Mass spectrometric analysis of putative capa-gene products in Musca domestica and Neobellieria bullata

Neuropeptides of the capa-gene are typical of the abdominal neurosecretory system of insects. In this study, we investigated these peptides in two widely distributed and large pest flies, namely Musca domestica and Neobellieria bullata. Using a combination of MALDI-TOF and ESI-QTOF mass spectrometry, periviscerokinins and a pyrokinin were analyzed from single perisympathetic organ preparations. The species-specific peptide sequences differ remarkably between the related dipteran species. These differences could make it possible to develop peptide-analogs with group- or species-specific efficacy.     

An active insect kinin analog with 4-aminopyroglutamate, a novel cis-peptide bond, type VI beta-turn motif

The insect kinins are potent diuretic peptides that preferentially form a cis-Pro, type VI beta-turn. An insect kinin analog containing (2S,4S)-4-aminopyroglutamate, a novel cis-peptide bond, type VI beta-turn motif, demonstrates significant activity in the physiological range in a cricket diuretic assay. This is the first instance of a 4-aminopyroglutamate analog of a peptide with a preference for a type VI turn that demonstrates significant bioactivity. The results provide further confirmatory evidence for the active conformation of the insect kinins, and a new scaffold with which to design biostable, peptidomimetic analogs capable of disrupting critical insect kinin-regulated processes in insects.     

An improved method for isolating high-quality DNA from<Emphasis Type="Italic">Vitis vinifera</Emphasis> nuclei

We have developed a simple and highly efficient protocol for isolating large quantities (150–400 μg/g leaf tissue) of high-quality DNA from fresh and frozenVitis vinifera leaves. Isolated DNA is essentially free of polysaccharides, polyphenols, and other major contaminants as judged by viscosity, clear color, A_260/280 ratio, digestibility by restriction enzymes for Southern blot analysis, and PCR suitability.     

Estimate of the mutation rate per nucleotide in humans

Many previous estimates of the mutation rate in humans have relied on screens of visible mutants. We investigated the rate and pattern of mutations at the nucleotide level by comparing pseudogenes in humans and chimpanzees to (i) provide an estimate of the average mutation rate per nucleotide, (ii) assess heterogeneity of mutation rate at different sites and for different types of mutations, (iii) test the hypothesis that the X chromosome has a lower mutation rate than autosomes, and (iv) estimate the deleterious mutation rate. Eighteen processed pseudogenes were sequenced, including 12 on autosomes and 6 on the X chromosome. The average mutation rate was estimated to be approximately 2.5 x 10(-8) mutations per nucleotide site or 175 mutations per diploid genome per generation. Rates of mutation for both transitions and transversions at CpG dinucleotides are one order of magnitude higher than mutation rates at other sites. Single nucleotide substitutions are 10 times more frequent than length mutations. Comparison of rates of evolution for X-linked and autosomal pseudogenes suggests that the male mutation rate is 4 times the female mutation rate, but provides no evidence for a reduction in mutation rate that is specific to the X chromosome. Using conservative calculations of the proportion of the genome subject to purifying selection, we estimate that the genomic deleterious mutation rate (U) is at least 3. This high rate is difficult to reconcile with multiplicative fitness effects of individual mutations and suggests that synergistic epistasis among harmful mutations may be common.     

The contribution of the y chromosome to hybrid male sterility in house mice

Hybrid sterility in the heterogametic sex is a common feature of speciation in animals. In house mice, the contribution of the Mus musculus musculus X chromosome to hybrid male sterility is large. It is not known, however, whether F(1) male sterility is caused by X-Y or X-autosome incompatibilities or a combination of both. We investigated the contribution of the M. musculus domesticus Y chromosome to hybrid male sterility in a cross between wild-derived strains in which males with a M. m. musculus X chromosome and M. m. domesticus Y chromosome are partially sterile, while males from the reciprocal cross are reproductively normal. We used eight X introgression lines to combine different X chromosome genotypes with different Y chromosomes on an F(1) autosomal background, and we measured a suite of male reproductive traits. Reproductive deficits were observed in most F(1) males, regardless of Y chromosome genotype. Nonetheless, we found evidence for a negative interaction between the M. m. domesticus Y and an interval on the M. m. musculus X that resulted in abnormal sperm morphology. Therefore, although F(1) male sterility appears to be caused mainly by X-autosome incompatibilities, X-Y incompatibilities contribute to some aspects of sterility.     

Optimized expression and purification for high-activity preparations of algal [FeFe]-hydrogenase

Background

Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographic studies.

Principal Findings

We describe the construction, overexpression and high-yield purification of a fusion protein consisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized through improvements in culture conditions and media components used for expression. We also demonstrated that fusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus, led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinity of Fd for DEAE allowed for two-step purification by ion exchange and StrepTactin affinity chromatography. In addition, the incorporation of a TEV protease site in the Fd-HydA1 linker allowed for the proteolytic removal of Fd after DEAE step, and purification of HydA1 alone by StrepTactin. In combination, this process resulted in HydA1 purification yields of 5 mg L⁻¹ of culture from E. coli with specific activities of 1000 U (U = 1 µmol hydrogen evolved mg⁻¹ min⁻¹).

Significance

The [FeFe]-hydrogenases are highly efficient enzymes and their catalytic sites provide model structures for synthetic efforts to develop robust hydrogen activation catalysts. In order to characterize their structure-function properties in greater detail, and to use hydrogenases for biotechnological applications, reliable methods for rapid, high-yield expression and purification are required.     

Active diuretic peptidomimetic insect kinin analogs that contain β-turn mimetic motif 4-aminopyroglutamate and lack native peptide bonds

The multifunctional 'insect kinins' of arthropods share the evolutionarily conserved C-terminal pentapeptide core sequence Phe-X(1)-X(2)-Trp-Gly-NH(2), where X(1)=His, Asn, Ser, or Tyr and X(2)=Ser, Pro, or Ala. Insect kinins regulate diuresis in many species of insects, including the house cricket, Acheta domesticus. Insect kinins, however, are susceptible to fast enzymatic degradation by endogenous peptidases that severely limit their potential use as tools for pest control or for endocrinological studies. To enhance resistance to peptidases, the core insect kinin sequence was structurally modified in this study to replace native peptide bonds susceptible to proteolytic degradation. These modifications include incorporation of two stereochemical variants of the β-turn mimetic motif 4-aminogutamate in place of the X(1)-X(2) residues, insertion of a reduced peptide bond between residues Trp-Gly, and replacement of the Phe residue with a hydrocinnamyl group. The resulting biostable, peptidomimetic analogs contain no native peptide bonds and yet retain significant diuretic activity in an in vitro cricket Malpighian tubule fluid secretion assay, matching the efficacy of a native A. domesticus kinin (Achdo-KI). These novel analogs represent ideal new tools for endocrinologists studying arthropod kinin regulated processes in vivo, and provide leads in the development of novel, environmentally friendly pest insect management agents capable of disruption of the critical processes that kinins regulate.     

Phylloquinone is the principal Mehler reaction site within photosystem I in high light

Photosynthesis is a vital process, responsible for fixing carbon dioxide, and producing most of the organic matter on the planet. However, photosynthesis has some inherent limitations in utilizing solar energy, and a part of the energy absorbed is lost in the reduction of O₂ to produce the superoxide radical (O2•−) via the Mehler reaction, which occurs principally within photosystem I (PSI). For decades, O₂ reduction within PSI was assumed to take place solely in the distal iron–sulfur clusters rather than within the two asymmetrical cofactor branches. Here, we demonstrate that under high irradiance, O₂ photoreduction by PSI primarily takes place at the phylloquinone of one of the branches (the A-branch). This conclusion derives from the light dependency of the O₂ photoreduction rate constant in fully mature wild-type PSI from Chlamydomonas reinhardtii, complexes lacking iron–sulfur clusters, and a mutant PSI, in which phyllosemiquinone at the A-branch has a significantly longer lifetime. We suggest that the Mehler reaction at the phylloquinone site serves as a release valve under conditions where both the iron–sulfur clusters of PSI and the mobile ferredoxin pool are highly reduced.

In high light, O₂ reduction to superoxide radical primarily occurs at the phylloquinone cofactors of photosystem I rather than its terminal [4Fe–4S] clusters.