Variation in the mating system of Sitka spruce (Picea sitchensis): evidence for partial assortative mating

Five allozyme polymorphisms were used to analyze the mating system in a Sitka spruce seed orchard in Saanichton, British Columbia. Allelic frequencies differed between the pollen and maternal pools at three of the five loci, with alleles rare in the maternal pool being even rarer in the effective pollen pool. Minor differences in pollen allelic frequencies were observed in the upper vs. lower crown. The multilocus outcrossing rate of the upper crown (t_m = 0.909) exceeded that of the lower crown (t_m = 0.764). Single-locus estimates of the outcrossing rate were significantly heterogeneous, with the lowest estimate of outcrossing, t = 0.773, observed for PGM-2 locus. Analyses of the mating system for the three maternal PGM-2 genotypes revealed heterogeneous pollen allelic frequencies and heterogeneous outcrossing rates, possibly due to assortative mating at this locus.     

Isolated cytoskeletons of human blood platelets: dark-field imaging of coiled and uncoiled microtubules

Detergent extraction of human blood platelets pre-treated with Taxol to stabilize microtubules allows isolation of marginal band (MB) cytoskeletons. We studied MB cytoskeleton structure using dark-field light microscopy and negative stain electron microscopy (EM). Dark-field illumination clearly demonstrated the "hoop" shape of MB cytoskeletons in unfixed suspensions where the microtubule coils had a mean diameter of 2.87 microns (+/- 0.18 micron, SD). Microtubules were uncoiled by brief exposure to trypsin (2 ng/micrograms protein) or by NaCl (154-600 mM) but not by DNase I, which removed approximately 40% of total actin, but had no effect on dark-field images of microtubule coils. As microtubules uncoiled, a single fiber emerged from the hoop and gradually lengthened as the brightness of the hoop diminished; these fibers correspond to the single microtubules seen by EM. Polypeptides of coiled and uncoiled MB cytoskeletons were analyzed by SDS-PAGE. When microtubules became uncoiled, no changes in the major components (alpha- and beta-tubulin, IEF-51K, or actin) were found. However, a number (greater than 10) of minor polypeptides, each less than 5% of total cytoskeletal protein and with an Mr ranging from 80,000- greater than 260,000, were decreased in "uncoiled" MB cytoskeletons. These results implicate one or more of these minor polypeptides in maintenance of hoop integrity. Dark-field light microscopy thus provides an approach toward investigating the mechanism(s) involved in maintaining the microtubule coil of the platelet marginal band.     

In vivo inactivation of glycerol dehydrogenase in Klebsiella aerogenes: properties of active and inactivated proteins.

Glycerol:oxidized nicotinamide adenine dinucleotide (NAD+) 2-oxidoreductase (EC 1.1.1.6), an inducible enzyme for anaerobic glycerol catabolism in Klebsiella aerogenes, was purified and found to have a molecular weight of 79,000 by gel electrophoresis. The protein seemed to be enzymatically active either as a dimer of a 40,000-dalton peptide at pH 8.6 or as a tetramer of 160,000 molecular weight at pH 7.0. The enzyme activity was present at high levels in cells growing anaerobically on glycerol, but disappeared with a half-life of about 45 min if molecular oxygen was introduced to the culture. In contrast, no such phenomenon occurred with dihydroxyacetone kinase activity, the second enzyme in the pathway. Immunochemical analysis showed that the inactivation of the oxidoreductase did not involve degradation of the protein. Furthermore, subunits of the active and inactive forms of the enzyme were indistinguishable in size on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and had similar isoelectric points (pH 4.7). Inactivation did, however, alter the gel filtration properties of the enzyme protein and, more importantly, reduced its affinity for the dye Cibacron F3GA and the coenzyme NAD+.     

Elucidating the Crucial Role of Poly N-Acetylglucosamine from Staphylococcus aureus in Cellular Adhesion and Pathogenesis

Staphylococcus aureus is an important pathogen that forms biofilms on the surfaces of medical implants. Biofilm formation by S. aureus is associated with the production of poly N-acetylglucosamine (PNAG), also referred to as polysaccharide intercellular adhesin (PIA), which mediates bacterial adhesion, leading to the accumulation of bacteria on solid surfaces. This study shows that the ability of S. aureus SA113 to adhere to nasal epithelial cells is reduced after the deletion of the ica operon, which contains genes encoding PIA/PNAG synthesis. However, this ability is restored after a plasmid carrying the entire ica operon is transformed into the mutant strain, S. aureus SA113Δica, showing that the synthesis of PIA/PNAG is important for adhesion to epithelial cells. Additionally, S. carnosus TM300, which does not produce PIA/PNAG, forms a biofilm and adheres to epithelial cells after the bacteria are transformed with a PIA/PNAG-expressing plasmid, pTXicaADBC. The adhesion of S. carnosus TM300 to epithelial cells is also demonstrated by adding purified exopolysaccharide (EPS), which contains PIA/PNAG, to the bacteria. In addition, using a mouse model, we find that the abscess lesions and bacterial burden in lung tissues is higher in mice infected with S. aureus SA113 than in those infected with the mutant strain, S. aureus SA113Δica. The results indicate that PIA/PNAG promotes the adhesion of S. aureus to human nasal epithelial cells and lung infections in a mouse model. This study elucidates a mechanism that is important to the pathogenesis of S. aureus infections.     

Ultrastructure of Spermatogenesis in the Testis of Paragonimus heterotremus

Lung fluke, Paragonimus heterotremus, is a flatworm causing pulmonary paragonimiasis in cats, dogs, and humans in Southeast Asia. We examined the ultrastructure of the testis of adult P. heterotremus with special attention to spermatogenesis and spermiogenesis using scanning and transmission electron microscopy. The full sequence of spermatogenesis and spermiogenesis, from the capsular basal lamina to the luminal surface, was demonstrated. The sequence comprises spermatogonia, spermatocytes with obvious nuclear synaptonemal complexes, spermatids, and eventual spermatozoa. Moreover, full steps of spermatid differentiation were shown which consisted of 1) early stage, 2) differentiation stage representing the flagella, intercentriolar body, basal body, striated rootlets, and electron dense nucleus of thread-like lamellar configuration, and 3) growing spermatid flagella. Detailed ultrastructure of 2 different types of spermatozoa was also shown in this study.     

Population structure,population history and DNA barcoding of fruit fly Bactrocera latifrons (Hendel) (Diptera: Tephritidae)

The fruit fly Bactrocera latifrons (Hendel) is an important pest of commercially significant plants such as chili, tomato and eggplant. The species is native to South and Southeast Asia, but has now invaded Japan, Hawaii and Africa. In this study, mitochondrial DNA sequences were used to infer genetic structure and demographic history of B. latifrons. The efficiency of DNA barcodes for identification of B. latifrons was also tested. Ninety‐three specimens infesting four host‐plant species were obtained from 11 sampling locations in Thailand. The mitochondrial haplotype network revealed no major divergent lineage, which was consistent with a phylogenetic analysis that found strong support for the monophyly of B. latifrons. Population pairwise F_ST revealed that most (65%) comparisons were not significantly different, suggesting a high rate of gene flow. Analysis of molecular variance (amova ) found no significant genetic differentiation among populations from different host‐plant species. Sharing of several haplotypes among flies from different host‐plants indicates that the flies were moved freely across the plant species. Demographic history analysis revealed that the population has undergone recent expansion dating back to the end of the last glaciation. Thus, the results indicate that both ongoing and historical factors have played important roles in determining the genetic structure and diversity of B. latifrons. DNA barcoding analysis revealed that B. latifrons specimens were clearly differentiated from other species with 100% correct identification. Therefore, cytochrome oxidase I (COI) barcoding sequences could be effectively used to identify this important pest species, which could encourage monitoring and control efforts for this species.     

Inducible and constitutive HSP70s confer synergistic resistance against metabolic challenges

Chaperonic proteins, including inducible HSP70 (HSP70i) and constitutive HSP70 (HSC70), have been implicated as essential players in the cellular adaptive protection. Ensuing studies demonstrated that overexpression of either protein individually protects against thermal and oxidative challenges. The present study aimed to determine whether a concurrent overexpression of both HSC70 and HSP70i confers a better metabolic protection than the expression of each protein alone. Using a rat heart-derived H9c2 cardiac myoblast cell line, we found that HSP70i was rapidly induced within 2–8 h following a mild thermal preconditioning (43 °C for 20 min) in both parental cells and an established H9/70c clonal sub-line overexpressing HSC70. The level of HSP70i protein in heat pretreated H9/70c clonal cells reached only 50% of that in heat pretreated H9c2 parental cells. Nevertheless, protection against lethal hyperthermia, menadione (an oxidant) and hydrogen peroxide (H₂O₂) exposure in the pretreated H9/70c clonal cells was significantly higher than the sum of protection afforded by the early induction of HSP70i in the pretreated parental cells and protection afforded by the pre-existing HSC70 in the H9/70c cells without preconditioning. Using dosimetric analysis, we also found that menadione resistance in the pretreated parental cells increased linearly with cellular HSP70i level (10–300 ng/mg total protein). However, the resistance in the pretreated H9/70c cells showed a biphasic relationship with cellular HSP70i level; when HSP70i concentration reached >250 ng/mg protein, survivability after menadione exposure was markedly enhanced. Similar results were observed in H9c2 cells genetically manipulated to overexpress both HSC70 and HSP70i. The survival benefit against lethal hyperthermia, oxidant treatment, and hypoxia/reoxygenation conferred by a concerted HSC70 and HSP70i overexpression was greater than the sum of benefits contributed by individual protein overexpression. Together, these findings suggest that HSC70 and HSP70i may complement each other in a synergistic manner to preserve cellular integrity during metabolic challenges.