Cloning and characterization of the HPr kinase/phosphorylase gene from Bacillus stearothermophilus No. 236

The Bacillus stearothermophilus no. 236 gene encoding the bifunctional enzyme HprK/P, the key regulator of carbon catabolite repression/activation (CCR/CCA) in most Gram-positive bacteria, was cloned and the (His)(6)-tagged gene product was characterized in detail. The nucleotide sequence of the hprK/P gene corresponded to an open reading frame of 951 bp that encoded a polypeptide of 316 amino acid residues with a calculated molecular mass of 35,458 Da. The deduced amino acid sequence of the B. stearothermophilus no. 236 HprK/P showed 64.5% identity with the B. subtilis enzyme, allowing us to identify two highly conserved motifs, the nucleotide binding P-loop (Walker motif A) and the HprK/P family signature sequence in the C-terminal half of the protein. Furthermore, complementation experiments showed that the cloned hprK/P gene product was functionally active in the B. subtilis cells. The purified (His)(6)-tagged B. stearothermophilus no. 236 HprK/P migrated on SDS-PAGE gel as a single species with a molecular mass of about 36 kDa, and behaved in gel filtration like a hexameric protein. The recombinant protein catalyzes the pyrophosphate (PPi)-dependent (highest activity at pH 7.0 and 40 degrees C) as well as the ATP-dependent phosphorylation of Ser46 in HPr (maximum activity at pH 8.0 and 45 degrees C). It also catalyzes the inorganic phosphate-dependent dephosphorylation (phosphorolysis) of seryl-phosphorylated HPr, optimally at pH 6.5 and 40 degrees C. BIAcore surface resonance analysis confirmed that a divalent cation, preferentially Mg(2+), was an indispensable cofactor for the three activities of the HprK/P. Fructose-1,6-bisphosphate (FBP) was observed to stimulate ATP-dependent kinase activity, while inorganic phosophate (Pi) inhibited ATP-dependent kinase activity. Mutations in the Walker motif A simultaneously abolished both types of kinase and phosphorylase activities. On the other hand, the conserved signature residues were confirmed to be involved in the PPi-dependent kinase and phosphorylase reactions.     

Analysis of tissue-specific expression of human type II collagen cDNA driven by different sizes of the upstream region of the beta-casein promoter

To investigate the ability of 1.8 kb or 3.1 kb bovine beta-casein promoter sequences for the expression regulation of transgene in vivo, transgenic mice were produced with human type II collagen gene fused to 1.8 kb and 3.1 kb of bovine beta-casein promoter by DNA microinjection. Five and three transgenic founder mice were produced using transgene constructs with 1.8 kb and 3.1 kb of bovine beta-casein promoters respectively. Founder mice were outbred with the wild type to produce F1 and F2 progenies. Total RNAs were extracted from four tissues (mammary gland, liver, kidney, and muscle) of female F1 transgenic mice of each transgenic line following parturition. RT-PCR and Northern blot analysis revealed that the expression level of transgene was variable among the transgenic lines, but transgenic mice containing 1.8 kb of promoter sequences exhibited more leaky expression of transgene in other tissues compared to those with 3.1 kb promoter. Moreover, Western blot analysis of transgenic mouse milk showed that human type II collagen proteins secreted into the milk of lactating transgenic mice contained 1.8 kb and 3.1 kb of bovine beta-casein promoter. These results suggest that promoter sequences of 3.1 kb bovine beta-casein gene can be used for induction of mammary gland-specific expression of transgenes in transgenic animals.     

Comparative analysis of two biliproteins, BP1 and BP2, from haemolymph of cabbage white butterfly, Pieris rapae

Two blue-pigment binding proteins, BP1 and BP2, are present in larval and pupal haemolymph of cabbage white butterfly, Pieris rapae, and fluctuate in expression during development. Both BP1 and BP2 are found in pupal haemolymph in varying proportions as well as in adult haemolymph, while only small amounts of BP2 are found in larval haemolymph. BPs are separated by 75% ammonium sulfate, and then purified effectively by ion exchange column chromatography and preparative gel electrophoresis. It was shown that BP1 and BP2 have molecular masses of 20,244 and 19,878 Da, and isoelectric points of 7.0 and 6.8, respectively. Considering their amino acid compositions and N-terminal amino acid sequences, the two proteins are almost identical except the first N-terminal amino acid. The first amino acid of BP1 is asparagine, whereas the initial residue of BP2 is aspartic acid. Anti-BP1 cross-reacts with BP2, indicating that they have immunological homogeneity. Western blotting analyses revealed that only BP1 was present in the larval tissues such as fat body, integument, muscle, and hindgut. However, BP1 was not found in midgut, Malphigian tubules, and silk gland. BP1 was also present in the protein bodies, and both cuticle and hemocoel sides of larval epidermis cells by the transmission electron microscopic observation. The information in this report will facilitate studies on the molecular biology and biological significance of insect BPs.     

Histone deacetylase 1-mediated histone modification regulates osteoblast differentiation

Osteogenesis is a complex process associated with dramatic changes in gene expression. To elucidate whether modifications in chromatin structure are involved in osteoblast differentiation, we examined the expression levels of histone deacetylases (HDACs) and the degree of histone acetylation at the promoter regions of osteogenic genes. During osteogenesis, total HDAC enzymatic activity was decreased with significant reduction in HDAC1 expression. Consistently, recruitment of HDAC1 to the promoters of osteoblast marker genes, including osterix and osteocalcin, was down-regulated, whereas histone H3 and H4 were hyperacetylated at those promoters during osteoblast differentiation. Moreover, suppression of HDAC activity with a HDAC inhibitor, sodium butyrate, accelerated osteogenesis by inducing osteoblast marker genes including osteopontin and alkaline phosphatase. Consistently, knockdown of HDAC1 by the short interference RNA system stimulated osteoblast differentiation. Taken together, these data propose that down-regulation of HDAC1 is an important process for osteogenesis.     

Characterization of a fungal strain capable of degrading chlorpyrifos and its use in detoxification of the insecticide on vegetables

A fungal strain capable of utilizing chlorpyrifos as sole carbon and energy sources was isolated from soil by enrichment cultivation approach. The half-lives of degradation (DT₅₀) for chlorpyrifos at concentrations of 1, 10, and 100 mg l⁻¹ by the fungal strain DSP in mineral salt medium were measured to be 2.03, 2.93, and 3.49 days, respectively. Two cell-free extracts [E (1:10) and E (1:20)] from the fungal strain DSP in bran–glucose medium were prepared and used to enhance chlorpyrifos degradation on vegetables. Compared with the controls, the DT₅₀ of chlorpyrifos were reduced by 70.3%, 65.6%, 80.6%, 80.6%, and 86.1%, and by 53.8%, 43.2%, 66.0%, 54.3%, and 67.7% on E (1:20) and E (1:10) treated pakchoi, water spinach, Malabar spinach, haricot beans, and pepper, respectively. The 7-day residual values (R ₇) of chlorpyrifos on E (1:10) treated vegetables were all lower than the corresponding maximum residue levels of European Union (EU MRLs), except that the R ₇ value on haricot beans was slightly higher than the corresponding EU MRLs. The results indicate that cell-free extracts could rapidly degrade chlorpyrifos residues on vegetables.     

Phenol Degradation by Immobilized Cells of Arthrobacter citreus

An aerobic microorganism with an ability to utilize phenol as carbon and energy source was isolated from a hydrocarbon contamination site by employing selective enrichment culture technique. The isolate was identified as Arthrobacter citreus based on morphological, physiological and biochemical tests. This mesophilic organism showed optimal growth at 25°C and at pH of 7.0. The phenol utilization studies with Arthrobacter citreus showed that the complete assimilation occurred in 24 hours. The organism metabolized phenol up to 22 mM concentrations whereas higher levels were inhibitory. Thin layer chromatography, UV spectral and enzyme analysis were suggestive of catechol, as a key intermediate of phenol metabolism. The enzyme activities of phenol hydroxylase and catechol 2,3-dioxygenase in cell free extracts of Arthrobacter citreus were indicative of operation of a meta-cleavage pathway for phenol degradation. The organism had additional ability to degrade catechol, cresols and naphthol. The degradation rates of phenol by alginate and agar immobilized cells in batch fermentations showed continuous phenol metabolism for a period of eight days.     

Protection of Staphylococcus aureus-infected septic mice by suppression of early acute inflammation and enhanced antimicrobial activity by ginsan

Ginsan, an acidic polysaccharide prepared from Panax ginseng, demonstrated multiple immunomodulatory effects in previous studies. This study was conducted to elucidate the antiseptic mechanism induced by ginsan in mice infected with Staphylococcus aureus. When mice were treated with ginsan before the bacterial challenge with S. aureus, they were highly protected from sepsis-induced death. The numbers of S. aureus recovered from ginsan-treated mice were considerably lower than those recovered from nontreated mice. The in vivo depletion of monocytes/macrophages caused more S. aureus to be recovered from the bacteria-infected mice. Nevertheless, mice treated with both etoposide and ginsan were able to maintain an antibacterial activity. In addition, the phagocytic activity of ginsan-treated macrophage against S. aureus was considerably enhanced. The synthesis of inflammatory cytokines, such as tumor necrosis factor-alpha interleukin (IL)-1beta, IL-6, IFN-gamma, IL-12, IL-18 and interferon gamma, was significantly downregulated at the early phase of sepsis in mice that were treated with ginsan before the bacterial challenge. Expression of Toll-like receptors (TLRs), including TLR2, TLR4, and TLR9, as well as the adaptor molecule MyD88, was considerably reduced in peritoneal macrophages that were treated with ginsan before a subsequent contact with S. aureus. These data indicated that ginsan protected mice from S. aureus-induced sepsis through the suppression of acute inflammatory responses at an early phase and the enhancement of antimicrobial activities at subsequent phases of infection.     

High glucose increase cell cycle regulatory proteins level of mouse embryonic stem cells via PI3-K/Akt and MAPKs signal pathways

This study examined the effects of high glucose on cell proliferation and its related signal pathways using mouse embryonic stem (ES) cells. Here, we showed that high glucose level significantly increased [3H]thymidine incorporation, BrdU incorporation, the number of cells, [3H]leucine, and [3H]proline incorporation in a time-( >3 hr) and dose-(> 25 mM) dependent manner. Moreover, high glucose level increased the cellular reactive oxygen species (ROS), Akt, and mitogen-activated protein kinases (MAPKs) phosphorylation. Subsequently, these signaling molecules involved in high glucose-induced increase of [3H]thymidine incorporation. High glucose level also increased cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK 4 protein levels, which is cell cycle regulatory proteins acting in G1-S phase of cell cycle. Inhibition of phosphatidylinositol 3-kinase (PI3-K) (LY 294002: PI3-kinase inhibitor, 10(-6) M), Akt (Akt inhibitor, 10(-5) M), and p44/42 MAPKs (PD 98059: MEK inhibitor, 10(-5) M) decreased these proteins. High glucose level phosphorylated the RB protein, which was decreased by inhibition of PI3-K and Akt. In conclusion, high glucose level stimulates mouse ES cell proliferation via the PI3-K/Akt and MAPKs pathways.     

Developmental regulation of K accumulation in pollen, anthers, and papillae: are anther dehiscence, papillae hydration, and pollen swelling leading to pollination and fertilization in barley (Hordeum vulgare L.) regulated by changes in K concentration?

The presence of potassium (K) in pollen, anthers and papillaefrom barley (Hordeum vulgare L.) flowers with different levelsof developmental stages starting from boot stage to fully matureflower, was studied by using the K-sensitive fluorescent dyePBFI (potassium-binding benzofuran isophthalate) and confocallaser scanning microscopy. The presence of heavy K fluorescencewas detected only at the aperture area of the mature pollen.Similarly, the presence of K increased with the progressionfrom immature to mature anther and papillae. In addition, ahigher concentration of K was observed only at the stomium area(the place of anther dehiscence) of mature anthers. Keepingin view the role of K as an active osmoticum and the consistentand synchronized appearance of K in mature pollen, anthers,and papillae, it was concluded that K may regulate anther dehiscence,pollen imbibition, and papillae hydration leading to pollinationand fertilization. Key words: Anther, confocal microscopy, papillae, PBFI, pollen, potassium