Purification and primary structure of two isoforms of arenicin, a novel antimicrobial peptide from marine polychaeta Arenicola marina

Two novel 21-residue antimicrobial peptides, arenicin-1 and arenicin-2, exhibiting activity against Gram-positive and Gram-negative bacteria and fungi, were purified from coelomocytes of marine polychaeta Arenicola marina (lugworm) by preparative gel electrophoresis and RP-HPLC. Molecular masses (2758.3 and 2772.3 Da) and complete amino acid sequences (RWCVYAYVRVRGVLVRYRRCW and RWCVYAYVRIRGVLVRYRRCW) were determined for each isoform. Each arenicin has one disulfide bond (Cys3-Cys20). The total RNA was isolated from the lugworm coelomocytes, RT-PCR and cloning were performed, and cDNA was sequenced. A 202-residue preproarenicin contains a putative signal peptide (25 amino acids) and a long prodomain. Arenicins have no structure similarity to any previously identified antimicrobial peptides.     

Abnormal scale morphogenesis: relationship of polypeptide patterns to action of the scaleless gene.

The polyacrylamide gradient gel electrophoresis (PAGGE) pattern of polypeptides isolated from normal scuttate scale epidermis of 1-week-old chicks was different from that of the anterior shank epidermis from 1-week-old scaleless mutant chicks. The PAGGE patterns of polypeptides isolated from normal and scaleless reticulate scale epidermis (from 1-week-old chicks) differed by only one band, whereas comparison of mutant's scuttate and reticulate patterns showed three band differences. These data are discussed in relation to the action of the scaleless gene on early morphogenesis of the two types of scales.     

Morphological and biochemical aspects of monofunctional phosphoramide mustard teratogenicity in rat embryos cultured in vitro

Day 10 rat embryos were exposed in vitro to a monofunctional analog of phosphoramide mustard (MPM) at concentrations of 25 to 200 micrograms/ml (144 to 1,156 X 10(-6) M). After a 24-hour exposure, embryos exhibited a dose-dependent decrease in growth parameters (crown-rump length, number of somites, and protein content) as well as incidence of malformations. Abnormal embryos were characterized by hypoplasia of the prosencephalon as well as hypoplasia of the mandibular arches, tail, and limb buds. Histological analysis revealed abnormal levels of necrotic cells, particularly in the neuroepithelium and surrounding mesenchyme. In all respects embryos exposed to MPM could not be distinguished from embryos exposed to phosphoramide mustard. We also determined using mouse L1210 cells that at the maximum nonlethal concentration used in our embryo exposure experiments, MPM did not cause DNA cross-linking but did cause single-strand DNA breaks. Phosphoramide mustard, at concentrations teratogenic to embryos in vitro, did produce DNA cross-linking. Taken together, our results indicate that although cyclophosphamide (CP)-induced DNA cross-linking may play a role in CP teratogenesis, DNA cross-linking is not an absolute requirement.     

Assessment of Caspian Seal By-Catch in an Illegal Fishery Using an Interview-Based Approach

The Caspian seal (Pusa caspica) has declined by more than 90% since 1900 and is listed as endangered by IUCN. We made the first quantitative assessment of Caspian seal by-catch mortality in fisheries in the north Caspian Sea by conducting semi-structured interviews in fishing communities along the coasts of Russia (Kalmykia, Dagestan), Kazakhstan and Turkmenistan. We recorded a documented minimum by-catch of 1,215 seals in the survey sample, for the 2008–2009 fishing season, 93% of which occurred in illegal sturgeon fisheries. Due to the illegal nature of the fishery, accurately quantifying total fishing effort is problematic and the survey sample could reflect less than 10% of poaching activity in the north Caspian Sea. Therefore total annual by-catch may be significantly greater than the minimum documented by the survey. The presence of high by-catch rates was supported independently by evidence of net entanglement from seal carcasses, during a mass stranding on the Kazakh coast in May 2009, where 30 of 312 carcasses were entangled in large mesh sturgeon net remnants. The documented minimum by-catch may account for 5 to 19% of annual pup production. Sturgeon poaching therefore not only represents a serious threat to Caspian sturgeon populations, but may also be having broader impacts on the Caspian Sea ecosystem by contributing to a decline in one of the ecosystem’s key predators. This study demonstrates the utility of interview-based approaches in providing rapid assessments of by-catch in illegal small-scale fisheries, which are not amenable to study by other methods.     

Isolated Potato Virus A coat protein possesses unusual properties and forms different short virus-like particles

In our previous study, we have observed that the isolated coat proteins (CP) of the Potyvirus Potato Virus A (PVA) virions exhibit an intrinsic tendency to self-associate into various multimeric forms containing some fractions of cross-β-structure. In this report, we studied the effect of solution conditions on the structure and dissociation of isolated PVA CP using a number of complementary physicochemical methods. Analysis of the structure of PVA CP in solution was performed by limited proteolysis with MALDI-TOF mass spectrometry analysis, transmission electron microscopy, intrinsic fluorescence spectroscopy, and synchrotron small angle X-ray scattering (SAXS). Overall structural characteristics of PVA CP obtained by combination of these methods and ab initio shape reconstruction by SAXS show that PVA CP forms large multi-subunit particles. We demonstrate that a mixture of compact virus-like particles (VLP) longer than 30 nm is assembled on dialysis of isolated CP into neutral pH buffer (at low ionic strength). Under conditions of high ionic strength (0.5 M NaCl) and high pH (pH 10.5), PVA dissociates into low compactness oval-shaped particles of approximately 30 subunits (20–30 nm). The results of limited trypsinolysis of these particles (enzyme/substrate ratio 1:100, 30 min) showed the existence of non-cleavable core-fragment, consisting of 137 amino acid residues. Trypsin treatment removed only a short N-terminal fragment in the intact virions. These particles are readily reassembled into regular VLPs by changing pH back to neutral. It is possible that these particles may represent some kind of intermediate in PVA assembly in vitro and in vivo.     

Novel aryl and heteroaryl substituted N-[3-(4-phenylpiperazin-1-yl)propyl]-1,2,4-oxadiazole-5-carboxamides as selective GSK-3 inhibitors

Synthesis, biological evaluation, and SAR dependencies for a series of novel aryl and heteroaryl substituted N-[3-(4-phenylpiperazin-1-yl)propyl]-1,2,4-oxadiazole-5-carboxamide inhibitors of GSK-3beta kinase are described. The inhibitory activity of the synthesized compounds is highly dependent on the character of substituents in the phenyl ring and the nature of terminal heterocyclic fragment of the core molecular scaffold. The most potent compounds from this series contain 3,4-di-methyl or 2-methoxy substituents within the phenyl ring and 3-pyridine fragment connected to the 1,2,4-oxadiazole heterocycle. These compounds selectively inhibit GSK-3beta kinase with IC(50) value of 0.35 and 0.41 microM, respectively.     

Gene and microRNA expression in p53‐deficient day 8.5 mouse embryos

BACKGROUND: Neural tube defects (NTDs) are one of the most common human birth defects, with a prevalence of approximately 1 in 1000 live births in the United States. In animal studies, deletion of p53 leads to a significant increase in embryos that exhibit exencephaly. Whereas several studies have closely investigated the morphologic changes of p53‐deficient embryos, no study has reported the molecular‐level alteration in p53‐deficient embryos. Here we attempt to identify genes and microRNAs (miRNAs) modified by deletion of p53 in day 8.5 mouse embryos. METHODS: Mouse embryos from p53 heterozygous crosses were collected, genotyped, and embryos of similar genotype (+/+; +/?; ?/?) were pooled. RNA from the pooled samples was isolated to determine mRNA and miRNA expression levels using Whole Genome Bioarrays and Low Density Arrays, respectively. RESULTS: In p53 ?/? embryos, 388 genes showed statistically significant alteration in gene expression of more than twofold compared to p53 +/+ embryos. Expression of p53 and well known p53 target genes, such as p21 and cyclin G1, were significantly down‐regulated in p53 ?/? embryos. In contrast, expression of other p53 target genes, such as Mdm2, Noxa, and Puma, were unchanged. We also identified six genes (Csk, Itga3, Jarid2, Prkaca, Rarg, and Sall4), known to cause NTDs when deleted, that are also down‐regulated in p53 ?/? embryos. Finally, five miRNAs (mir‐1, mir‐30e‐3p, mir‐142‐3p, mir‐301, and mir‐331) also showed statistically significant alterations in expression levels in p53 ?/? embryos compared to p53 +/+ embryos. Combined analysis of the experimental data using stepwise regression model and two publicly available algorithms identified putative target genes of these miRNAs. CONCLUSIONS: Our data have identified genes and miRNAs that may be involved in the mechanisms underlining NTDs and begin to define the developmental role of p53 in the etiology of NTDs. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

Hidden Chromosome Symmetry: In Silico Transformation Reveals Symmetry in 2D DNA Walk Trajectories of 671 Chromosomes

Maps of 2D DNA walk of 671 examined chromosomes show composition complexity change from symmetrical half-turn in bacteria to pseudo-random trajectories in archaea, fungi and humans. In silico transformation of gene order and strand position returns most of the analyzed chromosomes to a symmetrical bacterial-like state with one transition point. The transformed chromosomal sequences also reveal remarkable segmental compositional symmetry between regions from different strands located equidistantly from the transition point. Despite extensive chromosome rearrangement the relation of gene numbers on opposite strands for chromosomes of different taxa varies in narrow limits around unity with Pearson coefficient r = 0.98. Similar relation is observed for total genes'' length (r = 0.86) and cumulative GC (r = 0.95) and AT (r = 0.97) skews. This is also true for human coding sequences (CDS), which comprise only several percent of the entire chromosome length. We found that frequency distributions of the length of gene clusters, continuously located on the same strand, have close values for both strands. Eukaryotic gene distribution is believed to be non-random. Contribution of different subsystems to the noted symmetries and distributions, and evolutionary aspects of symmetry are discussed.     

Hair Trace Elements in Overweight and Obese Adults in Association with Metabolic Parameters

The objective of the present study was to investigate the level of toxic and essential trace elements in hair of adult overweight and obese persons as well as its association with metabolic parameters. Hair trace element levels were assessed using inductively coupled plasma mass-spectrometry in 112 overweight and obese patients and 106 lean controls. Serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), glucose, uric acid (UA) levels, and cholinesterase (CE) and gamma-glutamyltransferase (GGT) activity were also assessed. Excessive body weight significantly affected hair trace element levels. In particular, hair Co (33%), Cu (13%), I (30%), Mg (2-fold), Mn (25%), Zn (17%), and Ni (21%) levels were lower, whereas Al (14%) and As levels were higher in comparison to those in the control group. Correlation analysis demonstrated the most significant correlations for hair Mg with body weight, BMI, systolic and diastolic blood pressure, and UA, and for hair Al with body weight, BMI, TC, glucose, TG, CE, GGT, and UA. Multiple regression analysis demonstrated that trace elements were not associated with TC and LDL-C levels neither in crude nor in adjusted models. In turn, crude and adjusted models accounted for 25 and 43% of serum TG variance. The most significant associations were observed for hair Al, Fe, Si, and V in adjusted model. The obtained data demonstrate that obesity-related metabolic disorders may be at least partially mediated by altered trace element and mineral levels.