Spiraled origins

Recent studies have established that the eukaryotic actin-based cytoskeleton has prokaryotic origins. In addition to regulating cell shape and polarity, Gitai et al. (2005) provide convincing evidence that the Caulobacter actin homolog MreB also mediates the early segregation of the chromosomal origin, a typical functional role of the eukaryotic tubulin-based cytoskeleton.     

Fasciola hepatica: humoral and cytokine responses to a member of the saposin-like protein family following delivery as a DNA vaccine in mice

The humoral and cellular responses to DNA vaccination of BALB/c mice with a novel antigen from the Fasciola hepatica saposin-like protein family (FhSAP-2) have been investigated. Two constructs were produced containing the FhSAP-2 DNA sequence, one intended for extracellular secretion of FhSAP-2 protein, and one expressing FhSAP-2 in the cytoplasm of a transfected cell. The constructs were tested in HEK 293T cells, with the secretory construct producing less detectable FhSAP-2 relative to cytoplasmic construct when observed by fluorescence. The size of expressed protein was confirmed by Western blot of cell lysate, but FhSAP-2 was undetectable in cell supernatants. Both, secretory and cytoplasmic constructs as well as FhSAP-2 recombinant protein were tested in mice. The antibody response elicited in mice vaccinated with the rFhSAP-2 induced high levels of IgG(1), IgG(2), and IgE as well as high levels of IL-10 and IFNgamma indicating a mixed Th1/Th2 response. Vaccination of mice intramuscularly with the cytoplasmic FhSAP-2 construct resulted in a dominant IgG(2a) isotype antibody as well as a dominant IFNgamma cytokine, with significant IgE, IgG(1), and IL-10 responses also present, suggesting a mixed Th1/Th2 profile. Isotype and cytokine profiles elicited by the FhSAP-2 secretory construct were similar to those obtained with the cytoplasmic construct but at levels that were significantly lower. The results demonstrate that FhSAP-2 can be delivered as a DNA vaccine construct and induces a stronger Th1 response than the recombinant protein alone. This could result in an improvement in the immunoprophylactic potential of this candidate vaccine against F. hepatica.     

Heat shock protein 65-reactive T cells are involved in the pathogenesis of non-antigenic dimethyl dioctadecyl ammonium bromide-induced arthritis

Dimethyl dioctadecyl ammonium bromide (DDA) (C(38)H(80)NBr) is a nonantigenic lipoid material. DDA-induced arthritis (DIA) in the Lewis (LEW) (RT.1(l)) rat is a new experimental model for human rheumatoid arthritis (RA). DIA is a T cell-mediated autoimmune disease. However, the precise self/foreign Ags associated with the disease process in DIA are not yet known. We observed that LEW rats with DIA spontaneously raised a vigorous T cell response both to 65-kDa self (rat) heat shock protein (Rhsp65) and mycobacterial hsp65 (Bhsp65), but not to another arthritis-related Ag, bovine collagen type II. The T cell response to Rhsp65 was focused predominantly on determinant regions 120-134 and 213-227 of the self protein. Interestingly, pretreatment of adult LEW rats using either a mixture of peptides 120-134 and 213-227 of Rhsp65 or a low nonarthritogenic dose of DDA induced protection against subsequent DIA. Intriguingly, the protection induced by the latter was associated with spontaneous priming of T cells specific for peptide 213-227 of Rhsp65. Similarly, LEW rats neonatally tolerized against either Rhsp65 or Bhsp65 were significantly protected from subsequently induced DIA at adult stage, showing the disease-modulating attribute of the hsp65-specific T cells. Taken together, the above findings demonstrate that the hsp65-directed T cell repertoire is of significance in the pathogenesis of autoimmune arthritis induced by nonantigenic DDA. Like other animal models of RA involving hsp65, these first insights into the disease-associated Ags in the DIA model would pave the way for further understanding of the immunological aspects of induction and regulation of RA.     

Identification of an amino acid residue in the protein kinase C C1b domain crucial for its localization to the Golgi network

Protein kinase C (PKC) isoforms have been reported to be targeted to the Golgi complex via their C1 domains. We have shown recently that the regulatory domain of PKC induces apoptosis in neuroblastoma cells and that this effect is correlated to Golgi localization via the C1b domain. This study was designed to identify specific residues in the C1 domains that mediate Golgi localization. We demonstrate that the isolated C1b domains from PKCalpha, -delta, -epsilon, -eta, and - are targeted to the Golgi complex, whereas the corresponding C1a domains localize throughout the cell. Sequence alignment showed that amino acid residues corresponding to Glu-246 and Met-267 in PKC are conserved among C1b but absent from C1a domains. Mutation of Met-267, but not of Glu-246, to glycine abolished the Golgi localization of the isolated C1b domain and the regulatory domain of PKC. The mutated PKC regulatory domain constructs lacking Golgi localization were unable to induce apoptosis, suggesting a direct correlation between Golgi localization and apoptotic activity of PKC regulatory domain. Mutation of analogous residues in the C1b domain of PKCepsilon abrogated its Golgi localization, demonstrating that this effect is not restricted to one PKC isoform. The abolished Golgi localization did not affect neurite induction by PKCepsilon. However, the PKCepsilon mutant did not relocate to the Golgi network in response to ceramide and ceramide did not suppress the neurite-inducing capacity of the protein. Thus, the specific mutations in the C1b domain influence both the localization and function of full-length PKCepsilon.     

The phylogeny of Gaura (Onagraceae) based on ITS, ETS, and trnL-F sequence data

Gaura (Onagraceae: Onagreae) is a small North American genus of 21 species consisting mostly of night-blooming, moth-pollinated annuals and perennials. The current infrageneric classification based on differences in habit, floral symmetry, and fruit morphology recognizes eight sections within the genus. We examine the phylogenetic relationships of all 21 species of Gaura using DNA sequence data from the internal transcribed spacer region (ITS), the external transcribed spacer region (ETS), and the plastid trnL-F region. Combined analysis of these regions indicate Gaura is monophyletic only if it includes Stenosiphon, a monotypic genus comprised of S. linifolius. Within Gaura, our studies indicate that sections Gauridium, Schizocarya, Campogaura, Stipogaura, Xenogaura, and Gaura are monophyletic, but sections Xerogaura and Pterogaura are not and should be reevaluated. In addition, molecular data provide support for the hypothesis that G. sinuata and G. drummondii arose via interspecific hybridization followed by genome doubling; their influence on phylogenetic reconstruction is discussed.     

PCR detection of hemolysin (vhh) gene in Vibrio harveyi

The Vibrio harveyi hemolysin gene (vhh), which encodes for a virulence factor involved in pathogenicity to fish and shellfish species, may be targeted for species detection or strain differentiation. Primers designed for this gene were used in detection studies of V. harveyi strains from various hosts. One primer set among four tested, could amplify the expected gene fragment in PCR using templates from all 11 V. harveyi strains studied. Detection of the presence of the hemolysin gene could therefore serve as a suitable detection marker of Vibrio harveyi isolates potentially pathogenic to fish and shrimps.     

Effects of pollution on human growth and development: an introduction

Pollution is a worldwide problem and its potential to influence the physiology of human populations is great. Studies of human growth and development in relation to pollution have increased in number and quality since the mid-twentieth century. Many studies have found that some pollutants have detrimental effects on human growth, particularly prenatal growth. The heavy metal, lead, is commonly found in human populations and is related to smaller size at birth and studies have reported decrements that range up to about 200 grams. Noise stress from transportation sources also is related to reduced prenatal growth with somewhat smaller decrements reported. Studies of humans exposed to polychlorinated biphenyls, one of the persistent organic pollutants, have reduced size at birth, advanced sexual maturation and altered hormone levels related to thyroid regulation. Thus different pollutants exert effects through different physiological pathways. However, some studies have not observed these effects, which indicates that the situation is complex and requires further study with better study designs. Determining the effects of pollutants on human physiology and growth is difficult as it requires fairly large numbers of subjects who are not purposely exposed but for whom exposure can be measured. These effects of pollutants and the mechanisms of effect require further study to understand and, it is hoped, to blunt or block any detrimental effects on human health and well-being.     

Fluid Flow-induced Soluble Vascular Endothelial Growth Factor Isoforms Regulate Actin Adaptation in Osteoblasts

Although load-induced mechanical signals play a key role in bone formation and maintenance of bone mass and structure, the cellular mechanisms involved in the translation of these signals are still not well understood. Recent identification of a novel flow-induced mechanosignaling pathway involving VEGF in osteoblasts and the known VEGF regulation of actin reorganization in various cell types has led us to hypothesize that fluid shear stress-induced Vegf up-regulation underlies the actin cytoskeleton adaptation observed in osteoblasts during mechanotransduction. Our results show that MC3T3-E1 cells secrete significant VEGF in response to 5 h of pulsatile fluid shear stress (PFSS; 5 dynes/cm² at 1 Hz), whereas expression of VEGF receptors (VEGFR-1, VEGFR-2, or NRP1) is unaffected. These receptors, in particular VEGFR-2, participate in PFSS-induced VEGF release. Exposure to flow-conditioned medium or exogenous VEGF significantly induces stress fiber formation in osteoblasts that is comparable with PFSS-induced stress fiber formation, whereas VEGF knockdown abrogates this response to PFSS, thereby providing evidence that flow-induced VEGF release plays a role in actin polymerization. Using neutralizing antibodies against the receptors and VEGF isoforms, we found that soluble VEGFs, in particular VEGF₁₆₄, play a crucial role in transient stress fiber formation during osteoblast mechanotransduction, most likely through VEGFR-2 and NRP1. Based on these data we conclude that flow-induced VEGF release from osteoblasts regulates osteoblast actin adaptation during mechanotransduction and that VEGF paracrine signaling may provide potent cross-talk among bone cells and endothelial cells that is essential for fracture healing, bone remodeling, and osteogenesis.     

Autoantibodies Against an Extracellular Peptide of the GluR3 Subtype of AMPA Receptors Activate Both Homomeric and Heteromeric AMPA Receptor Channels

Autoantibodies to the GluR3-subtype of AMPA/glutamate receptors are found in the sera and cerebrospinal fluid of some individuals with epilepsy. They could possibly play a role in the pathophysiology of epilepsy since anti-GluR3 sera display glutamatergic agonist activity. We have investigated here the ability of affinity-purified antibodies (Abs) directed against the immunogenic peptide GluR3B (amino-acid 372–395) to interact with and activate recombinant GluR3-receptor channels expressed by Xenopus oocytes. We report here that the affinity-purified anti-GluR3B Abs directly activate GluR3-containing homomeric and heteromeric AMPA receptor complexes without the requirement of neuronal, glial or blood ancillary molecules. We present some of the properties of the purified anti-GluR3B Abs and discuss the possible physiological or pathological consequences of their activation of glutamate receptors.     

Antifouling activity of synthesized peptide analogs of the sponge metabolite barettin

Barettin (cyclo [(6-bromo-8-en-tryptophan) arginine]), a diketopiperazine isolated from the marine sponge Geodia barretti, is a potent inhibitor of barnacle larvae settlement with an EC50-value of 0.9 microM. In the present study, 14 analogs of barettin and its structural congener dipodazine were synthezised and tested for their ability to inhibit larval settlement. Two of the analogs have an intact barettin skeleton. The remaining analogs have a dipodazine skeleton (a diketopiperazine where arginine is replaced with glycine). Six of the tested synthetic analogs displayed significant settlement inhibition with the most potent inhibitor being benzo[g]dipodazine, which displayed even stronger activity than barettin (EC50-value 0.034 microM). The effect of benzo[g]dipodazine was also shown to be readily reversible, when cyprids were transferred to filtered seawater (FSW).