Late-quaternary spruce decline and rise in Japan and Sakhalin

The late-glacial logistic decline of spruce (Picea) populations (the intrinsic growth rate,r in yr^?1=?0.0015) was progressively delayed toward cooler sites in Japan; spruce populations expanded asymptotically (r=0.0040?0.0045) in Sakhalin early in the Holocene. Such a time-transgressive range shift in spruce distribution, covering a wide latitudinal range (ca. 35–50°N), implies that the regional temperature rise was not instantaneous, but was rapid, being 0.0015–0.0025 C yr^?1 from 13,000 to 10,000 years B.P. Spruce populations increased in response to Neoglacial cooling only at the margin of its distribution (r=0.0036), but then declined logistically 1,000 years B.P. (r=?0.0022).     

Protein kinase C of a human megakaryoblastic leukemic cell line (MEG-01). Analysis of subspecies and activation by diacylglycerol and free fatty acids

Protein kinase C (PKC) from a human megakaryoblastic leukemic cell line (MEG-01) was resolved into two fractions by hydroxyapatite column chromatography, which are indistinguishable from the brain type II (beta I/beta II) and type III (alpha) subspecies, by biochemical and immunoblot analysis. In the presence of both phosphatidylserine and diacylglycerol, several free unsaturated fatty acids (FFA's), such as arachidonic, oleic, linoleic and linolenic acids, further enhanced the enzyme activation, and allowed the enzyme to exhibit almost full activity at nearly basal levels of Ca2+ concentration. The concentration of unsaturated FFA's giving rise to the maximum enzyme activation was around 2 x 10(-5) M. Palmitic and stearic acids were inactive. The result implies that, in addition to diacylglycerol, the receptor-mediated release of unsaturated FFA's from membrane phospholipids may also take part in the activation of PKC.     

N-terminally extended form of C-type natriuretic peptide (CNP-53) identified in porcine brain

C-type natriuretic peptide of 22 residues (CNP-22) is very recently identified in porcine brain as a third member of the mammalian natriuretic peptide family (1). Using a radioimmunoassay system newly established for CNP-22, we searched for CNP-related peptides in porcine brain. In addition to CNP-22, one major form of immunoreactive CNP was detected in porcine brain extracts, being isolated by immunoaffinity chromatography and reverse phase high performance liquid chromatography. By microsequence analysis, the peptide was deduced to be a 53-amino acid peptide carrying a CNP-22 sequence at the C-terminus, and was designated C-type natriuretic peptide-53 (CNP-53). CNP-53 was found to be a major molecular form of CNP in porcine brain.     

mTORC1-independent translation control in mammalian cells by methionine adenosyltransferase 2A and S-adenosylmethionine

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM). As the sole methyl-donor for methylation of DNA, RNA, and proteins, SAM levels affect gene expression by changing methylation patterns. Expression of MAT2A, the catalytic subunit of isozyme MAT2, is positively correlated with proliferation of cancer cells; however, how MAT2A promotes cell proliferation is largely unknown. Given that the protein synthesis is induced in proliferating cells and that RNA and protein components of translation machinery are methylated, we tested here whether MAT2 and SAM are coupled with protein synthesis. By measuring ongoing protein translation via puromycin labeling, we revealed that MAT2A depletion or chemical inhibition reduced protein synthesis in HeLa and Hepa1 cells. Furthermore, overexpression of MAT2A enhanced protein synthesis, indicating that SAM is limiting under normal culture conditions. In addition, MAT2 inhibition did not accompany reduction in mechanistic target of rapamycin complex 1 activity but nevertheless reduced polysome formation. Polysome-bound RNA sequencing revealed that MAT2 inhibition decreased translation efficiency of some fraction of mRNAs. MAT2A was also found to interact with the proteins involved in rRNA processing and ribosome biogenesis; depletion or inhibition of MAT2 reduced 18S rRNA processing. Finally, quantitative mass spectrometry revealed that some translation factors were dynamically methylated in response to the activity of MAT2A. These observations suggest that cells possess an mTOR-independent regulatory mechanism that tunes translation in response to the levels of SAM. Such a system may acclimate cells for survival when SAM synthesis is reduced, whereas it may support proliferation when SAM is sufficient.     

Site-specific labeling of cytoplasmic peptide:N-glycanase by N,N'-diacetylchitobiose-related compounds

Peptide:N-glycanase (PNGase) is the deglycosylating enzyme, which releases N-linked glycan chains from N-linked glycopeptides and glycoproteins. Recent studies have revealed that the cytoplasmic PNGase is involved in the degradation of misfolded/unassembled glycoproteins. This enzyme has a Cys, His, and Asp catalytic triad, which is required for its enzymatic activity and can be inhibited by "free" N-linked glycans. These observations prompted us to investigate the possible use of haloacetamidyl derivatives of N-glycans as potent inhibitors and labeling reagents of this enzyme. Using a cytoplasmic PNGase from budding yeast (Png1), Man9GlcNAc2-iodoacetoamide was shown to be a strong inhibitor of this enzyme. The inhibition was found to be through covalent binding of the carbohydrate to a single Cys residue on Png1, and the binding was highly selective. The mutant enzyme in which Cys191 of the catalytic triad was changed to Ala did not bind to the carbohydrate probe, suggesting that the catalytic Cys is the binding site for this compound. Precise determination of the carbohydrate attachment site by mass spectrometry clearly identified Cys191 as the site of covalent attachment. Molecular modeling of N,N'-diacetylchitobiose (chitobiose) binding to the protein suggests that the carbohydrate binding site is distinct from but adjacent to that of Z-VAD-fmk, a peptide-based inhibitor of this enzyme. These results suggest that cytoplasmic PNGase has a separate binding site for chitobiose and other carbohydrates, and haloacetamide derivatives can irreversibly inhibit that catalytic Cys in a highly specific manner.     

Immunoreactive atrial natriuretic polypeptides in the adrenal medulla and sympathetic ganglia

Atrial natriuretic polypeptide (ANP)-like immunoreactivity was found in the rat adrenal gland by using indirect immunofluorescence and peroxidase-antiperoxidase techniques. ANP-like immunostaining was present in most of chromaffin cells with varying degrees of immunoreactivity. The majority of medullary cells displayed very intense immunostaining, and several clusters revealed weaker immunostaining. No staining was found in the adrenal cortex or in the nerve fibers in this organ. In the consecutive sections treated for dopamine-beta-hydroxylase (DBH), apparently all medullary cells had intense immunofluorescence for DBH and its distribution pattern was very similar to that for ANP-like immunoreactivity. While phenylethanolamine N-methyltransferase (PNMT) immunoreactive cells largely corresponded to the intensely stained ANP-like immunoreactive cells, suggesting that adrenaline cells contained a large amount of ANP-like substance, noradrenaline cells contained a smaller amount of this substance than adrenaline cells. Ultrastructural study showed that end-products due to the immunoreaction with the ANP antiserum were primarily associating with chromaffin granules. In addition, the presence of ANP-like immunoreactivity was investigated in several sympathetic ganglia of the rat. No principal ganglion cells were ANP-positive, whereas a few small intensely fluorescent (SIF) cells were ANP-immunoreactive. The present findings suggest that catecholamines coexist with ANP which has a natriuretic and vasodilating effect, in adrenal medullary cells and SIF cells in several rat sympathetic ganglia, but not in principal ganglion cells.     

Efflux of sphingomyelin, cholesterol, and phosphatidylcholine by ABCG1

Cholesterol and phospholipids are essential to the body, but an excess of cholesterol or lipids is toxic and a risk factor for arteriosclerosis. ABCG1, one of the half-type ABC proteins, is thought to be involved in cholesterol homeostasis. To explore the role of ABCG1 in cholesterol homeostasis, we examined its subcellular localization and function. ABCG1 and ABCG1-K120M, a WalkerA lysine mutant, were localized to the plasma membrane in HEK293 cells stably expressing ABCG1 and formed a homodimer. A stable transformant expressing ABCG1 exhibited efflux of cholesterol and choline phospholipids in the presence of BSA, and the cholesterol efflux was enhanced by the presence of HDL, whereas cells expressing ABCG1-K120M did not, suggesting that ATP binding and/or hydrolysis is required for the efflux. Mass and TLC analyses revealed that ABCG1 and ABCA1 secrete several species of sphingomyelin (SM) and phosphatidylcholine (PC), and SMs were preferentially secreted by ABCG1, whereas PCs were preferentially secreted by ABCA1. These results suggest that ABCA1 and ABCG1 mediate the lipid efflux in different mechanisms, in which different species of phospholipids are secreted, and function coordinately in the removal of cholesterol and phospholipids from peripheral cells.     

Plasma protein binding study of oxybutynin by high-performance frontal analysis

Plasma protein binding of oxybutynin (OXY) was investigated quantitatively and enantioselectively using high-performance frontal analysis (HPFA). An on-line HPLC system which consists of HPFA column, extraction column and analytical column was developed to determine the unbound concentrations of OXY enantiomers in human plasma, in human serum albumin (HSA) solutions, and in human alpha1-acid glycoprotein (AGP) solutions. OXY is bound in human plasma strongly and enantioselectively. The bound drug fraction in human plasma containing 2-10 microM (R)- or (S)-OXY was higher than 99%, and the unbound fraction of (R)-OXY was 1.56 times higher than that of (S)-isomer. AGP plays the dominant role in this strong and enantioselective plasma protein binding. The total binding affinities (nK) of (R)- and (S)-OXY to AGP were 6.86 x 10(6) and 1.53 x 10(7) M(-1), respectively, while the nK values of (R)- and (S)-OXY to HSA were 2.64 x 10(4) and 2.19 x 10(-4) M(-1), respectively. The binding affinity of OXY to AGP is much higher than that to HSA, and shows high enantioselectivity (SIR ratio of nK values is 2.2). It was found that both enantiomers are bound competitively at the same binding site on an AGP molecule. The binding property between OXY and low density lipoprotein (LDL) was investigated by using the frontal analysis method incorporated in high-performance capillary electrophoresis (HPCE/FA). It was found the binding is non-saturable and non-enantioselective.     

Loss of the TOR kinase Tor2 mimics nitrogen starvation and activates the sexual development pathway in fission yeast

Fission yeast has two TOR (target of rapamycin) kinases, namely Tor1 and Tor2. Tor1 is required for survival under stressed conditions, proper G(1) arrest, and sexual development. In contrast, Tor2 is essential for growth. To analyze the functions of Tor2, we constructed two temperature-sensitive tor2 mutants. Interestingly, at the restrictive temperature, these mutants mimicked nitrogen starvation by arresting the cell cycle in G(1) phase and initiating sexual development. Microarray analysis indicated that expression of nitrogen starvation-responsive genes was induced extensively when Tor2 function was suppressed, suggesting that Tor2 normally mediates a signal from the nitrogen source. As with mammalian and budding yeast TOR, we find that fission yeast TOR also forms multiprotein complexes analogous to TORC1 and TORC2. The raptor homologue, Mip1, likely forms a complex predominantly with Tor2, producing TORC1. The rictor/Avo3 homologue, Ste20, and the Avo1 homologue, Sin1, appear to form TORC2 mainly with Tor1 but may also bind Tor2. The Lst8 homologue, Wat1, binds to both Tor1 and Tor2. Our analysis shows, with respect to promotion of G(1) arrest and sexual development, that the loss of Tor1 (TORC2) and the loss of Tor2 (TORC1) exhibit opposite effects. This highlights an intriguing functional relationship among TOR kinase complexes in the fission yeast Schizosaccharomyces pombe.     

Discovery of potent, selective, orally active benzoxazepine-based Orexin-2 receptor antagonists

During our efforts to identify a series of potent, selective, orally active human Orexin-2 Receptor (OX2R) antagonists, we elucidated structure-activity relationship (SAR) on the 7-position of a benzoxazepine scaffold by utilizing Hammett σ(p) and Hansch-Fujita π value as aromatic substituent constants. The attempts led to the discovery of compound 1m, possessing good in vitro potency with over 100-fold selectivity against OX1R, good metabolic stability in human and rat liver microsome, good oral bioavailability in rats, and in vivo antagonistic activity in rats by oral administration.