small idiosyncratic shocks or, in our case, against ordinary daily weather that is not extreme. However, the greater responsiveness of daily foraging output compared with daily farming output to today’s weather suggests that foraging might not protect food consumption as well as farming against adverse climate perturbations.     

Dimethyl sulfoxide at 2.5% (v/v) alters the structural cooperativity and unfolding mechanism of dimeric bacterial NAD+ synthetase

Dimethyl sulfoxide (DMSO) is commonly used as a cosolvent to improve the aqueous solubility of small organic compounds. Its use in a screen to identify novel inhibitors of the enzyme NAD(+) synthetase led to this investigation of its potential effects on the structure and stability of this 60-kD homodimeric enzyme. Although no effects are observed on the enzyme's catalytic activity, as low as 2.5% (v/v) DMSO led to demonstrable changes in the stability of the dimer and its unfolding mechanism. In the absence of DMSO, the dimer behaves hydrodynamically as a single ideal species, as determined by equilibrium analytical ultracentrifugation, and thermally unfolds according to a two-state dissociative mechanism, based on analysis by differential scanning calorimetry (DSC). In the presence of 2.5% (v/v) DMSO, an equilibrium between the dimer and monomer is now detectable with a measured dimer association constant, K(a), equal to 5.6 x 10(6)/M. DSC curve analysis is consistent with this finding. The data are best fit to a three-state sequential unfolding mechanism, most likely representing folded dimer <==> folded monomer <==> unfolded monomer. The unusually large change in the relative stabilities of dimer and monomer, e.g., the association equilibrium shifts from an infinitely large K(a) down to approximately 10(6)/M, in the presence of relatively low cosolvent concentration is surprising in view of the significant buried surface area at the dimer interface, roughly 20% of the surface area of each monomer is buried. A hypothetical structural mechanism to explain this effect is presented.     

Glycine betaine and proline are the principal compatible solutes ofStaphylococcus aureus

The foodborne pathogenStaphylococcus aureus is distinguished by its ability to grow within environments of extremely high osmolarity (e.g., foods with low water activity values). In the present study, we examined the accumulation of intracellular organic solutes withinS. aureus strain ATCC 12600 when cells were grown in a complex medium containing high concentrations of NaCl. Consistent with previous reports [Measures JC (1975) Nature 257:398–400; Koujima I, et al. (1978) Appl Environ Microbiol 35:467–470; and Anderson CB, Witter LD (1982) Appl Environ Microbiol 43:1501–1503], intracellular proline was found to accumulate to high concentrations. However, NMR spectroscopy of cell extracts revealed glycine betaine to be the predominant intracellular organic solute accumulated within cells grown at high osmolarity. In additional experiments, we examined the growth rate ofS. aureus in a defined medium of high osmolarity and found it to be stimulated significantly by the presence of either exogenous proline or glycine betaine. Highest growth rates were obtained when the defined medium was supplemented with glycine betaine.