How dry are anhydrous enzymes? Measurement of residual and buried 18O-labeled water molecules using mass spectrometry

There is continual debate over the central role of water in protein folding, structure, stability, and dynamics. Catalytic activity has been demonstrated in organic media with, apparently “anhydrous” enzymes. Hence there is considerable discussion over whether there are a few residual water molecules or if the enzymes are demonstrating activity in the complete absence of water. Here we present measurements designed to test this hypothesis based on the detection of ¹⁸O-labeled water by mass spectrometry. This extremely sensitive technique avoids many of the potential errors associated with published methods for measuring water content such as gravimetry or Karl Fischer titrations. We have also explored the mass spectrometric detection of ²H-enriched water and found that lyophilization of deuteron-labeled protein can lead to extensive loss of the isotopic label during the drying process. “Anhydrous” protein was produced by extended drying, over P₂O₅, of lyophilized powders hydrated through the vapor phase with ¹⁸O-labeled water. Redissolution in standard water released the remaining protein-bound ¹⁸O-labeled water molecules, and the isotopic enrichment of the water was used to calculate the number of bound molecules per mole of protein. In the cases of lysozyme and subtilisin Carlsberg, 4 ± 2 and 15 ± 2 waters per mole were found, respectively. Comparisons with crystal structures showed these values correspond closely to the expected number of buried water molecules in these proteins. This is consistent with the idea that water physically entrapped within the rigid protein structure is retained but all the other more accessible surface-bound hydration molecules can be removed by the drying process. Such anhydrous subtilisin Carlsberg preparations have been found to be weakly catalytic and therefore it appears that additional water molecules on the surface of the enzyme are not essential for this level of enzyme activity. © 1997 John Wiley & Sons, Inc.     

Exercise training in heart failure

Chronic heart failure (CHF) is a common condition with a poor prognosis. It is associated with poor exercise tolerance and debilitating symptoms. These symptoms appear to be associated with pathophysiological changes that occur systemically in the patient with CHF. Exercise training in carefully selected patients has been shown to be safe and to improve exercise capacity. Many of the pathophysiological abnormalities of CHF are improved by training. Some studies have suggested a possible improvement in morbidity and mortality with training. This review analyzes the controlled clinical trials of exercise training in CHF published to date.     

Combined stable isotope and gut contents analysis of food webs in plant-dominated, shallow lakes

Summary 1. To determine feeding links between primary producers, invertebrates and fish, stable isotope analyses and gut content analyses of fish were conducted on the components of four shallow, eutrophic to hypertrophic, plant-dominated lakes.
2. Although separation of basal resources was possible, the diets of both fish and invertebrates were broad, comprising food from different compartments (planktonic, epiphytic/benthic), as well as from different trophic levels.
3. Mixing models were used to determine the extent to which periphyton production supported higher trophic levels. Only one species of invertebrate relied upon periphyton production exclusively.
4. Fish density affected the diets of invertebrates. The response was different for planktonic and epiphytic/benthic invertebrates. The proportion of periphyton production in the diets of zooplankton appeared to increase with fish density, whilst it decreased for other invertebrates.
5. As all zooplankton samples were collected in the open water at dusk, these results are further evidence for the diurnal horizontal migration of zooplankton. Although not conclusive, they are consistent with a behavioural response by invertebrates and zooplankton in the presence of fish.     

Copper microenvironments in the human body define patterns of copper adaptation in pathogenic bacteria

Copper is an essential micronutrient for most organisms that is required as a cofactor for crucial copper-dependent enzymes encoded by both prokaryotes and eukaryotes. Evidence accumulated over several decades has shown that copper plays important roles in the function of the mammalian immune system. Copper accumulates at sites of infection, including the gastrointestinal and respiratory tracts and in blood and urine, and its antibacterial toxicity is directly leveraged by phagocytic cells to kill pathogens. Copper-deficient animals are more susceptible to infection, whereas those fed copper-rich diets are more resistant. As a result, copper resistance genes are important virulence factors for bacterial pathogens, enabling them to detoxify the copper insult while maintaining copper supply to their essential cuproenzymes. Here, we describe the accumulated evidence for the varied roles of copper in the mammalian response to infections, demonstrating that this metal has numerous direct and indirect effects on immune function. We further illustrate the multifaceted response of pathogenic bacteria to the elevated copper concentrations that they experience when invading the host, describing both conserved and species-specific adaptations to copper toxicity. Together, these observations demonstrate the roles of copper at the host–pathogen interface and illustrate why bacterial copper detoxification systems can be viable targets for the future development of novel antibiotic drug development programs.     

Modification of cell-wall polymers of onion waste: III. Effect of extrusion-cooking on cell-wall material of outer fleshy tissues

The polymers of onion cell walls are known to be modified by heating, but there is little information on the effects of extrusion-cooking. This work investigates the effects of extrusion-cooking on the physico-chemical characteristics and microstructure of cell walls of onion waste in relation to cell-wall chemistry. Cell-wall material from white fleshy outer scale leaves of waste onions was extruded at a range of moisture contents, barrel temperatures and screw speeds through a co-rotating twin-screw extruder. Extrusion-cooking had little effect on the carbohydrate composition of cell-wall material. However, it resulted in an increase in the solubility of pectic polymers and hemicelluloses, and this was accompanied by an increase in swelling of the cell-wall material. The degree of solubility of the pectic polysaccharides was largely dependent on the barrel temperature, and involved depolymerisation.     

Spatial and temporal expression of histone demethylase,Kdm2a,during murine molar development

The histone demethylase, lysine (K)-specific demethylase 2A (Kdm2a), is highly conserved and expressed ubiquitously. Kdm2a can regulate cell proliferation and osteo/dentinogenic, adipogenic and chondrogenic differentiation of mesenchymal stem cells (MSCs) derived from dental tissue. We used quantitative real-time RT-PCR analysis and immunohistochemistry to detect Kdm2a expression during development of the murine molar at embryonic days E12, E14, E16 and E17 and postnatal days P3 and P14. Immunohistochemistry results showed no positive staining of Kdm2a at E12. At E14, Kdm2a was expressed weakly in the inner enamel epithelium, stellate reticulum cells and dental sac. At E16, Kdm2a was expressed mainly in the inner and outer enamel epithelium, stratum intermedium and dental sac, but weaker staining was found in cervical loop and dental papilla cells adjacent to the basement membrane. At E17, the strongest Kdm2a staining was detected in the ameloblasts and stronger Kdm2a staining also was detected in the stratum intermedium, outer enamel epithelium and dental papilla cells compared to the expression at E16. Postnatally, we found that Kdm2a was localized in secretory and mature ameloblasts and odontoblasts, and dentin was unstained. Real-time RT-PCR showed that Kdm2a mRNA levels in murine germ cells increased from E12 to E14 and from E14 to E16; no significant change occurred at E16, E17 or P3, then the levels decreased at P14 compared to P3. Kdm2a expression may be closely related to cell proliferation, to ameloblast and odontoblast differentiation and to the secretion of extracellular enamel and dentin during murine tooth development.     

Butenyl-spinosyns, a natural example of genetic engineering of antibiotic biosynthetic genes

Spinosyns, a novel class of insect active macrolides produced by Saccharopolyspora spinosa, are used for insect control in a number of commercial crops. Recently, a new class of spinosyns was discovered from S. pogona NRRL 30141. The butenyl-spinosyns, also called pogonins, are very similar to spinosyns, differing in the length of the side chain at C-21 and in the variety of novel minor factors. The butenyl-spinosyn biosynthetic genes (bus) were cloned on four cosmids covering a contiguous 110-kb region of the NRRL 30141 chromosome. Their function in butenyl-spinosyn biosynthesis was confirmed by a loss-of-function deletion, and subsequent complementation by cloned genes. The coding sequences of the butenyl-spinosyn biosynthetic genes and the spinosyn biosynthetic genes from S. spinosa were highly conserved. In particular, the PKS-coding genes from S. spinosa and S. pogona have 91–94% nucleic acid identity, with one notable exception. The butenyl-spinosyn gene sequence codes for one additional PKS module, which is responsible for the additional two carbons in the C-21 tail. The DNA sequence of spinosyn genes in this region suggested that the S. spinosa spnA gene could have been the result of an in-frame deletion of the S. pogona busA gene. Therefore, the butenyl-spinosyn genes represent the putative parental gene structure that was naturally engineered by deletion to create the spinosyn genes.     

Habitat specificity and home-range size as attributes of species vulnerability to extinction: a case study using sympatric rattlesnakes

Large home-range size and habitat specificity are two commonly cited ecological attributes that are believed to contribute to species vulnerability. The eastern diamondback rattlesnake Crotalus adamanteus is a declining species that occurs sympatrically with the more abundant canebrake rattlesnake Crotalus horridus in a portion of the south-eastern Coastal Plain, USA. In this study, we use the ecological similarities of the two species as experimental controls to test the role of home-range size and habitat specificity in the imperilment of the eastern diamondback rattlesnake. We used analysis of variance to investigate differences in home-range size between the two species, and home-range selection was modeled as habitat use versus availability with a case control sampling design using logistic regression. We failed to detect differences in home-range size between the two species; therefore, we could not identify home-range size as an attribute contributing to the imperilment of eastern diamondback rattlesnakes. Eastern diamondback rattlesnakes selected pine savannas to a degree that suggests that the species is a habitat specialist. Of the two factors examined, habitat specificity to the imperiled longleaf pine ecosystem may be a significant contributor to the decline of the eastern diamondback rattlesnake.