Hee-Moon Park

Controlled delivery of a hydrophilic drug from a biodegradable microsphere system by supercritical anti-solvent precipitation technique.

The purpose of this study was to prepare microspheres loaded with hydrophilic drug, bupivacaine HCl using poly(D,L-lactic-co-glycolic acid) (PLGA) and poly(L-lactic acid) (PLLA). Microspheres were prepared with varying the PLGA/PLLA ratio with two different levels of bupivacaine HCl (5 and 10%) using a supercritical anti-solvent (SAS) technique. Microspheres ranging from 4–10 µm in geometric mean diameter could be prepared, with high loading efficiency. Powder X-ray diffraction (PXRD) revealed that bupivacaine HCl retained its crystalline state within the polymer and was present as a dispersion within the polymer phase after SAS processing. The release of bupivacaine HCl from biodegradable polymer microspheres was rapid up to 4 h, thereafter bupivacaine HCl was continuously and slowly released for at least 7 days according to the PLGA/PLLA ratio and the molecular weight of PLLA.

Inhibitory effect of nikkomycin Z on chitin synthases in Candida albicans.

Nikkomycin Z is a competitive inhibitor of chitin synthases in fungi. It has been reported that it inhibits chitin synthases (Chs) 1 and 3, but not 2, of Saccharomyces cerevisiae. In our study, we found that: (a) nikkomycin Z inhibited all three Chs isozymes of Candida albicans (CaChs). The IC50 value for CaChs1 is 15 µM, for CaChs2 0.8 µM, and for CaChs3 13 µM; (b) nikkomycin Z inhibits vegetative growth of C. albicans differently in different growth media; growth inhibition was observed on Spider and corn meal agar plate, but not on Lees plate; (c) growth inhibition by nikkomycin Z accompanied by the absence of septum and cell wall chitin, which in turn brought about cell lysis. Nikkomycin Z did not lyse cells in Lees media and lysis was partially prevented in the presence of sorbitol as an osmostabilizer in Spider medium. Therefore, nikkomycin Z prevented the formation of septum and cell wall chitin by inhibiting chitin synthase activities in a growth medium‐dependent manner. Copyright

The LAMMER kinase homolog, Lkh1, regulates Tup transcriptional repressors through phosphorylation in Schizosaccharomyces pombe.

Disruption of the fission yeast LAMMER kinase, Lkh1, gene resulted in diverse phenotypes, including adhesive filamentous growth and oxidative stress sensitivity, but an exact cellular function had not been assigned to Lkh1. Through an in vitro pull-down approach, a transcriptional repressor, Tup12, was identified as an Lkh1 binding partner. Interactions between Lkh1 and Tup11 or Tup12 were confirmed by in vitro and in vivo binding assays. Tup proteins were phosphorylated by Lkh1 in a LAMMER motif-dependent manner. The LAMMER motif was also necessary for substrate recognition in vitro and cellular function in vivo. Transcriptional activity assays using promoters negatively regulated by Tup11 and Tup12 showed 6 or 2 times higher activity in the Δlkh1 mutant than the wild type, respectively. Northern analysis revealed derepressed expression of the fbp1+ mRNA in Δlkh1 and in Δtup11Δtup12 mutant cells under repressed conditions. Δlkh1 and Δtup11Δtup12 mutant cells showed flocculation, which was reversed by co-expression of Tup11 and -12 with Ssn6. Here, we presented a new aspect of the LAMMER kinase by demonstrating that the activities of global transcriptional repressors, Tup11 and Tup12, were positively regulated by Lkh1-mediated phosphorylation.

The activation of p38 MAPK primarily contributes to UV-induced RhoB expression by recruiting the c-Jun and p300 to the distal CCAAT box of the RhoB promoter

The Ras-related small GTP-binding protein RhoB is rapidly induced in response to genotoxic stresses caused by ionizing radiation. It is known that UV-induced RhoB expression results from the binding of activating transcription factor 2 (ATF2) via NF-Y to the inverted CCAAT box (-23) of the RhoB promoter. Here, we show that the association of c-Jun with the distal CCAAT box (-72) is primarily involved in UV-induced RhoB expression and p38 MAPK regulated RhoB induction through the distal CCAAT box. UV-induced RhoB expression and apoptosis were markedly attenuated by pretreatment with the p38 MAPK inhibitor. siRNA knockdown of RhoB, ATF2 and c-Jun resulted in decreased RhoB expression and eventually restored the growth of UV-irradiated Jurkat cells. In the reporter assay using luciferase under the RhoB promoter, inhibition of RhoB promoter activity by the p38 inhibitor and knockdown of c-Jun using siRNA occurred through the distal CCAAT box. Immunoprecipitation and DNA affinity protein binding assays revealed the association of c-Jun and p300 via NF-YA and the dissociation of histone deacetylase 1 (HDAC1) via c-Jun recruitment to the CCAAT boxes of the RhoB promoter. These results suggest that the activation of p38 MAPK primarily contributes to UV-induced RhoB expression by recruiting the c-Jun and p300 proteins to the distal CCAAT box of the RhoB promoter in Jurkat cells.

Paenibacillus camelliae sp. nov., isolated from fermented leaves of Camellia sinensis

A novel bacterium, strain blls-2T was isolated from Pu’er tea. The isolate was Gram-positive, endospore-forming motile rod that grew at 15∼42°C and pH 6.0∼10.2. The DNA G+C content was 48.3 mol%, the predominant isoprenoid quinone was MK-7, and the predominant cellular fatty acid was anteiso-C15:0 (54.2%) followed by C16:0 (15.5%) and iso-C16:0 (8.2%). The polar lipid pattern of blls-2T was characterized by the presence of diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylglycerol. Phy-logenetic analysis based on 16S rRNA gene sequence showed that the strain was affiliated within the Paenibacillaceae. The strain was most closely related to Paenibacillus granivorans A30T, with a similarity of 97.1%. Based on the phylogenetic and phenotypic characteristics of strain blls-2T, the isolate is thought to represent a novel taxon in the genus Paenibacillus. The name Paenibacillus camelliae sp. nov. is proposed for the fermented tea isolate; the type strain is blls-2T (= KCTC 13220T= CECT 7361T).

Identification of a gene, SOO1, which complements osmo-sensitivity and defect in in vitro β1,3-glucan synthase activity in Saccharomyces cerevisiae

The soo1-1 (for suppressor of osmo-sensitivity) mutation results in a temperature-dependent osmo-sensitive phenotype at non-permissive temperature (37 degrees C), makes yeast cells more susceptible to Zymolyase and affects in vitro beta1,3-glucan synthase activity level. Transformation of soo1-1 mutant by SOO1 gene reverses these effects. Nucleotide sequencing of SOO1 revealed that this gene is identical to the recently reported alpha-COP that is involved in the intracellular protein translocation from endoplasmic reticulum to Golgi and vice versa. Although the soo1-1 mutant strain showed osmotically remediable growth at 37 degrees C, SOO1 disruptants could not grow in any culture conditions. Analysis of cell wall fractions revealed that the soo1-1 mutation causes a decrease in in vitro beta1,3-glucan synthase activity, and, thus, a subsequent alteration of the cell wall composition.

RNA-binding protein Csx1 is phosphorylated by LAMMER kinase, Lkh1, in response to oxidative stress in Schizosaccharomyces pombe

Recent studies have shown that global gene expression during oxidative stress in Schizosaccharomyces pombe is regulated by stress‐induced activation and binding of Csx1 to atf1 + mRNA. However, the kinase responsible for the activation of Csx1 has not been identified. Here, we describe, for the first time, that Csx1 is phosphorylated by S. pombe LAMMER kinase, Lkh1, under oxidative conditions and that the stress‐activated binding of the Csx1 to the atf1 + mRNA was also affected by Lkh1 and Spc1. These data indicate that concerted actions of Spc1 and Lkh1 are required for the activation of Csx1 during oxidative condition in the fission yeast S. pombe.

Gryllotalpicola gen. nov., with descriptions of Gryllotalpicola koreensis sp. nov., Gryllotalpicola daejeonensis sp. nov. and Gryllotalpicola kribbensis sp. nov. from the gut of the African mole cricket, Gryllotalpa africana, and reclassification of Curtobacterium ginsengisoli as Gryllotalpicola ginsengisoli comb. nov.

Strains RU-16(T), RU-28, RU-04(T) and PU-02(T) were isolated from the gut of the African mole cricket, Gryllotalpa africana. Phylogenetic analyses based on 16S rRNA gene sequences revealed that the strains belonged to the family Microbacteriaceae. All four strains were most closely related to Curtobacterium ginsengisoli DCY26(T) (below 97% 16S rRNA gene sequence similarity). These isolates were Gram-stain-positive, motile (by gliding), rod-shaped and exhibited ivory-coloured colonies. Their chemotaxonomic properties included MK-11 as the major respiratory quinone, ornithine as the cell-wall diamino acid, acetyl as the acyl type of the peptidoglycan, cyclohexyl-C(17:0) as the major fatty acid and phosphatidylglycerol and diphosphatidylglycerol as the major polar lipids. On the basis of phenotypic, chemotaxonomic and phylogenetic analyses, we propose a new genus in the family Microbacteriaceae, Gryllotalpicola gen. nov., with three novel species, Gryllotalpicola daejeonensis sp. nov. (type strain RU-04(T)  =KCTC 13809(T)  =JCM 17590(T)), Gryllotalpicola koreensis sp. nov. (type strain RU-16(T)  = KCTC 13810(T)  =JCM 17591(T)) and Gryllotalpicola kribbensis sp. nov. (type strain PU-02(T)  =KCTC 13808(T)  =JCM 17593(T)). Gryllotalpicola koreensis is the type species of the genus. Additionally, we propose that Curtobacterium ginsengisoli should be reclassified in the genus as Gryllotalpicola ginsengisoli comb. nov. (type strain DCY26(T)  =KCTC 13163(T)  = JCM 14773(T)).

Small heat-shock protein Hsp9 has dual functions in stress adaptation and stress-induced G2-M checkpoint regulation via Cdc25 inactivation in Schizosaccharomyces pombe.

The small heat-shock protein Hsp9 from Schizosaccharomyces pombe was previously reported to be a homologue of Saccharomyces cerevisiae HSP12. Although Hsp9 is expressed in response to heat shock and nutritional limitation, its function is still not completely understood. Here, we explored the biological function of Hsp9 in S. pombe. The hsp9 gene might play a role in stress adaptation; hsp9 deletion caused heat sensitivity and overexpression induced heat tolerance. In addition, Hsp9 also contribute to cell cycle regulation in the nucleus. Δhsp9 cells grew more quickly and were shorter in length than wild-type cells. Moreover, Δhsp9 cells did not achieve checkpoint arrest under stress conditions, leading to cell death, and exhibited a short doubling time and short G2 phase. Overexpression of hsp9 induced cell cycle delay, increased the population of G2 phase cells, and rescued the phenotypes of cdc2-33, cdc25-22, Δrad24, and Δrad25 mutants, suggesting that Hsp9 probably regulates Cdc2 phosphorylation by modulating the Cdc25 activity. Indeed, immunoprecipitation experiments revealed that Hsp9 is associated with 14-3-3 and Cdc25. In Δhsp9 cells, the association of 14-3-3 with Cdc25 was weakened and Cdc2 phosphorylaton was reduced. Together, our data suggest that Hsp9 has dual functions in stress adaptation and regulating a G2-M checkpoint by the Cdc25 inactivation; this differs from S. cerevisiae HSP12, which maintains cell membrane stability under stress conditions.

LAMMER Kinase LkhA Plays Multiple Roles in the Vegetative Growth and Asexual and Sexual Development of Aspergillus nidulans

LAMMER kinase plays pivotal roles in various physiological processes in eukaryotes; however, its function in filamentous fungi is not known. We performed molecular studies on the function of the Aspergillus nidulans LAMMER kinase, LkhA, and report its involvement in multiple developmental processes. The gene for LkhA was highly expressed during reproductive organ development, such as that of conidiophores and cleistothecia. During vegetative growth, the patterns of germ tube emergence and hyphal polarity were changed and septation was increased by lkhA deletion. Northern analyses showed that lkhA regulated the transcription of brlA, csnD, and ppoA, which supported the detrimental effect of lkhA-deletion on asexual and sexual differentiation. LkhA also affected expression of cyclin-dependent kinase NimXcdc2, a multiple cell cycle regulator, and StuA, an APSES family of fungal transcription factors that play pivotal roles in multiple differentiation processes. Here, for the first time, we present molecular evidence showing that LAMMER kinase is involved in A. nidulans development by modulating the expression of key regulators of developmental processes.