Genetic evidence that GTP is required for transposition of IS903 and Tn552 in Escherichia coli

Surprisingly little is known about the role of host factors in regulating transposition, despite the potentially deleterious rearrangements caused by the movement of transposons. An extensive mutant screen was therefore conducted to identify Escherichia coli host factors that regulate transposition. An E. coli mutant library was screened using a papillation assay that allows detection of IS903 transposition events by the formation of blue papillae on a colony. Several host mutants were identified that exhibited a unique papillation pattern: a predominant ring of papillae just inside the edge of the colony, implying that transposition was triggered within these cells based on their spatial location within the colony. These mutants were found to be in pur genes, whose products are involved in the purine biosynthetic pathway. The transposition ring phenotype was also observed with Tn552, but not Tn10, establishing that this was not unique to IS903 and that it was not an artifact of the assay. Further genetic analyses of purine biosynthetic mutants indicated that the ring of transposition was consistent with a GTP requirement for IS903 and Tn552 transposition. Together, our observations suggest that transposition occurs during late stages of colony growth and that transposition occurs inside the colony edge in response to both a gradient of exogenous purines across the colony and the developmental stage of the cells.     

Induction of intrauterine growth restriction by reducing placental vascular growth with the angioinhibin TNP-470

The placenta is a specialized vascular interface between the maternal and fetal circulations that increases in size to accommodate the nutritional and metabolic demands of the growing fetus. Vascular proliferation and expansion are critical components of placental development and, consequently, interference with vascular growth has the potential to severely restrict concurrent development of both the placenta and fetus. In this study, we describe the effects of an antiangiogenic agent, TNP-470, on placental vascular development and the induction of a form of intrauterine growth restriction (IUGR) in mice. Administration of TNP-470 to dams in the second half of pregnancy resulted in a smaller maternal weight gain accompanied by decreased placental and fetal sizes in comparison with control animals. Total numbers of fetuses per litter were not affected significantly. Stereological analysis of placentas revealed no changes in the combined lengths of vessels. However, the mean cross-sectional areas of maternal and fetal vessels in the labyrinth of TNP-470-treated mice were reduced at Embryonic Day 13.5 (E13.5) but not at E18.5. Further analysis showed reduced placental endothelial proliferation at E13.5 and E18.5 in TNP-470-treated animals. No other structural or morphometric differences in placentas were detected between TNP-470-treated and control mice at E18.5. This study provides conclusive evidence that administration of TNP-470 interferes with placental vascular proliferation and vessel caliber and results in a reproducible model of IUGR.     

Structure and tropomyosin binding properties of the N-terminal capping domain of tropomodulin 1

Two families of actin regulatory proteins are the tropomodulins and tropomyosins. Tropomodulin binds to tropomyosin (TM) and to the pointed end of actin filaments and "caps" the pointed end (i.e., inhibits its polymerization and depolymerization). Tropomodulin 1 has two distinct actin-capping regions: a folded C-terminal domain (residues 160-359), which does not bind to TM, and a conserved, N-terminal region, within residues 1-92 that binds TM and requires TM for capping activity. NMR and circular dichroism were used to determine the structure of a peptide containing residues 1-92 of tropomodulin (Tmod1(1-92)) and to define its TM binding site. Tmod1(1-92) is mainly disordered with only one helical region, residues 24-35. This helix forms part of the TM binding domain, residues 1-35, which become more ordered upon binding a peptide containing the N-terminus of an alpha-TM. Mutation of L27 to E or G in the Tmod helix reduces TM affinity. Residues 49-92 are required for capping but do not bind TM. Of these, residues 67-75 have the sequence of an amphipathic helix, but are not helical. Residues 55-62 and 76-92 display negative 1H-15N heteronuclear Overhauser enhancements showing they are flexible. The conformational dynamics of these residues may be important for actin capping activity.     

Structure and anticoagulant activity of a sulfated galactan from the red alga, Gelidium crinale. Is there a specific structural requirement for the anticoagulant action?

Marine red algae are an abundant source of sulfated galactans with potent anticoagulant activity. However, the specific structural motifs that confer biological activity remain to be elucidated. We have now isolated and purified a sulfated galactan from the marine red alga, Gellidium crinale. The structure of this polysaccharide was determined using NMR spectroscopy. It is composed of the repeating structure -4-alpha-Galp-(1-->3)-beta-Galp1--> but with a variable sulfation pattern. Clearly 15% of the total alpha-units are 2,3-di-sulfated and another 55% are 2-sulfated. No evidence for the occurrence of 3,6-anhydro alpha-galactose units was observed in the NMR spectra. We also compared the anticoagulant activity of this sulfated galactan with a polysaccharide from the species, Botryocladia occidentalis, with a similar saccharide chain but with higher amounts of 2,3-di-sulfated alpha-units. The sulfated galactan from G. crinale has a lower anticoagulant activity on a clotting assay when compared with the polysaccharide from B. occidentalis. When tested in assays using specific proteases and coagulation inhibitors, these two galactans showed significant differences in their activity. They do not differ in thrombin inhibition mediated by antithrombin, but in assays where heparin cofactor II replaces antithrombin, the sulfated galactan from G. crinale requires a significantly higher concentration to achieve the same inhibitory effect as the polysaccharide from B. occidentalis. In contrast, when factor Xa instead of thrombin is used as the target protease, the sulfated galactan from G. crinale is a more potent anticoagulant. These observations suggest that the proportion and/or the distribution of 2,3-di-sulfated alpha-units along the galactan chain may be a critical structural motif to promote the interaction of the protease with specific protease and coagulation inhibitors.     

EGb761 Pretreatment Reduces Monoamine Oxidase Activity in Mouse Corpus Striatum During 1-Methyl-4-Phenylpyridinium Neurotoxicity

EGb761 produces reversible inhibition of both monoamine oxidase (MAO) isoforms in the central nervous system. 1-Methyl-4-phenylpyridinium (MPP+) neurotoxicity is prevented by treatment with the MAO inhibitor pargyline. We investigated EGb761's effect on striatal MAO activity during MPP+ neurotoxicity. C-57 black mice were pretreated with EGb761 (10 mg/kg) daily for 17 days followed by administration of MPP+ (0.72 mg/kg). MPP+ enhanced striatal MAO (30%) activity at 6 h, and EGb761 prevented this effect. MAO-B activity in striatum was enhanced (70%) 6 h after MPP+ administration and was reduced to almost normal levels in EGb761 + MPP+ group compared to MPP+ group. Pretreatment with EGb761 partially prevented (32%) the striatal dopamine-depleting effect of MPP+ and prevented the reduction in striatal tyrosine hydroxylase activity (100%). Results suggest that EGb761 supplements may be effective in reducing MAO activity as well as enhancement in dopamine metabolism, thereby preventing MPP+-neurotoxicity.     

Viable but nonculturable Vibrio cholerae O1 in the aquatic environment of Argentina

In Argentina, as in other countries of Latin America, cholera has occurred in an epidemic pattern. Vibrio cholerae O1 is native to the aquatic environment, and it occurs in both culturable and viable but nonculturable (VNC) forms, the latter during interepidemic periods. This is the first report of the presence of VNC V. cholerae O1 in the estuarine and marine waters of the Rio de la Plata and the Argentine shelf of the Atlantic Ocean, respectively. Employing immunofluorescence and PCR methods, we were able to detect reservoirs of V. cholerae O1 carrying the virulence-associated genes ctxA and tcpA. The VNC forms of V. cholerae O1 were identified in samples of water, phytoplankton, and zooplankton; the latter organisms were mainly the copepods Acartia tonsa, Diaptomus sp., Paracalanus crassirostris, and Paracalanus parvus. We found that under favorable conditions, the VNC form of V. cholerae can revert to the pathogenic, transmissible state. We concluded that V. cholerae O1 is a resident of Argentinean waters, as has been shown to be the case in other geographic regions of the world.     

Modulation of chondrocyte adhesion to collagen by echistatin

Primary chondrocytes from quail embryo epiphysis (quail epiphyseal chondrocytes, QEC) can grow either in suspension or in monolayer. In this study, the adhesion of QEC to collagen II was used as a model to study the regulation of the ligand-binding activity of integrin receptors that allows these cells to undergo a rapid transition from suspension to an adherent state. Preincubation of suspension QEC (QECSP) with the disintegrin echistatin increased by 40% their adhesion to collagen II. An inverse relationship between immobilized collagen density and echistatin-induced increase of chondrocyte adhesion was observed, thus suggesting that the disintegrin acts by increasing the ligand-binding affinity of collagen receptor(s). Further, echistatin activity does not appear to depend upon a direct binding of the disintegrin to collagen receptor(s). In fact, immobilized anti-beta1 antibodies, but not immobilized echistatin, served as effective binding sites for QECSP. Echistatin failed to stimulate chondrocyte adhesion to collagen in the presence of metabolic inhibitors, while an activating anti-beta1 antibody was still effective. Thus, echistatin may promote cell adhesion by interfering with energy-dependent signals that keep the collagen receptor(s) in a low-affinity state. Adhesion experiments performed in the presence of pharmacological inhibitors indicate that phosphatidyl inositol 3-kinase (PI3-K)/protein kinase C (PKC) and protein kinase A (PKA) pathways may transmit opposing signals on chondrocyte adhesion, and that collagen receptors are kept in a low-affinity state by PI3-kinase/PKC signalling. Since echistatin is a high-affinity ligand for alphavbeta3 integrin, the effect of the function-blocking anti-alphavbeta3 antibody LM609 was investigated. Like echistatin, LM609 stimulated chondrocyte adhesion to collagen and failed to support their attachment. Therefore, our data suggest that alphavbeta3-antagonists might regulate the binding activity of the beta1 collagen receptor, which in turn leads to the rapid transition of chondrocytes from suspension to an adherent state.     

<Emphasis Type="Italic">Lepidium meyenii</Emphasis> (Maca) increases litter size in normal adult female mice

Background  

Lepidium meyenii, known as Maca, grows exclusively in the Peruvian Andes over 4000 m altitude. It has been used traditionally to increase fertility. Previous scientific studies have demonstrated that Maca increases spermatogenesis and epididymal sperm count. The present study was aimed to investigate the effects of Maca on several fertility parameters of female mice at reproductive age.     

Alpha-tocopherol improves biochemical and dynamic parameters in cryopreserved boar semen

Cryopreservation is associated with the production of reactive oxygen species which lead to lipid peroxidation of sperm membranes. The objective was to determine an alpha-tocopherol concentration capable of improving the quality of cryopreserved porcine semen. Boar spermatozoa frozen with 200, 500 or 1000 microg/mL alpha-tocopherol were thawed and incubated at 37 degrees C for 4 h. Routine parameters of semen quality, susceptibility to lipid peroxidation 2-thiobarbituric acid (TBARS) and oxygen uptake were evaluated. Motility was higher (P<0.05) in samples treated with different concentrations of alpha-tocopherol up to 2 h of incubation. Viability and acrosome integrity significantly decreased during incubation (no significant differences between treatments). Two hundred micrograms per milliliter alpha-tocopherol protected spermatozoa against lipid peroxidation during incubation, but 1000 microg/mL failed to protect after 2 h of incubation. There was a negative association between TBARS and motility, suggesting that lipid peroxidation affected sperm motility. Both control and 200 microg/mL alpha-tocopherol samples preserved the capacity to generate oxidative energy up to 1 h of incubation. The addition of 200 microg/mL alpha-tocopherol in the semen extender could be useful to preserve boar spermatozoa against the oxidative stress generated by cryopreservation.     

Acceleration of healing,reduction of fibrotic scar,and normalization of tissue architecture by an angiotensin analogue,NorLeu3-A(1-7)

Angiotensin peptides have been demonstrated to modulate cellular proliferation, angiogenesis, and dermal repair. In this report, the effects of an analogue of the active angiotensin peptide angiotensin(1-7), namely norLeu3-angiotensin(1-7) (NorLeu3-A(1-7)), on the healing of epithelial wounds are presented. Three models were used to evaluate the normal (rats) and delayed (diabetic mice) healing responses of full-thickness excision wounds and the healing responses of full-thickness incision wounds (rats). NorLeu3-A(1-7) was superior to the naturally occurring angiotensin peptide angiotensin(1-7) and to Regranex (Ortho McNeil, Somerville, N.J.) (a formulation of recombinant platelet-derived growth factor used clinically for the treatment of diabetic ulcers) in accelerating tissue repair. By day 9 (normal rats) and day 11 (diabetic mice), the differences in the rates of closure of full-thickness excision wounds between NorLeu3-A(1-7) and Regranex were statistically significant (n = 5 per group). Full healing was observed for 60 percent of the diabetic mice treated topically with NorLeu3-A(1-7) by day 18 after injury, at which time full healing of wounds on placebo-treated or Regranex-treated diabetic mice was not observed. In the rat incision model, accelerated healing and reduced gross appearance of scarification were observed. Administration of NorLeu3-A(1-7) reduced fibrosis and scarring in the healing wounds. This action was more pronounced with longer administration of the peptide after injury. In fact, if systemic administration of the peptide (NorLeu3-A(1-7)) was continued during the remodeling phase, then the formation of new adnexal structures at the center of full-thickness excision wounds was observed, with an increase in the appearance of small immature hair follicles at the sites of the excision wounds. The action of this peptide was blocked by the AT receptor antagonist d-Ala7-angiotensin(1-7), which suggests that this receptor is involved in the healing responses to exogenous NorLeu3-A(1-7). These data suggest that this novel angiotensin peptide has the potential to be of benefit in accelerating wound repair and reducing scar formation.