Conformational change of bovine serum albumin by heat treatment

The thermal denaturation of bovine serum albumin (BSA) was studied at pH 2.8 and 7.0 in the range of 2–65°C. The relative proportions of -helix, -structure, and disordered structure in the protein conformation were determined as a function of temperature, by the curve-fitting method of circular dichroism spectra. With the rise of temperature at pH 7.0, the proportion of -helix decreased above 30°C and those of -structure and disordered structure increased in the same temperature range. The structural change was reversible in the temperature range below 45°C. However, the structural change was partially reversible upon cooling to room temperature subsequent to heating at 65°C. On the other hand, the structural change of BSA at pH 2.3 was completely reversible in the temperature range of 2–65°C, probably because the interactions between domains and between subdomains might disappear due to the acid expansion. The secondary structure of disulfide bridges-cleaved BSA remained unchanged during the heat treatment up to 65°C at pH 2.8 and 7.0.     

Background selection by the peppered moth (Biston betularia Linn.): individual differences

The hypothesis that dimorphically coloured, cryptic moths select appropriate rest sites by comparing their body scales to substrate reflectance was tested using typical and melanic morphs of the peppered moth, Biston betularia (L.). Experiments designed to block the individual's inspection of its inherited colour phenotype do not support Kettlewell's contrast/conflict (self-inspection) hypothesis. Instead, tracking of marked moths over successive days revealed individual differences in rest-site selection which were not related to treatments, experience (imprinting), nor closely to a moth's inherited colour pattern. Differences between family broods indicate that some genetic bias in background selection exists. The production of artificially selected lines with consistent but opposing preferences will allow us to investigate the co-evolution of pattern and behaviour.     

Cis proline mutants of ribonuclease A. I. Thermal stability.

A chemically synthesized gene for ribonuclease A has been expressed in Escherichia coli using a T7 expression system (Studier, F.W., Rosenberg, A.H., Dunn, J.J., & Dubendorff, J.W., 1990, Methods Enzymol. 185, 60-89). The expressed protein, which contains an additional N-terminal methionine residue, has physical and catalytic properties close to those of bovine ribonuclease A. The expressed protein accumulates in inclusion bodies and has scrambled disulfide bonds; the native disulfide bonds are regenerated during purification. Site-directed mutations have been made at each of the two cis proline residues, 93 and 114, and a double mutant has been made. In contrast to results reported for replacement of trans proline residues, replacement of either cis proline is strongly destabilizing. Thermal unfolding experiments on four single mutants give delta Tm approximately equal to 10 degrees C and delta delta G0 (apparent) = 2-3 kcal/mol. The reason is that either the substituted amino acid goes in cis, and cis<==>trans isomerization after unfolding pulls the unfolding equilibrium toward the unfolded state, or else there is a conformational change, which by itself is destabilizing relative to the wild-type conformation, that allows the substituted amino acid to form a trans peptide bond.     

Characterization of the stabilizing effect of point mutations of pyruvate oxidase from Lactobacillus plantarum: protection of the native state by modulating coenzyme binding and subunit interaction.

Point mutations in the gene of pyruvate oxidase from Lactobacillus plantarum, with proline residue 178 changed to serine, serine 188 to asparagine, and alanine 458 to valine, as well as a combination of the three single point mutations, lead to a significant functional stabilization of the protein. The enzyme is a tetrameric flavoprotein with tightly bound cofactors, FAD, TPP, and divalent metal ions. Thus, stabilization may be achieved either at the level of tertiary or quaternary interactions, or by enhanced cofactor binding. In order to discriminate between these alternatives, unfolding, dissociation, and cofactor binding of the mutant proteins were analyzed. The point mutations do not affect the secondary and tertiary structure, as determined by circular dichroism and protein fluorescence. Similarly, the amino acid substitutions neither modulate the enzymatic properties of the mutant proteins nor do they stabilize the structural stability of the apoenzymes. This holds true for both the local and the global structure with unfolding transitions around 2.5 M and 5 M urea, respectively. On the other hand, deactivation of the holoenzyme (by urea or temperature) is significantly decreased. The most important stabilizing effect is caused by the Ala-Val exchange in the C-terminal domain of the molecule. Its contribution is close to the value observed for the triple mutant, which exhibits maximum stability, with a shift in the thermal transition of ca. 10 degrees C. The effects of the point mutations on FAD binding and subunit association are interconnected. Because FAD binding is linked to oligomerization, the stability of the mutant apoenzyme-FAD complexes is increased. Accordingly, mutants with maximum apparent FAD binding exhibit maximum stability. Analysis of the quaternary structure of the mutant enzymes in the absence and in the presence of coenzymes gives clear evidence that both improved ligand binding and subunit interactions contribute to the observed thermal stabilization.     

Acclimatory responses in enzymatic activity and metabolite concentrations in the tropical scorpion Heterometrus fulvipes

1. 1.|Changes in tissue metabolite concentrations and enzyme activities in the pedipalpal (PM) and heart (HM) muscles of the tropical scorpion Heterometrus fulvipes show that the metabolism in PM and HM is fundamentally reorganized following low (18°C) and high (38°C) temperature acclimation.

2. 2.|Changes in metabolite concentrations show that metabolite biosynthesis showed increases after cold acclimation but decreases after warm acclimation.

3. 3.|Similarly, changes in enzyme activities show a preponderance of glycolysis and HMP shunt activity after cold acclimation, while after warm acclimation glycogenolysis, oxidative metabolism and gluconeogenesis predominated.

4. 4.|Higher metabolite concentrations and enzyme activities both before and after thermal acclimation in HM reflect its greater compensatory abilities.

Differential winter mortality and seasonal selection in the polymorphic ladybird Adalia bipunctata (L) in the Netherlands

Seasonal selection acting on the melanic polymorphism in the two-spot ladybird Adalia bipunctata was investigated in The Netherlands. An increase in melanic frequency over the spring-summer reproductive period was quantified. The selective advantage gained by melanics averaged 9%, but significant heterogeneity occurred between populations. Adult hibernation behaviour is described. The beetles when outdoors show a highly clumped distribution both between and within trees. The distribution of the morph classes between aggregations is random. Survivorship in a hibernating cohort (initial n= 1898) on a grid of 70 lime trees near Utrecht was monitored by making three counts over the winter of 1981–1982. Intense selection favouring each melanic morph occurred during December and January. The relative fitness of non-melanics was 0.55 (melanics =1). The discovery of dead beetles in late January (about 5% of total losses) and the absence of spatially density-dependent mortality were consistent with a climatic stress rather than selective predation. The period of selection was associated with very cold temperatures averaging up to 4°C below normal and an overall mortality of nearly 75%. There was no change in morph frequency, near normal temperatures and a lower mortality from February to early April. Examination of groups of nearby trees in late January strongly suggested that similar differential mortality had occurred except on some willows. This difference was probably due to the more protected hibernation sites available on these trees. Samples of hibernating cohorts at three other sites showed no evidence of differential mortality. Laboratory experiments with hibernating beetles found no difference in survivorship or rate of weight loss between starved non-melanics and melanics in temperature regimes with and without periods of adult activity. It is concluded that the intense winter selection on the study limes is probably exceptional. Examination of changes in morph frequency through the annual cycle suggests that at some sites the selection favouring melanics during reproduction is counterbalanced by selection against melanics in late summer or early autumn. The results are discussed in relation to mathematical models of cyclical selection and to other field studies including that of Timoféeff-Ressovsky (1940), who found large decreases in melanic frequency during hibernation in Berlin.     

Industrial melanism in the spittlebug Philaenus spumarius (L) (Homoptera: Aphrophoridae)*

The visible polymorphism of the spittlebug, Philaenus spumarius, has been investigated in the vicinity of a smokeless fuel factory in the Cynon Valley of south Wales. The factory is a significant source of local particulate air pollution. A striking relationship exists between the combined frequencies of the eight dark (melanic) morphs and proximity to the factory. Maximum melanic phenotype frequencies of over 95“, occur in both sexes immediately adjacent to it and decline to levels normal for south Wales 1.5–6 km away, depending on direction. This relationship is largely confined to sites within the valley; samples from adjacent localities outside it have melanic frequencies within normal limits for the south Wales area. Maximum melanic phenotype frequencies in the Cynon Valley are far higher than any known from elsewhere in the species range in Europe, Asia and North America. No consistent difference is apparent in total melanic frequency between males and females at any of the sites in this study. However, marked differences exist between the sexes in the relative contributions of the eight melanic phenotypes to the overall association with the factory. For females the industrial melanism is entirely attributable to the group flanicollis + gibbus + leucocephalus (mainly leucocephalus) whereas in males both this group and the group quadnmaculatus + albomaculatus + leucopthalmus contribute to the relationship. It is suggested that this relationship is due to the selective effects of the local air pollution from the factory. The exact nature of the selection involved is as yet uncertain; it would appear to be strong since the local adaptation involved has developed in a maximum of 40 generations since the factory was opened. Finally, comparison is made with two other insects, a ladybird and a moth, in which high frequencies of melanic forms are also associated with this pollution source.     

The efficacy of biobehavioral and compliance interventions in the adjunctive treatment of mild pregnancy-induced hypertension

This investigation assessed the efficacy of a biobehavioral intervention in the adjunctive treatment of mild pregnancy-induced hypertension (PIH), a potentially serious complication of pregnancy in which normotensive women develop hypertension, proteinuria, and edema of unknown etiology late in gestation. Forty-five women with symptoms of PIH were randomly assigned to one of three treatment conditions: bed rest alone (the most common obstetrical treatment), bed rest with individualized compliance enhancement training, or a four-session biobehavioral treatment consisting of bed rest, compliance enhancement training, and individualized thermal biofeedback-assisted relaxation training. Results indicated that while blood pressure for the bed rest and compliance enhancement groups continued to rise and pose an increasing health risk to maternal and fetal well-being, subjects in the biobehavioral group maintained their blood pressure at a significantly lower, and presumably safer, level. The biobehavioral treatment is hypothesized to affect blood pressure levels in subjects with mild PIH through the mediation of the sympathetic nervous system, decreasing peripheral vascular resistance and cardiac output. The results of this investigation suggest that the biobehavioral intervention may be an effective adjunct to bed rest in the treatment of mild PIH remote from term.     

Notes on the use of wood refugia by Physella heterostropha (say) in a thermally disturbed swamp

Physella heterostropha size distributions and densities were measured in Pen Branch delta, a thermally altered swamp of the Savannah River, South Carolina. During cessation of thermal input, measurements were taken in a highly impacted area and more natural area. Of the three substrates sampled, logs, small woody material and benthos, snail density was highest on small pieces of submerged wood and < 20% of the snails on logs were out of the water. Following resumption of thermal input, snail density was four times higher on logs than previously measured. Snails responded to elevated water temperatures by congregating in a narrow band slightly above the water. Snail size did not appear to affect their response. However, the thermal history of the snails influenced their behaviour and survival rates. Logs may be a potential refuge for snails when water temperature are greater greater than 38°C. However, our results indicate that long-term survival in this manner may be impossible.