Conformational distributions of melittin in water/methanol mixtures from frequency-domain measurements of nonradiative energy transfer

We used fluorescence energy transfer to examine the effects of solvent composition on the distribution of distances between the single tryptophan residue of melittin (residue 19) to the N-terminal alpha-amino group, which was labeled with a dansyl residue. The tryptophan intensity decays, with and without the dansyl acceptor, were measured by the frequency-domain method. The data were analyzed by a least-squares algorithm which accounts for correlation between the parameters. A wide distribution of tryptophan to dansyl distances was found for the random-coil state, with a Gaussian half-width of 25 A. Increasing concentrations of methanol, which were shown to induce and alpha-helical conformation, resulted in a progressive decrease in the width of the distribution, reaching a limiting half-width of 3 A at 80% (v/v) methanol. The distance from the indole moiety of Trp-19 to the dansyl group in 80% (v/v) methanol/water was found to be 25 A, as assessed from the center of the distance distribution. A distance of 24-25 A was recovered from the X-ray crystal structure of the tetramer, which is largely alpha-helical. At low ionic strength (less than 0.01) the CD spectra revealed a small fraction or amount of alpha-helix for melittin in water, which implies a small fraction of residual structure. This residual structure is apparently lost in guanidine hydrochloride as demonstrated by a further broadening in the distribution of distances. These results demonstrate the usefulness of frequency-domain measurements of resonance transfer for resolution of conformational distributions of proteins.     

Changing household composition,labor patterns,and fertility in a highland New Guinea population

The European demographic transition of the nineteenth century is often proposed as a model for demographic change in twentieth century developing nations, and economic development is seen as leading to an inevitable reduction in total fertility in these nations. This paper examines data from the Gainj of Papua New Guinea, a natural fertility population with very low reproductive output, and suggests that the effects of development on fertility change are much more complex than a simple demographic transition model would suggest. Looking at two variables known to contribute significantly to low total fertility among the Gainj, late age at first birth for women and long interbirth intervals, the paper suggests that households, in their recruitment and allocation of labor, may exert a mediating influence in the relationship between economic development and fertility.     

The effect of glycogen depletion and supercompensation on the physical working capacity at the fatigue threshold

The purpose of this investigation was to determine the effect of glycogen depletion and supercompensation on the physical working capacity at the fatigue threshold (PWCFT). Ten adult males (mean age 23 years, SD 3) volunteered as subjects for this study. During the first laboratory visit the subjects performed a maximal bicycle ergometer test for the determination of maximum oxygen consumption (VO2max). Between 48 and 72 h later, the subjects pedaled to exhaustion at a power output which corresponded to a mean of 76% of VO2max (range, 72-80%) for the purpose of glycogen depletion. For the next 3 days, the subjects were fed a 10.5 MJ.day-1 low carbohydrate diet which consisted of 7.5% carbohydrates, 22.0% protein and 70.5% fat. The subjects then performed an incremental cycle ergometer test to the onset of fatigue or PWCFT, which was estimated from integrated electromyographic voltages of the vastus lateralis muscle. For the next 3 days the subjects were fed a 10.5 MJ high carbohydrate diet which consisted of 72.2% carbohydrates, 12.4% protein and 15.4% fats for the purpose of glycogen supercompensation. The subjects then performed a second PWCFT test. A paired t-test indicated that there was no significant (p greater than 0.05) difference between the means of the PWCFT values (depletion 246 W, SD 30; supercompensation 265 W, SD 28) and they were highly correlated at r = 0.884. The results of this investigation suggested that the methods commonly used to affect glycogen depletion or supercompensation had no effect on PWCFT.     

A novel RNA helicase gene tightly linked to the Triplo-lethal locus of Drosophila.

The Triplo-lethal (Tpl) locus of Drosophila is the only known locus which is lethal when present in three copies rather than the normal two. After recovering a hybrid-dysgenesis-induced mutation of Tpl we used a rapid combination of transposon tagging, chromosome microdissection and PCR to clone the P element that had transposed into the Tpl region. That P element is located within the gene for a new and unique member of the RNA helicase family. This new helicase differs from all others known by having glycine-rich repeats at both the amino and carboxyl termini. Curiously, genetic analysis shows that the P element inserted into this gene is not responsible for the Tpl mutant phenotype. We present possible explanations for these findings.     

The cyanelle genome of Cyanophora paradoxa, unlike the chloroplast genome, codes for the ribosomal L3 protein.

We describe a 1132 bp sequence of the cyanelle genome of Cyanophora paradoxa containing the rpl3 gene. This gene, which is not chloroplast encoded in plants, is the first of a long cyanelle ribosomal operon whose organization resembles that of the S10 operon of E. coli. We have shown that the rpl3 gene is transcribed in cyanelles as a 7500 nucleotide precursor and that the 5'-end of the mRNA starts approximately 90 nucleotides upstream from the initiation codon. However, no typical procaryotic promoter could be found for this gene. We have detected, using anti E. coli L3 antibodies, the cyanelle L3 protein in cyanelle extracts and in E. coli cells transformed with the cyanelle rpl3 gene.     

Cloning and expression of the toxin B gene ofClostridium difficile

Results from our cloning studies on toxin A indicated that the gene for toxin B resided approximately 1 kb upstream of the toxin A gene. Clone pCD19, which contains the 5-end of the toxin A gene and a small open reading frame, was found to contain 1.2 kb of DNA which, when subcloned, expressed a nontoxic peptide that reacted with toxin B antibodies. The rest of the toxin B gene was located on the 6.8 kb cloned fragment of plasmid pCD19L. The two fragments overlapped 0.8 kb. Lysates containing protein expressed by the 6.8 fragment were cytotoxic and lethal, and were neutralized by toxin B antibody. The two fragments were ligated to give the complete toxin B gene. The protein expressed by the complete gene was cytotoxic and lethal, and showed complete immunological identity with toxin B. Further analysis of the expressed protein and the toxin B gene confirmed our earlier findings showing that toxin B has a molecular weight of 240,000 or greater.     

Neutral endopeptidase from nuchal ligament of fetal calves

The nuchal ligament of unborn calves contains a neutral endopeptidase that is biochemically and immunologically similar to the neutral endopeptidase (NEP), or enkephalinase, from human kidney. Enzymatic activity was inhibited more than 90% by phosphoramidon (1 microM). The specific activity in membrane fractions, as determined by hydrolysis of the dansylated substrate, DAPGN, was similar in tissue from fetuses of gestational ages ranging from 100 to 280 days. NEP activity in adult ligament tissue, however, was less than 10% of that in fetal tissue. Fibroblasts dissociated from ligament tissue by collagenase displayed less NEP activity than did preparations of intact ligament, and activity was even lower in cultured cells. By contrast, fibroblasts cultured from fetal calf lungs had NEP activity comparable to that in the ligament tissue. When ligament fibroblasts were cultured on subcellular matrices derived from fetal lung fibroblasts the NEP activity increased relative to those cultured on plastic alone. These studies confirm the presence of neutral endopeptidase (NEP) in the nuchal ligament of the fetal calf. The consistent activity through a range of gestational ages and the influence of the subcellular matrix suggest that this enzyme might be involved in growth of the ligament during fetal life.     

Isolated microspore culture of maize: effects of isolation technique,reduced temperature,and sucrose level

Improvements in ab initio microspore culture of maize are presented using a modified isolation technique, reduced temperature during early stages of culture, and an elevated sucrose level in the culture medium. Blending-isolation, using excised anthers, was less stressful on microspores than pressing anthers against a stainless steel sieve and resulted in a 3-fold increase in the yield of embryo-like structures (ELS). Exposure to reduced temperature (15°C) during the first 4 days of culture improved microspore viability and increased by 2-fold the number of ELS produced. Higher levels of sucrose (8.0–9.5%) also resulted in improved response. Maximum yield in the present study was 92 ELS per 100 anther equivalents, exceeding previously reported values of 15 ELS per 100 anther equivalents for ab initio microspore culture of maize. The increase in the total number ELS produced had no observable effect on their quality as evidenced by the frequency of formation of callus capable of regenerating plants.