Differential expression of protein phosphatase type 1 isotypes and nucleolin during cell cycle arrest

In the present study, we examined the expression and cytolocalization of protein phosphatase type 1 (PP1) isoforms and nucleolin in human osteoblastic cell line MG63 cells at two boundaries in the cell cycle. We treated MG63 cells with hydroxyurea and nocodazole to arrest the cells at the G(1)/S and G(2)/M boundaries, respectively. As judged from the results of Western blot analysis, PP1 isoforms were expressed differently at each boundary of the cell cycle. Nucleolin was also shown to have a different expression pattern at each boundary. In the hydroxyurea-treated cells, nucleolus-like bodies were bigger in size and decreased in number compared with those in asynchronized cells. However, the subcellular localization of PP1s and nucleolin was not changed. Anti-nucleolin antibody interacted with 110-kDa and 95-kDa proteins present in asynchronized cells and in the cells treated with hydroxyurea. Treatment of the cells with nocodazole decreased the level of the 95-kDa form of nucleolin. In the nocodazole-treated cells, it was impossible to distinguish the distribution of each protein. The phosphorylation status of nucleolin in the cell cycle arrested samples was examined by 2D-IEF-PAGE followed by Western blot analysis. In the case of asynchronized cells or hydroxyurea-treated ones, nucleolin was located at a basic isoelectric point (dephosphorylated status); whereas in the G(2)/M arrest cells, the isoelectric point of nucleolin shifted to an acidic status, indicating that nucleolin was phosphorylated. The present results indicate that PP1 and nucleolin were differently expressed at G(1)/S and G(2)/M boundaries of the cell cycle and acted in a different fashion during cell-cycle progression.     

Citric acid production by a novel autochthonous Candida zeylanoides isolate: optimization of process parameters

Biotechnology Letters - In this study, citric acid (CA) production by autochthonous Candida zeylanoides 7.12 was investigated and optimized. Response surface methodology (RSM) was used for...     

<Emphasis Type="Italic">In vitro</Emphasis> propagation from young and mature explants of thyme (<Emphasis Type="Italic">Thymus vulgaris</Emphasis> and <Emphasis Type="Italic">T. longicaulis</Emphasis>) resulting in genetically stable shoots

An efficient in vitro propagation protocol, applicable both to young and mature explants of two Thymus spp., results in genetically stable plantlets. In vitro-grown shoot tips of Thymus vulgaris L. were exposed to cytokinins (6-benzyladenine, kinetin, and thidiazuron) alone or in combination with auxins, gibberellic acid (GA₃) and/or silver nitrate in order to optimize in vitro shoot proliferation. Optimum shoot proliferation (97% regeneration rate, with 8.6 shoots produced per explant) was obtained when semi-solid Murashige and Skoog (MS) medium was supplemented with 1 mg L⁻¹ kinetin and 0.3 mg L⁻¹ GA₃. Rooting of the shoots was easily obtained on semi-solid MS medium that was either hormone-free or supplemented with auxins. However, the best root apparatus (92.5% rooting rate, with 19 adventitious roots per shoot) developed on MS medium supplemented with 0.05 mg L⁻¹ 2,4-dichlorophenoxyacetic acid. Genetic stability was confirmed in the in vitro-germinated mother plant as well as the shoots that underwent two, four, six, eight, or ten cycles of in vitro subculturing by random amplified polymorphic DNA (RAPD) analysis. When applied to the micropropagation of mature shoot tips of T. longicaulis C. Presl subsp. longicaulis var. subisophyllus (Borbás) Jalas, the optimized in vitro propagation protocol resulted in a 97.5% shoot regeneration rate, with five shoots formed per explant, and 100% rooting. Rooted plantlets of both species were transferred to 250-mL plastic pots and successfully acclimatized by gradually reducing the relative humidity.     

Ala-9Val polymorphism of Mn-SOD gene in sickle cell anemia

Oxidative stress may be contributory to the pathophysiology of the abnormalities that underlie the clinical course of sickle cell anemia. We looked for a possible genetic association between the functional polymorphism Ala-9Val in the human Mn-SOD gene and sickle cell anemia. One hundred and twenty-seven patients with sickle cell anemia and 127 healthy controls were recruited into the study. Alanine versus valine polymorphism in the signal peptide of the Mn-SOD gene was evaluated using a primer pair to amplify a 107-bp fragment followed by digestion with the restriction enzyme NgoMIV. In the sickle cell anemia patients, the frequency of Val/Val genotype was approximately 1.4-fold lower and that of Ala/Val was 1.3-fold higher compared to the controls. No significant difference in genotype frequencies was found between patients and controls (χ(2) = 4.561, d.f. = 2, P = 0.101). The Val-9 was the most common allele in patient and healthy subjects. No significant difference in allele frequencies was found between patients and controls (χ(2) = 1.496, d.f. = 1, P = 0.221). We conclude that the Mn-SOD gene polymorphism is not associated with sickle cell anemia.     

Biological control agents for white grubs (Coleoptera: Scarabaeidae) in anticipation of the establishment of the Japanese beetle in California

We tested biological control agents for the control of 3rd-instar scarab turfgrass pests, both for the masked chafer Cyclocephala hirta LeConte and the Japanese beetle, Popillia japonica Newman. The former species is endemic in California whereas the latter, although not yet established, constitutes a permanent serious threat to agriculture and horticulture in California. We conducted experiments using C. hirta in California and P. japonica in New Jersey. A field trial conducted in 2 different California turfgrass sites compared the field persistence in the absence of hosts of Bacillus thuringiensis Berliner subspecies japonensis Buibui strain, the milky disease bacterium, Paenibacillus (=Bacillus) popilliae (Dutky), and the entomopathogenic nematodes Steinernema kushidai Mamiya and Heterorhabditis bacteriophora Poinar to that of the organophosphate diazinon. Soil samples taken 0-70 d after applications were bio-assayed with P. japonica. Only diazinon and the entomopathogenic nematode S. kushidai caused substantial mortality and S. kushidai activity persisted significantly longer than diazinon activity. In greenhouse experiments, combinations of entomopathogenic nematode species usually resulted in additive mortality of scarab larvae. Combinations of S. kushidai and diazinon also resulted in additive mortality. In field trials, the efficacy of H. bacteriophora and especially S. kushidai and S. glaseri, was comparable to that of diazinon over 14-18 d. However, it is likely that at least S. kushidai would have outperformed diazinon over an extended period because of its longer persistence and potential for recycling in the hosts. S. kushidai, should it become commercially available, deserves further examination as an alternative to chemical white grub control especially as a highly compatible component of sustainable turfgrass management.     

Genetic differentiation between clone collections and natural populations of European black poplar (<Emphasis Type="Italic">Populus nigra</Emphasis> L.) in turkey

The European black poplar (Populus nigra L.) is an ecologically and economically important tree species for Turkey. The important and major genetic resources of species for future breeding and ex situ conservation purposes have been archived in a clone bank in Ankara by selecting clones from natural populations and old plantations throughout Turkey. There is no study to date assessing genetic composition these materials. Two-hundred-thirty-three P. nigra clones from six geographic region of Turkey (clone collection populations), and 32 trees from two natural populations (Tunceli and Melet) were genotyped by using 12 nuclear microsatellite DNA markers. There were nine clones which duplicated in various frequencies. The analysis carried out with removal of the duplicated clones revealed a moderately high genetic diversity in studied populations. The observed heterozygosities ranged from 0.59 in Tunceli natural to 0.69 in Central Anatolia clone collection populations. In general, there was excess of heterozygosity in the studied populations. Populations composed of clone collections were significantly differentiated from natural populations (F _ST = 0.17), while there was little differentiation among those populations in the clone collection (F _ST = 0.03). Two distantly located natural populations with small sizes also differed from each other (F _ST = 0.17). Genetic structure analysis revealed two distinct groups (clone collection vs natural populations) with very high membership values (>92%). Clone collection populations had high level of admixture while natural populations had homogenous genetic structure. The presence of large number of clonal duplication, reduced genetic differentiation, and high level of admixture in clone collection populations indicate that genetic resources of European black poplar were highly degraded through genetic erosion and pollution caused by intensive cultural practices and extensive dispersal of clonal materials. To understand genetic diversity and its structural pattern thoroughly in the six clone collection populations, a further study with extensive and systematic sampling of European black poplar populations in major river ecosystems in Turkey will be useful.     

Covalent labeling of nuclear vitamin D receptor with affinity labeling reagents containing a cross-linking probe at three different positions of the parent ligand: Structural and biochemical implications

Structure-functional characterization of vitamin D receptor (VDR) requires identification of structurally distinct areas of VDR-ligand-binding domain (VDR-LBD) important for biological properties of 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃). We hypothesized that covalent attachment of the ligand into VDR-LBD might alter ‘surface structure’ of that area influencing biological activity of the ligand. We compared anti-proliferative activity of three affinity alkylating derivatives of 1,25(OH)₂D₃ containing an alkylating probe at 1,3 and 11 positions. These compounds possessed high-affinity binding for VDR; and affinity labeled VDR-LBD. But, only the analog with probe at 3-position significantly altered growth in keratinocytes, compared with 1,25(OH)₂D₃. Molecular models of these analogs, docked inside VDR-LBD tentatively identified Ser237 (helix-3: 1,25(OH)₂D₃-1-BE), Cys288 (β-hairpin region: 1,25(OH)₂D₃-3-BE,) and Tyr295 (helix-6: 1,25(OH)₂D₃-11-BE,) as amino acids that are potentially modified by these reagents. Therefore, we conclude that the β-hairpin region (modified by 1,25(OH)₂D₃-3-BE) is most important for growth inhibition by 1,25(OH)₂D₃, while helices 3 and 6 are less important for such activity.