Amide-transforming activity of Streptomyces: possible application to the formation of hydroxy amides and aminoalcohols

To develop an efficient bioconversion process for amides, we screened our collection of Streptomyces strains, mostly obtained from soil, for effective transformers. Five strains, including the SY007 (NBRC 109343) and SY435 (NBRC 109344) of Streptomyces sp., exhibited marked conversion activities from the approximately 700 strains analyzed. These strains transformed diverse amide compounds such as N-acetyltetrahydroquinoline, N-benzoylpyrrolidine, and N-benzoylpiperidine into alcohols or N,O-acetals with high activity and regioselectivity. N,O-acetal was transformed into alcohol by serial tautomerization and reduction reactions. As such, Streptomyces spp. can potentially be used for the efficient preparation of hydroxy amides and aminoalcohols.     

Intracellular distribution of functional M(1) -muscarinic acetylcholine receptors in N1E-115 neuroblastoma cells

Signaling by muscarinic agonists is thought to result from the activation of cell surface acetylcholine receptors (mAChRs) that transmit extracellular signals to intracellular systems. In N1E-115 neuroblastoma cells, we detected both plasma membrane and intracellular M(1) -mAChRs using both biochemical and pharmacological methods. In intact cells, both plasma membrane and intracellular M(1) -mAChRs were detected by the hydrophobic ligand probe, 1-quinuclidinyl-[phenyl-4-(3) H]-benzilate ([(3) H]-QNB) whereas the hydrophilic probe, 1-[N-methyl-(3) H] scopolamine ([(3) H]-NMS), detected only cell surface receptors. These probes detected comparable numbers of receptors in isolated membrane preparations. Immunohistochemical studies with M(1) -mAChR antibody also detected both cell-surface and intracellular M(1) -mAChRs. Carbachol-stimulated phosphatidylinositol hydrolysis and Ca(2+) mobilization were completely inhibited by a cell-impermeable M(1) antagonist, muscarinic toxin -7 and the G(q/11) inhibitor YM-254890. However, carbachol-stimulated extracellular-regulated kinase 1/2 activation was unaffected by muscarinic toxin-7, but was blocked by the cell-permeable antagonist, pirenzepine. extracellular regulated kinase 1/2 phosphorylation was resistant to blockade of G(q/11) (YM-254890) and protein kinase C (bisindolylmaleimide I). Our data suggest that the geographically distinct M(1) -mAChRs (cell surface versus intracellular) can signal via unique signaling pathways that are differentially sensitive to cell-impermeable versus cell-permeable antagonists. Our data are of potential physiological relevance to signaling that affects both cognitive and neurodegenerative processes.     

Differential role of DNA-PKcs phosphorylations and kinase activity in radiosensitivity and chromosomal instability

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is the key functional element in the DNA-PK complex that drives nonhomologous end joining (NHEJ), the predominant DNA double-strand break (DSB) repair mechanism operating to rejoin such breaks in mammalian cells after exposure to ionizing radiation. It has been reported that DNA-PKcs phosphorylation and kinase activity are critical determinants of radiosensitivity, based on responses reported after irradiation of asynchronously dividing populations of various mutant cell lines. In the present study, the relative radiosensitivity to cell killing as well as chromosomal instability of 13 DNA-PKcs site-directed mutant cell lines (defective at phosphorylation sites or kinase activity) were examined after exposure of synchronized G(1) cells to (137)Cs γ rays. DNA-PKcs mutant cells defective in phosphorylation at multiple sites within the T2609 cluster or within the PI3K domain displayed extreme radiosensitivity. Cells defective at the S2056 cluster or T2609 single site alone were only mildly radiosensitive, but cells defective at even one site in both the S2056 and T2609 clusters were maximally radiosensitive. Thus a synergism between the capacity for phosphorylation at the S2056 and T2609 clusters was found to be critical for induction of radiosensitivity.     

Ubiquitous distribution of phosphatidylinositol phosphate synthase and archaetidylinositol phosphate synthase in Bacteria and Archaea,which contain inositol phospholipid

In Eukarya, phosphatidylinositol (PI) is biosynthesized from CDP-diacylglycerol (CDP-DAG) and inositol. In Archaea and Bacteria, on the other hand, we found a novel inositol phospholipid biosynthetic pathway. The precursors, inositol 1-phosphate, CDP-archaeol (CDP-ArOH), and CDP-DAG, form archaetidylinositol phosphate (AIP) and phosphatidylinositol phosphate (PIP) as intermediates. These intermediates are dephosphorylated to synthesize archaetidylinositol (AI) and PI. To date, the activities of the key enzymes (AIP synthase, PIP synthase) have been confirmed in only three genera (two archaeal genera, Methanothermobacter and Pyrococcus, and one bacterial genus, Mycobacterium). In the present study, we demonstrated that this novel biosynthetic pathway is universal in both Archaea and Bacteria, which contain inositol phospholipid, and elucidate the specificity of PIP synthase and AIP synthase for lipid substrates. PIP and AIP synthase activity were confirmed in all recombinant cells transformed with the respective gene constructs for four bacterial species (Streptomyces avermitilis, Propionibacterium acnes, Corynebacterium glutamicum, and Rhodococcus equi) and two archaeal species (Aeropyrum pernix and Sulfolobus solfataricus). Inositol was not incorporated. CDP-ArOH was used as the substrate for PIP synthase in Bacteria, and CDP-DAG was used as the substrate for AIP synthase in Archaea, despite their fundamentally different structures. PI synthase activity was observed in two eukaryotic species, Saccharomyces cerevisiae and Homo sapiens; however, inositol 1-phosphate was not incorporated. In Eukarya, the only pathway converts free inositol and CDP-DAG directly into PI. Phylogenic analysis of PIP synthase, AIP synthase, and PI synthase revealed that they are closely related enzymes.     

Preparation of optically-active 3-pyrrolidinol and its derivatives from 1-benzoylpyrrolidinol by hydroxylation with Aspergillus sp. and stereo-selective esterification by a commercial lipase

An effective preparation scheme for optically-active 3-pyrrolidinol and its derivatives based on biological transformation is proposed. Aspergillus sp. NBRC 109513 hydroxylated 1-benzoylpyrrolidine, yielding (S)-1-benzoyl-3-pyrrolidinol with 66 % ee. Kinetic resolution of 1-benzoyl-3-pyrrolidinol by Amano PS-IM lipase formed optically-active 1-benzoyl-3-pyrrolidinol with >99 % ee. (S)-1-Benzoyl-3-pyrrolidinol was successfully converted to 3-pyrrolidinol and its derivatives with by chemical reactions (>99 % ee).     

Recovery of Flow-Mediated Vasodilatation after Repetitive Measurements Is Involved in Early Vascular Impairment: Comparison with Indices of Vascular Tone

In repetitive measurements of flow-mediated dilatation (FMD), the duration of the interval between measurements remains controversial. In this pilot study, we conducted three sequential measurements of low–flow-mediated constriction (L-FMC), FMD and flow-mediated total dilation (FMTD; L-FMC+ FMD) at baseline and intervals of 15 and 60 min in 30 healthy males. FMD₁₅, L-FMC₁₅, and FMTD₁₅ were significantly lower than the respective first measurements, but all indices showed full recovery at 60 min in all subjects. The baseline diameter was slightly increased at 15 min and restored at 60 min, but the maximum diameter, and the baseline and reactive flow velocity unchanged. We examined the relationship between recovery rate of FMTD at 15 min (FMTD-R) and cardio-ankle vascular index (CAVI). Univariate analysis showed moderate correlation between FMTD-R, and CAVI and L-FMC₀. Patients were divided according to FMTD-R value; the low-FMTD-R group [below the median value (−26.2%)] included a significantly higher proportion of smokers and higher CAVI values than the high-FMTD-R group. The reproducibility of FMTD and FMTD-R was evaluated in another group of 25 healthy subjects. The range of variation across measurements was 1.1% for FMTD and 4.6% for FMTD-R; with intraclass correlation coefficients of 0.93 and 0.95, respectively. The present study demonstrated blunted recovery of FMD within 15 min, suggesting the need for selection of a more adequate interval between measurements to avoid underestimation of FMD in subsequent measurements. The findings demonstrated the reproducibility of FMTD-R and FMTD measurements, and that FMTD-R might be involved in arterial stiffness and early vascular impairment in the healthy subjects.     

Expression,purification, and characterization of highly active endo-α-N-acetylgalactosaminidases expressed by silkworm-baculovirus expression system

The O-glycosidase, endo-α-N-acetylgalactosaminidase from Enterococcus faecalis (endoEF) catalyzes the cleavage of core 1 and core 3 type O-linked disaccharides between GalNAc and serine or threonine residues from glycoproteins. The endoEF has broad substrate specificity and thus is extensively utilized for the structural and functional analysis of the O-linked glycans. In this study, we expressed and purified the recombinant endoEF (rEndoEF) by using the silkworm-baculovirus expression vector system (Silkworm-BEVS) and confirmed the deglycosylation activity of rEndoEF targeting reporter glycoproteins, which was equivalent to the commercial O-glycosidase. Thus, our study provides important clues to produce highly active rEndoEF O-glycosidases employing silkworm-BEVS as an alternative.     

Giant invasive pituitary adenoma extending into the sphenoid sinus and nasopharynx: report of a case with intraoperative cytologic diagnosis

BACKGROUND: Invasive pituitary adenomas involving the skull base are difficult to distinguish from other, more aggressive tumors. Intraoperaive diagnoses are crucial for deciding the course of treatment. CASE: A large mass extending from the sella turcica to the sphenoid sinus and nasopharynx was identified in a 42-year-old male. Because of the lack of endocrine abnormalities and lack of an apparent rise in pituitary hormones, preoperative diagnoses included chordoma, chondrosarcoma, meningioma and pituitary adenoma. Tumor fragments were easily squeezed into a thin layer of cells for cytologic specimens. Uniform, round tumor cells were arranged in minimally cohesive cell sheets and possessed regular, ovoid nuclei with a fine chromatin pattern and granular cytoplasm with prominent Golgi areas. The cytologic features indicated a probable diagnosis of pituitary adenoma and excluded other possibilities. Immunohistochemical demonstration of prolactin and ultrastructural features established the final diagnosis of prolactinoma. With the administration of bromocriptine, a large reduction in tumor size occurred. As compared to frozen sections, cytologic preparations are more effective for the intraoperative diagnosis of pituitary adenomas. Such neoplasms should always be included in the differential diagnosis of tumors involving the skull base.