Using fluorescence correlation spectroscopy to study conformational changes in denatured proteins

Fluorescence correlation spectroscopy (FCS) is a sensitive analytical tool that allows dynamics and hydrodynamics of biomolecules to be studied under a broad range of experimental conditions. One application of FCS of current interest is the determination of the size of protein molecules in the various states they sample along their folding reaction coordinate, which can be accessed through the measurement of diffusion coefficients. It has been pointed out that the analysis of FCS curves is prone to artifacts that may lead to erroneous size determination. To set the stage for FCS studies of unfolded proteins, we first show that the diffusion coefficients of small molecules as well as proteins can be determined accurately even in the presence of high concentrations of co-solutes that change the solution refractive index significantly. Indeed, it is found that the Stokes-Einstein relation between the measured diffusion coefficient and solution viscosity holds even in highly concentrated glycerol or guanidinium hydrochloride (GuHCl) solutions. These measurements form the basis for an investigation of the structure of the denatured state of two proteins, the small protein L and the larger, three-domain protein adenylate kinase (AK). FCS is found useful for probing expansion in the denatured state beyond the unfolding transition. It is shown that the denatured state of protein L expands as the denaturant concentration increases, in a process akin to the transition from a globule to a coil in polymers. This process continues at least up to 5 M GuHCl. On the other hand, the denatured state of AK does not seem to expand much beyond 2 M GuHCl, a result that is in qualitative accord with single-molecule fluorescence histograms. Because both the unfolding transition and the coil-globule transition of AK occur at a much lower denaturant concentration than those of protein L, a possible correlation between the two phenomena is suggested.     

An Escherichia coli mutant thermosensitive in the B subunit of DNA gyrase: effect on the structure and replication of the colicin E1 plasmid in vitro

Summary An E. coli strain which carries a mutation conferring clorobiocin resistance and temperature sensitivity for growth has recently been described and evidence has been presented suggesting that the mutation is located in the gyrB gene (Orr et al. 1979). The replication of the ColE1 plasmid was analysed in cell-free extracts from this thermosensitive strain. These extracts were totally deficient in the replication of exogenous plasmid DNA and were unable to maintain the superhelical structure of the plasmid DNA. Both defects could be fully complemented by addition of purified gyrB protein.     

Myogenic contractility is more dependent on myofilament calcium sensitization in term fetal than adult ovine cerebral arteries

Regulation of cytosolic calcium and myofilament calcium sensitivity varies considerably with postnatal age in cerebral arteries. Because these mechanisms also govern myogenic tone, the present study used graded stretch to examine the hypothesis that myogenic tone is less dependent on calcium influx and more dependent on myofilament calcium sensitization in term fetal compared with adult cerebral arteries. Term fetal and adult posterior communicating cerebral arteries exhibited similar myogenic responses, with peak tensions averaging 24 and 26% of maximum contractile force produced in any given tissue in response to an isotonic Krebs buffer containing 122 mM K(+) (K(max)) at optimum stretch ratios (working diameter/unstressed diameter) of 2.19 and 2.23, respectively. Graded stretch increased cytosolic Ca(2+) concentration at stretch ratios >2.0 in adult arteries, but increased Ca(2+) concentration only at stretch ratios >2.3 in fetal arteries. In permeabilized arteries, myogenic tone peaked at a stretch ratio of 2.1 in both fetal and adult arteries. The fetal %K(max) values at peak myogenic tone were not significantly different at either pCa 7.0 (23%) or pCa 5.5 (25%) but were significantly less at pCa 8.0 (8.4 +/- 2.3%). Conversely, adult %K(max) values at peak myogenic tone were significantly less at both pCa 8.0 (10.4 +/- 1.8%) and pCa 7.0 (16%) than at pCa 5.5 (27%). The maximal extents of stretch-induced increases in myosin light chain phosphorylation in intact fetal (20%) and adult (17%) arteries were similar. The data demonstrate that the cerebrovascular myogenic response is highly conserved during postnatal maturation but is mediated differently in fetal and adult cerebral arteries.     

Developmental response of <Emphasis Type="Italic">Euplectrus comstockii</Emphasis> to ascorbic acid in the diet of the larval host, <Emphasis Type="Italic">Heliothis virescens</Emphasis>

Recent developments in genetic engineering have paved the way for researchers to produce crops of high nutritional and yield value, in addition to being resistant to diseases and pests. Ascorbic acid content is one of the parameters researchers are trying to enhance in plants. This study investigated the effect of different levels of dietary ascorbic acid of a beneficial wasp, Euplectrus comstockii Howard (Hymenoptera: Eulophidae), by measuring life history parameters of the wasp when reared on lepidopteran larvae fed a basal diet containing low and high levels of ascorbic acid. Odds and odds ratio analyses showed that the probability of egg hatch and adult emergence for the wasp increased with the amount of ascorbic acid in the diet of the host, and that the rate of development and probability of female or male progeny was similar for most levels of ascorbic acid tested. This would indicate that as the ascorbic acid concentration increases in the pest insect the effectiveness of the wasp is likely to increase and when, by comparison with other published findings, the effectiveness of microbial pathogens is likely to decrease.     

Cannabigerol: a bibliometric overview and review of research on an important phytocannabinoid

Phytochemistry Reviews - Cannabigerol (CBG) is one of the major phytocannabinoids present in Cannabis sativa L. but is presumed to be an artefact or degradation product of cannabigerolic acid...     

Phenols and phenol oxidases are involved in cadmium accumulation in the water plants Nymphoides peltata (Menyanthaceae) and Nymphaeae (Nymphaeaceae)

This comparative study investigates the mechanism of cadmium accumulation in the semiaquatic plant Nymphoides peltata (Menyanthaceae) and the aquatic plant Nymphaea (Nymphaeaceae). It was conducted as part of an ongoing study of the use of water plants for phytoremediation. Epidermal structures, known as hydropotes, are located on the abaxial epidermis of the leaf laminae of Nymphoides peltata and are shown to contain phenols, peroxidase and polyphenol oxidase activities. When plants are subjected to 50 mg/l of cadmium in the growth medium, these hydropotes accumulate cadmium. Cadmium-induced increases in phenols, peroxidase and polyphenol oxidase activities were determined in plant extracts. Cadmium binding by polymerized phenols was demonstrated in vivo. In comparison with Nymphaeae epidermal glands, N. peltata hydropotes are larger, open, and create bigger crystal, the latter principally composed of calcium and, proportionally, less cadmium. Although both plants showed similar levels of cadmium accumulation, N. peltata was sensitive while Nymphaeae was resistant to this cadmium level. It is suggested that in these water plants the main mechanism for cadmium accumulation is based on the trapping of cadmium crystals by polymerized phenols in specialized epidermal structures and this is due to peroxidase and polyphenol oxidase activities. Nymphaeae, with greater peroxidase activity and more polyphenols, is more resistant to this heavy metal than N. peltata.     

Lipid metabolism in the cockroach, Periplaneta americana, is activated by the hypertrehalosemic peptide, HTH-I

Phosphatidylcholine and phosphatidylethanolamine are the major constituents of the phospholipid pool in cockroach (Periplaneta americana) fat body and hemolymph. Both species of phospholipid are significantly decreased 6h after injecting hypertrehalosemic hormone I (HTH-I) into the hemocoel. Loss of phospholipid is accompanied by an accumulation of the phospholipid degradation products glycerophosphorylcholine and glycerol. HTH-I also increases phospholipase activity in the hemolymph and this is thought to be responsible for the depletion of hemolymph phospholipid. Phospholipase activity peaks approximately 2h after injection of HTH-I and returns to normal at 6h. In vitro, total phospholipid in the fat body is decreased by HTH-I whereas the concentration of diacylglycerol displays a corresponding increase. HTH-I elevates free fatty acid levels but has no effect on triacylglycerol. These effects of HTH-I are blocked by the phospholipase inhibitor mepacrine.     

Genetic heterogeneity of Campylobacter concisus determined by pulsed field gel electrophoresis-based macrorestriction profiling

In order to investigate the genetic diversity of Campylobacter concisus to assist molecular typing studies, the use of macrorestriction profiling was examined. A suitable protocol was developed that included the use of formaldehyde pretreatment to prevent DNA degradation, and restriction enzyme NotI for pulsed field gel electrophoresis-based genotyping. Subsequently, 53 strains of C. concisus, principally from cases of diarrhoea in children, were examined. Fifty-one distinct patterns were obtained, indicating the high discriminatory potential of the method. Patterns comprised between one and 14 restriction fragments, with type and reference strains of two well-defined genomospecies of oral and faecal origin containing six and 12 fragments respectively. Our results show that C. concisus is genetically diverse and suggest the species as currently defined to be a taxonomic continuum comprised of several genomospecies. The pulsed field gel electrophoresis typing method described here has considerable potential for molecular epidemiological studies of C. concisus and may be a useful adjunctive method for helping to resolve key taxonomic issues for this species.