Actinocatenispora comari sp. nov., an endophytic actinomycete isolated from aerial parts of Comarum salesowianum.

A novel actinomycete, designated NUM-2625T, was isolated as an endophytic bacterium in aerial parts of Comarum salesowianum, an endemic species in the Altai, Himalaya mountain chain area, collected from Khasagt Khairkhan Mountain in Mongolia. The 16S rRNA gene sequence of strain NUM-2625T showed the highest similarity to Actinocatenispora thailandica TT2-10T (99.4 %), Actinocatenispora sera KV-744T (99.3 %), and Actinocatenispora rupis CS5-AC17T (97.7 %). Chemotaxonomic properties of strain NUM-2625T were essentially consistent with those of the genus Actinocatenispora, such as the presence of meso-diaminopimelic acid as the diagnostic diamino acid of the peptidoglycan, MK-9(H4) and MK-9(H6) as the major menaquinones, and iso-C16 : 0, iso-C15 : 0, iso-C14 : 0 3-OH, and anteiso-C17 : 0 as the major fatty acids. Meanwhile, digital DNA-DNA hybridization and average nucleotide identity values revealed a low relatedness between strain NUM-2625T and the other type strains of the genus Actinocatenispora. In addition, strain NUM-2625T exhibited several phenotypic properties that could be used to distinguish it from its closest relatives. Based on the results of polyphasic analyses, strain NUM-2625T represents a novel species in the genus Actinocatenispora, for which the name Actinocatenispora comari sp. nov. is proposed. The type strain is NUM-2625T (=NBRC 114660T=TBRC 13496T).

Structural basis of substrate specificity and regiochemistry in the MycF/TylF family of sugar O-methyltransferases.

Sugar moieties in natural products are frequently modified by O-methylation. In the biosynthesis of the macrolide antibiotic mycinamicin, methylation of a 6'-deoxyallose substituent occurs in a stepwise manner first at the 2'- and then the 3'-hydroxyl groups to produce the mycinose moiety in the final product. The timing and placement of the O-methylations impact final stage C-H functionalization reactions mediated by the P450 monooxygenase MycG. The structural basis of pathway ordering and substrate specificity is unknown. A series of crystal structures of MycF, the 3'-O-methyltransferase, including the free enzyme and complexes with S-adenosyl homocysteine (SAH), substrate, product, and unnatural substrates, show that SAM binding induces substantial ordering that creates the binding site for the natural substrate, and a bound metal ion positions the substrate for catalysis. A single amino acid substitution relaxed the 2'-methoxy specificity but retained regiospecificity. The engineered variant produced a new mycinamicin analog, demonstrating the utility of structural information to facilitate bioengineering approaches for the chemoenzymatic synthesis of complex small molecules containing modified sugars. Using the MycF substrate complex and the modeled substrate complex of a 4'-specific homologue, active site residues were identified that correlate with the 3' or 4' specificity of MycF family members and define the protein and substrate features that direct the regiochemistry of methyltransfer. This classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.

A new structural form in the SAM/metal-dependent o­methyltransferase family: MycE from the mycinamicin biosynthetic pathway.

O-linked methylation of sugar substituents is a common modification in the biosynthesis of many natural products and is catalyzed by multiple families of S-adenosyl-L-methionine (SAM or AdoMet)-dependent methyltransferases (MTs). Mycinamicins, potent antibiotics from Micromonospora griseorubida, can be methylated at two positions on a 6-deoxyallose substituent. The first methylation is catalyzed by MycE, a SAM- and metal-dependent MT. Crystal structures were determined for MycE bound to the product S-adenosyl-L-homocysteine (AdoHcy) and magnesium, both with and without the natural substrate mycinamicin VI. This represents the first structure of a natural product sugar MT in complex with its natural substrate. MycE is a tetramer of a two-domain polypeptide, comprising a C-terminal catalytic MT domain and an N-terminal auxiliary domain, which is important for quaternary assembly and for substrate binding. The symmetric MycE tetramer has a novel MT organization in which each of the four active sites is formed at the junction of three monomers within the tetramer. The active-site structure supports a mechanism in which a conserved histidine acts as a general base, and the metal ion helps to position the methyl acceptor and to stabilize a hydroxylate intermediate. A conserved tyrosine is suggested to support activity through interactions with the transferred methyl group from the SAM methyl donor. The structure of the free enzyme reveals a dramatic order-disorder transition in the active site relative to the S-adenosyl-L-homocysteine complexes, suggesting a mechanism for product/substrate exchange through concerted movement of five loops and the polypeptide C-terminus.

Characteristics of cesium accumulation in the filamentous soil bacterium Streptomyces sp. K202.

A filamentous soil bacterium, strain K202, was isolated from soil where an edible mushroom (Boletopsis leucomelas) was growing and identified as belonging to the genus Streptomyces on the basis of its morphological characteristics and the presence of LL-2, 6-diaminopimelic acid. We studied the existence states of Cs and its migration from extracellular to intracellular fluid in the mycelia of Streptomyces sp. K202. The results indicated that Cs accumulated in the cells through at least 2 steps: in the first step, Cs(+) was immediately and non-specifically adsorbed on the negatively charged cell surface, and in the second step, this adsorbed Cs(+) was taken up into the cytoplasm, and a part of the Cs entering the cytoplasm was taken up by an energy-dependent transport system(s). Further, we confirmed that a part of the Cs(+) was taken up into the mycelia competitively with K(+), because K(+) uptake into the intact mycelia of the strain was significantly inhibited by the presence of Cs(+) in the culture media. This suggested that part of the Cs is transported by the potassium transport system. Moreover, (133)Cs-NMR spectra and SEM-EDX spectra of the mycelia that accumulated Cs showed the presence of at least 2 intracellular Cs states: Cs(+) trapped by intercellular materials such as polyphosphate and Cs(+) present in a cytoplasmic pool.

Functional analysis of MycE and MycF, two O-methyltransferases involved in the biosynthesis of mycinamicin macrolide antibiotics.

Mg motors: We characterized the in vitro function of MycE and MycF, two O-methyltransferases involved in the biosynthesis of mycinamicin antibiotics. Each enzyme was confirmed to be an S-adenosyl-L-methionine (SAM)-dependent deoxysugar methyltransferase. Their optimal activities require the presence of Mg(2+). With the reconstituted in vitro assays, the order of mycinamicin VI-->III-->IV in the post-PKS (polyketide synthase) tailoring pathway of mycinamicin was unambiguously determined.

Accumulation and localization of cesium in edible mushroom (Pleurotus ostreatus) mycelia.

The characteristics of Cs accumulation and localization in edible mushrooms were examined using the mycelia of Pleurotus ostreatus-Y1. Scanning electron microscope images revealed the existence of white spots, and energy dispersive X-ray microanalyzer analysis indicated the presence of larger amounts of Cs and P in these spots in mycelia cultured on medium containing 25 mM CsCl. The (137)Cs activities in the mycelia were approximately 4-6 times higher than those in water used for (137)Cs elution. Higher Cs concentrations in the sediment fraction including vacuolar pellets were obtained compared to the upper fractions. It was observed that yellowish spots caused by the fluorescence of 4',6-diamidino-2-phenylindole (DAPI)-stained polyphosphate were localized in the mycelia. The higher fluorescence intensity of the yellowish-grained spots was measured in comparison with other regions in the mycelium. These results suggested that Cs in the mycelia was trapped by polyphosphate in vacuoles or other organelles.

Ion-channel formation assisted by electrostatic interhelical interactions in covalently dimerized amphiphilic helical peptides.

An ultimate goal of synthetic ion-channel peptide design is to construct stable and functional ion-conducting pores. It is expected that specific interhelical interactions would facilitate the association of helices in phospholipid membranes and the successive helix-bundle formation. In the present study, we rationally designed helix-bundle ion channels using the synthetic hybrid peptide K20E20, a disulfide dimer of cationic- and anionic-amphiphilic helices Ac-CGG-(BKBA) 5-NH 2 and Ac-CGG-(BEBA) 5-NH 2. Circular dichroism (CD) measurements in aqueous media implied helix stabilization in the peptide caused by the interhelical electrostatic interactions. In addition, CD spectra recorded in the presence of DPPC liposomes and dye-leakage measurements suggested a high degree of association of peptide monomers in phospholipid membranes as well as high affinities between peptide and lipid bilayers. These features allowed ion-channel formation at extremely low peptide concentrations (as low as 1 nM). According to electrophysiological analyses, stable helix bundles were constructed of six peptide helices by association of three K20E20 molecules. Helix-helix association in lipid membranes, peptide-membrane interactions, and ion-channel formation of K20E20 peptides were all facilitated by intramolecular electrostatic interactions between the helices of the hybrid peptide and were pH-dependent. Conductance through K20E20 ion channels decreased under acidic conditions because of the interruption of the salt bridges.

The delayed manifestation of T-cell receptor (TCR) variants in X-irradiated mice depends on Trp53 status.

The influence of Trp53 on the radiation-induced elevation of T-cell receptor (TCR) variant fractions was examined in splenic T lymphocytes of Trp53-proficient and -deficient mice. Wild-type Trp53+/+, heterozygous Trp53+/- and null Trp53-/- mice were exposed to 3 Gy of X rays at 8 weeks of age. The fraction of TCR-defective variants was measured at various times after irradiation. Initially, the TCR variant fraction increased rapidly and reached its maximum level at 9 days after irradiation before decreasing gradually. In Trp53+/+ and Trp53+/- mice, the TCR variant fraction fell to normal background levels at 16 and 20 weeks of age, respectively. In contrast, the TCR variant fraction of Trp53-/- mice failed to decrease to background levels during the observation period. Baseline levels were then maintained for approximately 60 weeks in the Trp53+/+ mice and approximately 40 weeks in the Trp53+/- mice. After the long flat period, a significant re-increase in the fraction of TCR variants was found after 72 weeks of age in the irradiated Trp53+/+ mice and after 44 weeks of age in the irradiated Trp53+/- mice. Measurement of the fraction of apoptotic cells in the spleen and thymus 4 h after X irradiation at these ages in Trp53+/+ and Trp53+/- mice demonstrated a reduction in apoptosis in the irradiated mice compared to the nonirradiated mice. This suggests that the delayed increase in TCR variants after irradiation is due to a reduction in Trp53-dependent apoptosis.

Pathoanatomy of posterior malleolar fractures of the ankle.

BACKGROUND: The functional outcome following ankle fractures that involve a posterior malleolar fragment is often not satisfactory, and treatment of this type of fracture remains controversial. Thorough knowledge of the pathologic anatomy of the posterior malleolar fracture is essential for planning appropriate treatment. Thus, we conducted a computed tomographic study to clarify the pathologic anatomy of the posterior malleolar fracture. METHODS: Between 1999 and 2003, fifty-seven consecutive patients with a unilateral ankle fracture with one or more posterior fragments were managed at our hospital. We reviewed the patients' preoperative computed tomographic scans to determine (1) the ratio of the posterior fragment area to the total cross-sectional area of the tibial plafond and (2) the angle between the bimalleolar axis and the major fracture line of the posterior malleolus. Each fracture was categorized according to the location of the major fracture line on the computed tomographic image at the level of the tibial plafond. RESULTS: The fifty-seven fractures were categorized into three types: (1) the posterolateral-oblique type (thirty-eight fractures; 67%), (2) the medial-extension type (eleven fractures; 19%), and (3) the small-shell type (eight fractures; 14%). Two of the eleven medial-extension fractures extended to the anterior part of the medial malleolus. A total of nine of the eleven medial-extension fractures actually consisted of two fragments [corrected] The conditions are not exclusive of one another; for example, in the case of one of the fractures exhibiting two fragments, the fracture also extended to the anterior part of the medial malleolus [corrected] The average area of the fragment comprised 11.7% of the cross-sectional area of the tibial plafond for posterolateral-oblique fractures and 29.8% for medial-extension fractures. In the cases of seven of the nine fractures that comprised >25% of the tibial plafond, the fracture line extended to the medial malleolus. The angles between the bimalleolar axis and the major fracture line of the posterior malleolus varied. CONCLUSIONS: The fracture lines associated with posterior malleolar fractures appear to be highly variable. A large fragment extending to the medial malleolus existed in almost 20% of the posterior malleolar fractures in the current study, and some fragments involved almost the entire medial malleolus. Because of the great variation in fracture configurations, preoperative use of computed tomography may be justified. The information obtained from this study will be helpful for conducting basic research of this condition and for determining appropriate surgical approaches.

[Effect of Cepharanthin to prevent radiation induced xerostomia in head and neck cancer].

We retrospectively examined the effect of Cepharanthin to prevent radiation xerostomia in 37 cases of head and neck cancer. In the Cepharanthin group, the degree of xerostomia was milder than in the non-Cepharanthin group in spite of higher normal tissue complication probability (NTCP) and mean dose (MD) of parotid glands. In the non-Cepharanthin group, the degree of xerostomia was significantly correlated with NTCP and MD of parotid glands. MD of parotid glands and use of Cepharanthin were significantly related to more severe xerostomia by multivariate analysis with logistic regression. Cepharanthin may prevent radiation xerostomia after radiotherapy for head and neck cancer.

[Effect of Cepharanthin in preventing radiation induced normal tissue damage in prostate cancer].

A total of 97 patients with prostate cancer who underwent radiotherapy were retrospectively reviewed to analyze the protective efficacy of Cepharanthin for acute or late toxicity to the bladder/urethra and rectum. Cepharanthin administrations were divided into 3 groups: intravenous, oral, and non-administration. Acute urinary toxicity was significantly milder for the intravenous group than for the oral and non-administration groups. The protective efficacy of Cepharanthin was not approved for acute rectal toxicity, but late rectal toxicity was significantly milder for the intravenous group than for the oral group. Intravenous Cepharanthin administration may prevent acute or late toxicity by radiotherapy for prostate cancer.

Effect of alpha,alpha-dialkyl amino acids on the protease resistance of peptides.

A tryptic [EC 3.4.21.4] digestion assay of 2-aminoisobutyric acid (Aib)-containing peptides was carried out to investigate the effect of alpha,alpha-dialkyl amino acid residues on the protease resistance. The introduction of Aib residues to the P1' positions exhibited a 19-fold higher protease resistance than the peptide with Aib residues introduced to the P2 position or the non-Aib peptide. The peptide having Aib residues introduced to the P1' and P2 positions resulted in complete resistance.

[Results of combined therapy for maxillary sinus squamous cell carcinoma].

The results of 54 cases of maxillary sinus squamous cell carcinoma treated between 1980 and 2002 were analyzed retrospectively. The T classification according to the 1997 UICC was as follows: 2 with stage T1, 29 with T3, and 23 with T4. Ten patients(18.5%) had lymph node metastases at diagnosis. All patients underwent combined therapy including radiotherapy, surgery, and regional or systemic chemotherapy. Fifteen patients received hyperfractionated twice-daily radiotherapy (1.2 Gy or 1.5 Gy/fraction), and the remaining 39 patients received a conventional once-daily regimen(1.5-2 Gy/fraction). The 5-year overall survival and 5-year disease-free survival for all patients were 56.0% and 46.7%, respectively. The N classification was the only significant prognostic factor for 5-year disease-free survival by univariate analysis (favoring N = 0, p = 0.04). There were no significant differences in other prognostic factors including gender, T classification (T1-3 vs. T4), hyperfractionated radiotherapy (yes vs. no), total dose (BED: < 69 Gy10 vs. > or = 69 Gy10), and intra-arterial chemotherapy(yes vs. no). Although radiation-induced cataract was observed in 9 patients, no other severe late complications developed.

Genetic and biochemical characterization of EshA, a protein that forms large multimers and affects developmental processes in Streptomyces griseus.

The 52-kDa protein, EshA, whose expression is controlled developmentally, is produced during the late growth phase of Streptomyces spp. We found that disruption of the eshA gene, which encodes the EshA protein, abolishes the aerial mycelium formation and streptomycin production in Streptomyces griseus when grown on an agar plate. The eshA disruptant KO-390 demonstrated a reduced amount of expression of the transcriptional activator strR, thus accounting for the failure to produce streptomycin. KO-390 was found to accumulate deoxynucleoside triphosphates at high levels, including dGTP, at late growth phase. The accumulation of dGTP was a cause for the impaired ability of KO-390 to produce aerial mycelium, because the ability to form aerial mycelium was completely repaired by addition of decoyinine, an inhibitor of GMP synthetase. The accumulation of dNTP in KO-390 coincided with a reduced rate of DNA synthesis. The developmental time frame of these phenomena in KO-390 matched a burst of EshA expression in the wild-type strain. In contrast to S. griseus, the eshA disruption did not affect the ability for Streptomyces coelicolor to form aerial mycelium and did not result in the aberrant accumulation of dNTP accompanied by arrest of DNA synthesis, implying qualitative differences in addition to quantitative differences between the two EshA proteins. We propose that the S. griseus EshA protein somehow positively affects (or regulates) the replication of DNA in wild-type cells at late growth phase but leads to aberrant phenotypes in mutant cells due to the disturbed DNA replication. The EshA protein was found to exist as a multimer ( approximately 20-mers) creating a cubic-like structure with a diameter of 27 nm and located predominantly in cytoplasm.

Corrective osteotomy for malunited fracture of the glenoid cavity: a case report.

Fractures of the glenoid cavity that are substantially displaced are rare. A patient with shoulder pain and dysfunction caused by a severely malunited fracture of the glenoid cavity was treated successfully with corrective osteotomy and bone grafting. Functional results 2 years after surgery were satisfactory, and radiographs showed no evidence of degenerative change. Although appropriate initial management should prevent the development of symptomatic malunion, results of the current study suggest that later reconstruction of the glenoid cavity restores satisfactory function, even if so much time has elapsed that glenoid osteotomy must be done to achieve reduction.

Role of p53 gene in apoptotic repair of genotoxic tissue damage in mice.

When DNA is damaged by exposure to a small amount of radiation, it is repaired efficiently by innate mechanisms. However, if cellular damage is more extensive, DNA repair cannot be adequately completed. To clarify the role of the p53 gene in apoptotic tissue repair, the incidence of in-vivo radiation-induced somatic mutation was evaluated by measuring the T cell receptor (TCR) gene expression in p53(+/+) and p53(-/-) mice. After gamma-irradiation with 3 Gy, the TCR mutation frequency (MF) was higher in p53(+/+) mice than in the controls. However, when the mice were exposed to 3 Gy at a low dose rate, the TCR MF did not increase in the p53(+/+) mice, whereas it increased and remained elevated in p53(-/-) mice, which are unable to induce apoptosis. In p53(+/+) mice, the TCR MF peaked 9 days after gamma-irradiation with 3 Gy at a high dose rate, and then gradually decreased with a half-life of about 13 days. However, in p53(-/-) mice, the peak level of the TCR MF did not decline significantly with time. Hence, complete repair of mutagenic damage in irradiated tissues requires the integration of DNA repair and p53-dependent apoptotic tissue repair.