Levels of genetic diversity at different stages of the domestication cycle of interior spruce in British Columbia

Concerns over the reductionist nature of the domestication of forest-tree species focus on the possibility of potential genetic erosion during this process. To address these concerns, genetic diversity assessments in a breeding zone the Province of British Columbia “interior” spruce (Picea glauca×engelmanni) program was conducted using allozyme markers. Genetic-variation comparisons were made between natural and production (seed orchard) populations as well as seed and seedling crops produced from the same breeding zone’s seed orchard. The natural population sample consisted of a total of 360 trees representing three stands within each of three watersheds present in the Shuswap-Adams low-elevation zone of interior British Columbia. Small amounts of genetic differentiation were observed among the nine natural populations (4%) and this was attributable to extensive gene flow Consequently, the sum of these nine populations was considered as a baseline for the genetic variation present in the breeding zone. The comparisons between the seed orchard and the breeding zone produced a similar percentage of polymorphic loci while the expected hetrozygosity (0.207 vs 0.210) and the average number of alleles per locus (2.7 vs 2.4) were slightly lower in the seed orchard. A total of seven natural populations’ rare alleles were not present in the orchard population, while one allele was unique to the orchard. The %P increased to 70.6% in the seedlot, but dropped to the natural populations level (64.7%) in the plantation. The observed increase in %P was a result of pollen contamination in the orchard. It is suspected that the reduction in the plantation was caused by an unintentional selection in the nursery. Simulated roguing in the orchard did not drastically reduce even if up to 50% of the orchard’s clones were rogued. However, roguing was associated with a reduction in the average number of alleles per locus (i.e., sampling effect). Received: 2 January 1996 / Accepted: 24 May 1996     

Murine Cytomegalovirus m02 Gene Family Protects against Natural Killer Cell-Mediated Immune Surveillance

The murine cytomegalovirus m02 gene family encodes putative type I membrane glycoproteins named m02 through m16. A subset of these genes were fused to an epitope tag and cloned into an expression vector. In transfected and murine cytomegalovirus-infected cells, m02, m04, m05, m06, m07, m09, m10, and m12 localized to cytoplasmic structures near the nucleus, whereas m08 and m13 localized to a filamentous structure surrounding the nucleus. Substitution mutants lacking the m02 gene (SMsubm02) or the entire m02 gene family (SMsubm02-16) grew like their wild-type parent in cultured cells. However, whereas SMsubm02 was as pathogenic as the wild-type virus, SMsubm02-16 was markedly less virulent. SMsubm02-16 produced less infectious virus in most organs compared to wild-type virus in BALB/c and C57BL/6J mice, but it replicated to wild-type levels in the organs of immunodeficient gamma(c)/Rag2 mice, lacking multiple cell types including natural killer cells, and in C57BL/6J mice depleted of natural killer cells. These results argue that one or more members of the m02 gene family antagonize natural killer cell-mediated immune surveillance.     

The relation between hip impact velocity and hip impact force differs between sideways fall techniques.

Fall techniques that reduce fall severity may decrease the risk of hip fractures. A fundamental variable for fall severity is impact force, but impact velocity is also used. The purpose of the study was to determine whether impact velocity is valid to determine differences in fall severity between different techniques. Five young adults with martial arts (MA) experience performed sideways falls from kneeling height using three techniques: Block with arm (Block) and MA techniques with and without use of the arm to break the fall. In addition, one subject also performed MA falls from standing height. Linear regression analysis showed a moderate relation between hip impact velocity and force, which was depended on technique. In falls with comparable impact velocities, forces in MA falls were lower than forces in Block falls. Hence, differences in impact force could not be predicted by velocity. In conclusion, hip impact velocity may be useful to make an approximate prediction of impact force within fall techniques. However, to determine differences between techniques it was not always a valid predictor. When direct impact force measurements are not possible, methods combining impact velocity with energy estimates before and after impact might be more valid.     

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.