Structural analysis of a non-contiguous second-site revertant in T4 lysozyme shows that increasing the rigidity of a protein can enhance its stability.

The mutation Glu108-->Val (E108V) in T4 lysozyme was previously isolated as a second-site revertant that specifically compensated for the loss of function associated with the destabilizing substitution Leu99-->Gly (L99G). Surprisingly, the two sites are 11 A apart, with Leu99 in the core and Glu108 on the surface of the protein. In order to better understand this result we have carried out a detailed thermodynamic, enzymatic and structural analysis of these mutant lysozymes as well as a related variant with the substitution Leu99-->Ala. It was found that E108V does increase the stability of L99G, but it also increases the stability of both the wild-type protein and L99A by essentially equal amounts. The effects of E108V on enzymatic activity are more complicated. The mutation slightly reduces the maximal rate of cell wall hydrolysis of wild-type, L99G and L99A. At the same time, L99G is an unstable protein and rapidly loses activity during the course of the assay, especially at temperatures above 20 degrees C. Thus, even though the double mutant L99G/E108V has a slightly lower maximal rate than L99G, over a period of 20-30 minutes it hydrolyzes more substrate. This decrease in the rate of thermal inactivation appears to be the basis of the action of E108V as a second-site revertant of L99G. Mutant L99A creates a cavity of volume 149 A(3). Instead of enlarging this cavity, mutant L99G results in a 4-5 A displacement of part of helix F (residues 108-113), creating a solvent-accessible declivity. In the double mutant, L99G/E108V, this helix returns to a position akin to wild-type, resulting in a cavity of volume 203 A(3). Whether the mutation Glu108-->Val is incorporated into either wild-type lysozyme, or L99A or L99G, it results in a decrease in crystallographic thermal factors, especially in the helices that include residues 99 and 108. This increase in rigidity, which appears to be due to a combination of increased hydrophobic stabilization plus a restriction of conformational fluctuation, provides a structural basis for the increase in thermostability.     

Mapping and analysis of protein sulfhydryl groups by modification with 2-bromoacetamido-4-nitrophenol

A method for identifying cysteine-containing peptides in proteins is presented using 2-bromoacetamido-4-nitrophenol (BNP) to introduce an easily detectable probe. The formation of a covalent bond between the protein sulfhydryl group and the acetamido moiety of BNP introduces a chromophore with an absorbance maximum at 410 nm. The modified protein can then be cleaved with appropriate proteases and the resulting peptides separated by chromatographic methods. Monitoring the effluent at a single wavelength (405 nm) provides a rapid and simple method of detecting and isolating only those peptides which contain cysteine residue(s). The nitrophenol derivative is stable under conditions required for protease cleavage. The reagent is therefore useful for locating cysteine-containing peptides in protein digests and can be used to explore the accessibility of different cysteines under a variety of conditions. The ease of modification, specificity of reaction, product stability, and simple detection of modified peptides make BNP ideal for investigation of cysteine residues.     

Construction and characterization of a single polypeptide chain containing two enzymatically active dihydrofolate reductase domains.

A single polypeptide chain containing two dihydrofolate reductase (DHFR) sequences from Escherichia coli was constructed to determine if a repeat sequence fusion protein could be expressed in an active form. The possibility that intersequence interactions could play a significant role for this enzyme is suggested by the results of Hall and Frieden (1989, Proc. Natl Acad. Sci. USA, 86, 3060-3064) who observed a substantial decrease in the yield of active enzyme when folded in the presence of a large C-terminal fragment. The fusion protein [DHFR(Cys152Glu)--Ile--DHFR (Met1Gln)] was efficiently expressed in E. coli cells and has an activity which is twice that of the wild-type enzyme in the standard assay. The Michaelis constants of the fusion protein for the substrate, dihydrofolate and the cofactor, NADPH, are essentially unchanged from those of the wild-type protein. The urea-induced in vitro unfolding reaction of the fusion protein at low concentrations was found to be fully reversible and follow a three state model, suggesting that the two domains unfold independently. At higher protein concentrations the unfolding transition broadened and shifted to a higher urea concentration. Size-exclusion chromatography results are consistent with the formation of aggregates at the higher protein concentration, even in the absence of denaturant.     

Is group progressive relaxation training as effective with hyperactive children as individual EMG biofeedback treatment?

This study examined whether group progressive relaxation training was as effective as individual EMG biofeedback training in facilitating the academic achievement and self-control of 45 hyperactive elementary school children. Academic achievement was assessed with the Gates-MacGinities Reading Tests, and self-control was measured with the Nowicki-Strickland and the Teacher Rating scales. Eight sessions were scheduled at weekly intervals. Progressive relaxation was conducted in groups of seven or eight and was induced with a commercial audiocassette program. EMG training augmented frontalis biofeedback with those taped exercises. A placebo group listened to taped children's stories. Multivariate analysis of variance indicated no significant differences among the three contrast groups when all dependent variables were considered together. However, univariate F values and discriminant analysis disclosed locus of control to be significantly more internal for the progressive relaxation condition. Also, differences between the two relaxation and the placebo groups, though not statistically significant, were all in the expected direction. While the relative efficacy of group progressive relaxation could not be established conclusively, the data appeared sufficiently positive to warrant further investigation of this cost-effective prospective intervention.The opinions expressed by the authors are not necessarily those of their respective institutions.     

Sex pheromones in maleMelittobia parasitic wasps: Female response to conspecific and congeneric males of 3 species

Presented a choice between conspecific males and 2 congeneric males, virgin females ofMelittobia australica andM. digitata chose conspecific males disproportionately more often, whereasM. femorata females distributed themselves evenly among the choices. Empty tubes, provided as the fourth choice in the test apparatus, were entered much less often than tubes containing live males. Females of all species chose “wrong” males about equally frequently. These observations suggest that even non-conspecific males possess some degree of attractiveness to virgin females. Chemicals in the sex pheromone of the males are presumed to be the source of the males' attractancy. The incomplete species specificity is interpreted in light of the life history of this genus, and it is suggested that specific recognition cues operate primarily after the sexes come together. Supported by a grant from Kagoshima Prefecture in Japan under the exchange program of faculty members between Kagoshima University and the University of Georgia.     

Two novel loci underlie natural differences in Caenorhabditis elegans abamectin responses

Parasitic nematodes cause a massive worldwide burden on human health along with a loss of livestock and agriculture productivity. Anthelmintics have been widely successful in treating parasitic nematodes. However, resistance is increasing, and little is known about the molecular and genetic causes of resistance for most of these drugs. The free-living roundworm Caenorhabditis elegans provides a tractable model to identify genes that underlie resistance. Unlike parasitic nematodes, C. elegans is easy to maintain in the laboratory, has a complete and well annotated genome, and has many genetic tools. Using a combination of wild isolates and a panel of recombinant inbred lines constructed from crosses of two genetically and phenotypically divergent strains, we identified three genomic regions on chromosome V that underlie natural differences in response to the macrocyclic lactone (ML) abamectin. One locus was identified previously and encodes an alpha subunit of a glutamate-gated chloride channel (glc-1). Here, we validate and narrow two novel loci using near-isogenic lines. Additionally, we generate a list of prioritized candidate genes identified in C. elegans and in the parasite Haemonchus contortus by comparison of ML resistance loci. These genes could represent previously unidentified resistance genes shared across nematode species and should be evaluated in the future. Our work highlights the advantages of using C. elegans as a model to better understand ML resistance in parasitic nematodes.     

M89V Sialic Acid Acetyl Esterase (SIAE) and All Other Non-Synonymous Common Variants of This Gene Are Catalytically Normal

Catalytically defective rare variants of Sialic acid Acetyl Esterase (SIAE) have previously been linked to autoimmunity. Studies presented here confirm that the M89V SIAE protein and all other products of common variant alleles of SIAE are catalytically normal. Although overexpressing transfected non-lymphoid cells secrete small amounts of SIAE that can associate with the cell surface, normal human lymphocytes do not exhibit cell surface SIAE, supporting genetic evidence in mice that indicates that this protein functions in a lymphocyte intrinsic manner. Analyses of the plasma proteome also indicate that SIAE is not secreted in vivo. A re-analysis exclusively of catalytically defective rare variant alleles of SIAE in subjects in which this gene was completely sequenced confirmed an association of SIAE with autoimmunity. A subset of catalytically defective rare variant SIAE alleles has previously been typed in a large genotyping study comparing a diverse group of disease subjects and controls; our re-analysis of this data shows that catalytically defective alleles are enriched in disease subjects. These data suggest that SIAE may be associated with autoimmunity and that further study of catalytically defective rare variant SIAE alleles in terms of autoimmune disease susceptibility is strongly warranted.