Injection of Dip-allatostatin or Dip-allatostatin pseudopeptides into mated female Diploptera punctata inhibits endogenous rates of JH biosynthesis and basal oocyte growth

Studies on the catabolism of allatostatins (ASTs) provided the rationale for the design of a series of Dip-allatostatin-derived pseudopeptide mimetic analogues. In vitro, the Dip-ASTs and pseudopeptides show varying degrees of resistance to catabolism and all show significant inhibition of juvenile hormone (JH) biosynthesis. This study was undertaken to determine whether potent Dip-ASTs and/or their pseudopeptide mimetic counterparts caused 'allatostatic' effects in vivo following injection into mated female Diploptera punctata. Animals injected with aqueous solvent or Dip-AST 7(1-7) N-terminal fragment, which excludes the active core region of the ASTs, were used as controls. An in vitro radiochemical assay revealed that injection of Dip-AST 5, 7 or pseudopeptide analogues 397-2 or AST(b)φ2 significantly inhibited the biosynthesis of JH (P<0.05). The results also indicate that basal oocyte growth was significantly inhibited by injection of these same compounds, with the exception of Dip-AST 7 (P<0.05). Analogues 396-1 and 419 did not significantly inhibit rates of JH biosynthesis but did significantly inhibit the growth of basal oocytes. Analyses of feeding, excretion and food absorption/utilization patterns of these same animals suggested that these compounds are not toxic to the insect; rather they directly inhibit the biosynthesis of JH by the corpora allata, and reduce the rate of growth of basal oocytes. Disruption of critical reproductive and/or developmental processes by pseudopeptide analogues of the ASTs could provide novel and selective strategies for future insect pest management.     

Searching for evidence of positive selection in the human genome using patterns of microsatellite variability

Both natural selection and nonequilibrium population-level processes can lead to a skew in the frequency distribution of polymorphisms. Population-level processes are expected to affect all loci in a roughly equal fashion, whereas selection will affect only some regions of the genome. We conducted a sliding-window analysis of the frequency distribution of microsatellite polymorphisms across the human genome to identify regions that may be under positive selection. The analysis was based on a published data set of 5,257 mapped microsatellites in individuals of European ancestry. Observed and expected numbers of alleles were compared under a stepwise mutation model (SMM) using analytical formulae. Observed and expected heterozygosities were compared under a SMM using coalescent simulations. The two sets of analyses gave similar results. Approximately one-fourth of all loci showed a significant deficit of heterozygosity, consistent with a recent population expansion. Forty-three windows were identified with extreme skews in the frequency distribution of polymorphisms (in the direction of a deficit of heterozygosity, given the number of alleles). If these extreme windows are tracking selection at linked sites, theory predicts that they should be more common in regions of the genome with less recombination. We tested this prediction by comparing recombination rates in these extreme windows and in other regions of the genome and found that extreme windows had a significantly lower recombination rate than the genomic average. The proportion of extreme windows was significantly higher on the X chromosome than on the autosomes. Moreover, all the windows with extreme skews on the X chromosome were found in two clusters near the centromere; both these clusters exhibit markedly reduced recombination rates. These analyses point to regions of the genome that may recently have been subject to positive selection. These results also suggest that the effects of positive selection may be more pronounced on the X chromosome than on the autosomes in humans.     

Isolation,identification, and synthesis of a disulfated sulfakinin from the central nervous system of an arthropods the white shrimp Litopenaeus vannamei

Two myotropic peptides displaying tyrosyl sulfation have been isolated from an extract of central nervous systems (brain, suboesophageal ganglion, thoracic ganglia, and ventral nerve cord) of the white shrimp Litopenaeus vannamei. Both peptides were identified by mass spectrometry and belong to the sulfakinin family of neuropeptides, which are characterized by the C-terminal hexapeptide Y(SO(3)H)GHMRF-NH(2) preceded by two acidic amino acid residues. Pev-SK 1 (AGGSGGVGGEY(SO(3)H)DDY(SO(3)H)GH(L/I) RF-NH(2)) has two sulfated tyrosyl residues and a unique (L/I) for M substitution in the C-terminal sequence. Pev-SK 2 (pQFDEY(SO(3)H)GHMRF-NH(2)) fully complies with the typical sulfakinin core sequence and is blocked by a pyroglutamyl residue. Synthetic analogs (sulfated and unsulfated) were synthesized and the tyrosyl sulfations were confirmed by myotropic activity studies and co-elution with the native fractions. Pev-SK 1 is the first disulfated neuropeptide elucidated in the phylum of the arthropoda, with the only other reported disulfated neuropeptide, called cionin, found in a protochordate. The similarities in amino acid sequence and posttranslational modifications of the crustacean sulfakinins and protochordate cionin provide further evidence for the hypothesis stating that gastrin/CCK, cionin, and sulfakinins originate from a common ancestral gastrin/CCK-like peptide.     

Newly discovered functions for some myotropic neuropeptides in locusts

The field of neuropeptide research in insects during the past twenty years can be characterized by the enormous number of peptides that have been identified. In the locusts, Locusta migratoria and Schistocerca gregaria only, structural information is now available for more than 60 peptides. Quite a number of these peptides were isolated on the basis of their effect on visceral muscle contraction in vitro. A very limited number of reports describe the 'in vivo' function of a myotropic neuropeptide. Moreover, for most of the brain neuropeptides, we ignore whether they have a hormonal function. In this paper, we describe the recently discovered in vivo effects of some of the myotropic peptides, identified in locusts in the past decade. Schistocerca-neuropeptide F accelerates egg development; locustasulfakinin inhibits food intake and [His(7)]-corazonin induces body color pigmentation.     

Synthesis of 3-(12-hydroxy-p-carboranyl)propionic acid, a hydrophobic, N-terminal tyrosine-mimetic for peptides

A new, p-carborane containing analog of tyrosine, namely 3-[1-hydroxy-1,12-dicarba-closo-dodecaboran(12)-12-yl]propionic acid was prepared from p-carborane in five steps involving hydroxypropylation of O-protected 1-hydroxy-p-carborane as the key transformation. The simple tyrosine mimetic can function as a hydrophobic surrogate for an N-terminal tyrosine residue in insect and mammalian neuropeptides to enhance the lipophilicity, and therefore, the cuticle and/or tissue permeability properties of mimetic analogs.     

Single nucleotide polymorphisms and recombination rate in humans

Levels of heterozygosity for single nucleotide polymorphisms vary by more than one order of magnitude in different regions of the human genome. Regional differences in the rate of recombination explain a substantial fraction of the variation in levels of nucleotide polymorphism, consistent with the widespread action of natural selection at the molecular level.     

Pharmacological characterization of STKR, an insect G protein-coupled receptor for tachykinin-like peptides

STKR is a G protein-coupled receptor that was cloned from the stable fly, Stomoxys calcitrans. Multiple sequence comparisons show that the amino acid sequence of this insect receptor displays several features that are typical for tachykinin (or neurokinin, NK) receptors. Insect tachykinin-related peptides, also referred to as "insectatachykinins," produce dose-dependent calcium responses in Drosophila melanogaster Schneider 2 cells, which are stably transfected with this receptor (S2-STKR). These responses do not depend on the presence of extracellular Ca(2+)-ions. A rapid agonist-induced increase of inositol 1,4,5-trisphosphate (IP(3)) is observed. This indicates that the agonist-induced cytosolic Ca(2+)-rise is caused by a release of Ca(2+) ions from intracellular calcium stores. The pharmacology of STKR is analyzed by studying the effects of the most important antagonists for mammalian NK-receptors on STKR-expressing insect cells. The results show that spantide II, a potent substance P antagonist, is a real antagonist of insectatachykinins on STKR. We have also tested the activity of a variety of natural insectatachykinin analogs by microscopic image analysis of calcium responses in S2-STKR cells. At a concentration of 1 microM, almost all natural analogs produce a significant calcium rise in stable S2-STKR cells. Interestingly, Stc-TK, an insectatachykinin that was recently discovered in the stable fly (S. calcitrans), also proved to be an STKR-agonist. Stc-TK, a potential physiological ligand for STKR, contains an Ala-residue (or A) instead of a highly conserved Gly-residue (or G). Arch.     

Effects of demographic parameters on metapopulation size and persistence: an analytical stochastic model

An analytical stochastic metapopulation model is developed. It describes how individuals will be distributed among patches as a function of density-dependent birth, death and emigration rates, and the probability of successful dispersal. The model includes demographic stochasticity, but not catastrophes, environmental stochasticity or variation in patch size and suitability. All patches are equally likely to be colonized by migrants. The model predicts: (a) mean and variance of the number of individuals per patch; (b) probability distribution of individuals per patch; (c) mean number of individuals in transit; and (d) turn-over rate and expected persistence time of a single patch. The model shows that (a) dispersal rates must be intermediate in order to ensure metapopulation persistence; (b) the mean number of individuals per patch is often well below the carrying capacity; (c) long transit times and/or high mortality during dispersal reduce the mean number of individuals per patch; (d) density-dependent emigration responses will usually increase metapopulation size and persistence compared with density-independent dispersal; (e) an increase in the per capita net growth rate can both increase and decrease metapopulation size and persistence depending on whether dispersal rates are high or low; (f) density-independent birth, death, and emigration rates lead to a spatial pattern described by the negative binomial distribution.     

Tissue classification with gene expression profiles.

Constantly improving gene expression profiling technologies are expected to provide understanding and insight into cancer-related cellular processes. Gene expression data is also expected to significantly aid in the development of efficient cancer diagnosis and classification platforms. In this work we examine three sets of gene expression data measured across sets of tumor(s) and normal clinical samples: The first set consists of 2,000 genes, measured in 62 epithelial colon samples (Alon et al., 1999). The second consists of approximately equal to 100,000 clones, measured in 32 ovarian samples (unpublished extension of data set described in Schummer et al. (1999)). The third set consists of approximately equal to 7,100 genes, measured in 72 bone marrow and peripheral blood samples (Golub et al, 1999). We examine the use of scoring methods, measuring separation of tissue type (e.g., tumors from normals) using individual gene expression levels. These are then coupled with high-dimensional classification methods to assess the classification power of complete expression profiles. We present results of performing leave-one-out cross validation (LOOCV) experiments on the three data sets, employing nearest neighbor classifier, SVM (Cortes and Vapnik, 1995), AdaBoost (Freund and Schapire, 1997) and a novel clustering-based classification technique. As tumor samples can differ from normal samples in their cell-type composition, we also perform LOOCV experiments using appropriately modified sets of genes, attempting to eliminate the resulting bias. We demonstrate success rate of at least 90% in tumor versus normal classification, using sets of selected genes, with, as well as without, cellular-contamination-related members. These results are insensitive to the exact selection mechanism, over a certain range.