Antimicrobial activity and cytotoxicity trait of a bioactive peptide purified from Lactococcus garvieae subsp. bovis BSN307T

A bioactive peptide of 8595 Da was purified from the cell free supernatant of Lactococcus garvieae subsp. bovis BSN307^T. MALDI MS/MS peptide mapping and the data base search displayed no significant similarity to any reported antimicrobial peptide of LAB. This peptide at a dose concentration of 200 µg ml⁻¹ inhibited the growth of both Gram-positive and Gram-negative bacteria by 58–89% and a dose of 500 µg ml⁻¹ scavenged 50% of DPPH-free radicals generated. Interestingly, cytotoxicity assay demonstrated that 17 µg ml⁻¹ of peptide selectively inhibited 50% proliferation of mammalian cancer cell lines HeLa and MCF-7 whereas normal H9c2 cells remained unaffected. Fluorescent microscopic analysis after DAPI nuclear staining of HeLa cells showed characteristics of apoptosis and activation of caspase-3 was ascertained by caspase-3 fluorescence assay.     

Inhibition of GSK-3β Rescues the Impairments in Bone Formation and Mechanical Properties Associated with Fracture Healing in Osteoblast Selective Connexin 43 Deficient Mice

Connexin 43 (Cx43) is the most abundant gap junction protein in bone and is required for osteoblastic differentiation and bone homeostasis. During fracture healing, Cx43 is abundantly expressed in osteoblasts and osteocytes, while Cx43 deficiency impairs bone formation and healing. In the present study we selectively deleted Cx43 in the osteoblastic lineage from immature osteoblasts through osteocytes and tested the hypothesis that Cx43 deficiency results in delayed osteoblastic differentiation and impaired restoration of biomechanical properties due to attenuated β-catenin expression relative to wild type littermates. Here we show that Cx43 deficiency results in alterations in the mineralization and remodeling phases of healing. In Cx43 deficient fractures the mineralization phase is marked by delayed expression of osteogenic genes. Additionally, the decrease in the RankL/ Opg ratio, osteoclast number and osteoclast size suggest decreased osteoclast bone resorption and remodeling. These changes in healing result in functional deficits as shown by a decrease in ultimate torque at failure. Consistent with these impairments in healing, β-catenin expression is attenuated in Cx43 deficient fractures at 14 and 21 days, while Sclerostin (Sost) expression, a negative regulator of bone formation is increased in Cx43cKO fractures at 21 days, as is GSK-3β, a key component of the β-catenin proteasomal degradation complex. Furthermore, we show that alterations in healing in Cx43 deficient fractures can be rescued by inhibiting GSK-3β activity using Lithium Chloride (LiCl). Treatment of Cx43 deficient mice with LiCl restores both normal bone formation and mechanical properties relative to LiCl treated WT fractures. This study suggests that Cx43 is a potential therapeutic target to enhance fracture healing and identifies a previously unknown role for Cx43 in regulating β-catenin expression and thus bone formation during fracture repair.     

Functional analysis of a single chain chimeric alpha/beta-granulocyte-macrophage colony-stimulating factor receptor. Importance of a glutamate residue in the transmembrane region.

To analyze the function of each subunit of the receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF), GMR, we previously generated a single-chain chimeric receptor by fusion of the extracellular and transmembrane domain from the alpha-subunit (alpha-GMR) to the intracellular part of the beta-subunit (beta-GMR) introducing an additional glutamate residue at the fusion site (alpha/beta-GMR). We demonstrated the capacity of alpha/beta-GMR to bind GM-CSF with low affinity and to induce GM-CSF-dependent activation of tyrosine kinase activity and proliferation in transfected Ba/F3 cells. To further compare the functions of wild type and chimeric receptors, we now report that this alpha/beta-GMR is sufficient to mediate morphological changes, expression of alpha(4)- and beta(1)-integrin receptor subunits, and serine-phosphorylation of Akt kinase. To analyze the function of the glutamate residue at the fusion region of alpha/beta-GMR various point mutants changing this amino acid and its position were expressed in Ba/F3 cells. None of these mutants was capable of supporting GM-CSF-dependent proliferation; however, when beta-GMR was coexpressed, GM-CSF mediated short and long term proliferation. Interestingly, some mutants but not alpha/beta-GMR can induce proliferation in the presence of an anti-alpha-GMR antibody. These data demonstrate the significance of a glutamate residue in the transmembrane region of alpha/beta-GMR for ligand-induced receptor activation.     

Promotion by phloroglucinol of micropropagation of Minuartia valentina, an endangered and endemic Spanish plant

Tissue culture techniques for the propagation and conservation of endemic or threatened plants can be used to complement the methods usually applied in ex situ conservation. Thus, Minuartia valentina (Caryophyllaceae), an endangered plant species endemic to the Valencia Community (Eastern Spain), was successfully regenerated through shoot proliferation from wild plants growing in their natural area. Nodal segments, 10~mm long, were cut from rametes of adult material, sterilised and established in vitro. Equally successful shoot multiplication was achieved on Murashige and Skoog (MS) medium with 80 mg l-1 phloroglucinol in combination with either 1 mg l-1 6-benzylaminopurine or 1 mg l-1 kinetin. Excised shoots were rooted in MS medium supplemented with an auxin (indole acetic acid, indole-3-butyric acid, or napththalene acetic acid). Shoots rooted well (96–100%) within three weeks in all auxin treatments. However, the use of napththalene acetic acid was discarded because this auxin delayed root differentiation, and induced adventitious root malformation. Rooted plantlets were transferred to pots and 85% of them acclimatized successfully four weeks after transfer to greenhouse conditions, where they exhibited normal morphology and growth.     

Genetic and evolutionary relationships among Asian Macaques

Published gene frequency data, checked for consistency of allele definitions across laboratories and for comparability of geographically identical samples, were pooled into a data set containing frequencies at nine loci for each of 20 populations that encompassed 10 macaque species. Genetic distances were calculated by the methods of Kidd and Cavalli-Sforza (1974). These distances were used to construct phylogenetic trees and to evaluate the relationships between divergence times and effective population sizes. Inter-and intraspecific genetic distances and the groupings defined by phenetic tree analyses support Fooden’s (1976) classification of the genus Macacainto four species groups. A paleozoogeographical model of Asia including the known times of major sea-level changes allows us to explain Macacainto four species groups. A paleozoogeographical model of Asia including the known times of major sea-level changes allows us to explain qualitatively the inferred evolutionary relationships among macaque species. Many assumptions are required in order to estimate the variables necessary in the quantitative prediction of genetic differences for a comparison between any two populations. Examination of those assumptions demonstrates the need for more accurate genetic as well as paleozoogeographic information. An erratum to this article is available at .     

Comparison of methods for estimating Erwinia carotovora numbers on potato tubers

Two methods for estimation of the numbers of Erwinia carotovora on potato tubers were compared using 17 naturally infected stocks. The 'wash' method estimates the surface count, whereas the 'peel' method is based on a subcuticular count. A correlation of 0.79 was obtained. Comparisons between methods were also made using a bactericide, Myacide As, and on stocks during chitting. Erwinia carotovora counts were reduced in treated tubers with the 'wash' method, but not with the 'peel' method.     

Lactose repressor protein modified with dansyl chloride: activity effects and fluorescence properties

Chemical modification using 5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride) has been used to explore the importance of lysine residues involved in the binding activities of the lactose repressor and to introduce a fluorescent probe into the protein. Dansyl chloride modification of lac repressor resulted in loss of operator DNA binding at low molar ratios of reagent/monomer. Loss of nonspecific DNA binding was observed only at higher molar ratios, while isopropyl beta-D-thiogalactoside binding was not affected at any of the reagent levels studied. Lysine residues were the only modified amino acids detected. Protection of lysines-33 and -37 from modification by the presence of nonspecific DNA correlated with maintenance of operator DNA binding activity, and reaction of lysine-37 paralleled operator binding activity loss. Energy transfer between dansyl incorporated in the core region of the repressor protein and tryptophan-201 was observed, with an approximate distance of 23 A calculated between these two moieties.