Growth hormone-releasing peptide-2 stimulates secretion and synthesis of adrenocorticotropic hormone in mouse pituitary

Growth hormone (GH)-releasing peptides (GHRPs) are synthetic peptides which induce strong GH release in both animals and humans. Among them, GHRP-2 is known to stimulate GH release by acting at both hypothalamic and pituitary sites, but also induces adrenocorticotropic hormone (ACTH) release in healthy subjects. GHRP-2 may stimulate ACTH release directly via GHRP receptor type 1a in ACTH-producing tumors. GHRP-2 increases ACTH secretion in rat in vivo, but not ACTH release from rat primary pituitary cells. In the present study, in order to elucidate the mechanism underlying ACTH secretion by GHRPs, mouse pituitary cells were stimulated by GHRP-2. GHRP receptor mRNA was expressed in the mouse pituitary, and GHRP-2 directly stimulated secretion and synthesis of ACTH in the mouse anterior pituitary cells. GHRP-2 increased intracellular cyclic AMP production. H89, a potent protein kinase A (PKA) inhibitor, and bisindolylmaleimide I, a selective protein kinase C (PKC) inhibitor, inhibited the GHRP-2-induced ACTH release, and that H89, but not bisindolylmaleimide I, inhibited the GHRP-2-induced proopiomelanocortin mRNA levels. Together, the GHRP-2-induced ACTH release was regulated via both PKA and PKC pathways in the mouse pituitary cells, while ACTH was synthesized by GHRP-2 only via the PKA pathway.     

Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium,Halomonas elongata

1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic β-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with l-glutamate. This enzyme required pyridoxal 5′-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25°C and had K_ms of 9.1 mM for l-glutamate and 4.5 mM for dl-ASA. DABA acetyltransferase catalyzed acetylation of DABA to γ-N-acetyl-α,γ-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a K_m of 8.4 mM in the presence of 0.77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30°C.Halotolerance is of considerable interest scientifically and from the perspective of wide application in fermentation industries and in agriculture. When eubacteria are exposed to hyperosmotic stress, they accumulate various low-molecular-weight organic compounds, the so-called “compatible solutes” such as polyols, amino acids, sugars, and betaines (7–9, 13, 19, 48), because maintenance of turgor pressure is a prerequisite for growth under the conditions of elevated external osmotic pressure. Since Galinski et al. (14) discovered 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) as a compatible solute in Ectothiorhodospira halochloris, an extremely halophilic phototrophic eubacterium, ectoine has been found to be distributed widely in nature, largely in moderately halophilic eubacteria (3, 11, 12, 26, 38, 50). In addition, ectoine has been investigated as a new excellent universal osmoprotectant in this decade, since incorporation of external ectoine under hyperosmotic stress has been observed to confer protection on various nonhalotolerant eubacteria (16, 21, 44).We previously isolated a moderately halophilic eubacterium, Halomonas elongata (31), from dry salty land in Thailand. We identified ectoine and γ-N-acetyl-α,γ-diaminobutyric acid (ADABA), which is one of the cleavage structures of ectoine, as osmotically responding compounds in the cells grown in a glucose-mineral medium containing NaCl in a concentration range of 3 to 15% (31). To understand the accumulation mechanism of the intracellular ectoine, characterization of enzymes involved in the biosynthesis of ectoine is indispensable. Therefore, we have focused on the biosynthetic enzyme of ectoine in this organism. We observed that radioactivity from [1-¹⁴C]aspartate was most efficiently incorporated into ectoine and that the signal intensity was enriched preferentially from [1-¹³C]acetate into the methyl carbon at position 2′ and from [2-¹³C]acetate into the methine carbon at position 2 of the ectoine skeleton, respectively, in ¹³C nuclear magnetic resonance (NMR) spectroscopy (22). From these findings, we also hypothesized the following pathway essentially similar to that described by Peters et al. (34): aspartic β-semialdehyde (ASA) is converted to 2,4-diaminobutyric acid (DABA) by transamination, and DABA is converted to ADABA by acetylation with acetyl coenzyme A (CoA), which in turn yields ectoine by circularization (Fig. ​(Fig.1).1). The three enzymes involved in this pathway are DABA aminotransferase, DABA acetyltransferase, and ectoine synthase in order of the reactions to ectoine. Peters et al. (34) detected the activity of the first and the second of the three steps by using crude extracts of E. halochloris and H. elongata. However, the characterization of these enzymes was limited; in particular, their responses to various salt concentrations remained unknown. Open in a separate windowFIG. 1Proposed biosynthetic pathway of ectoine in H. elongata {"type":"entrez-protein","attrs":{"text":"OUT30018","term_id":"1200192464","term_text":"OUT30018"}}OUT30018.In this study, we confirmed the biosynthetic pathway of ectoine by using purified enzymes in H. elongata {"type":"entrez-protein","attrs":{"text":"OUT30018","term_id":"1200192464","term_text":"OUT30018"}}OUT30018 and characterized the three enzymes involved in the conversion of ASA to ectoine for the first time.     

Ultrafine membrane compartments for molecular diffusion as revealed by single molecule techniques

Plasma membrane compartments, delimited by transmembrane proteins anchored to the membrane skeleton (anchored-protein picket model), would provide the membrane with fundamental mosaicism because they would affect the movement of practically all molecules incorporated in the cell membrane. Understanding such basic compartmentalized structures of the cell membrane is critical for further studies of a variety of membrane functions. Here, using both high temporal-resolution single particle tracking and single fluorescent molecule video imaging of an unsaturated phospholipid, DOPE, we found that plasma membrane compartments generally exist in various cell types, including CHO, HEPA-OVA, PtK2, FRSK, HEK293, HeLa, T24 (ECV304), and NRK cells. The compartment size varies from 30 to 230 nm, whereas the average hop rate of DOPE crossing the boundaries between two adjacent compartments ranges between 1 and 17 ms. The probability of passing a compartment barrier when DOPE is already at the boundary is also cell-type dependent, with an overall variation by a factor of approximately 7. These results strongly indicate the necessity for the paradigm shift of the concept on the plasma membrane: from the two-dimensional fluid continuum model to the compartmentalized membrane model in which its constituent molecules undergo hop diffusion over the compartments.     

A nucleus-encoded factor, CRR2, is essential for the expression of chloroplast ndhB in Arabidopsis

The chloroplast NDH complex, NAD(P)H dehydrogenase, reduces the plastoquinone pool non-photochemically and is involved in cyclic electron flow around photosystem I (PSI). A transient increase in chlorophyll fluorescence after turning off actinic light is a result of NDH activity. We focused on this subtle change in chlorophyll fluorescence to isolate nuclear mutants affected in chloroplast NDH activity in Arabidopsis by using chlorophyll fluorescence imaging. crr2-1 and crr2-2 (chlororespiratory reduction) are recessive mutant alleles in which accumulation of the NDH complex is impaired. Except for the defect in NDH activity, photosynthetic electron transport was unaffected. CRR2 encodes a member of the plant combinatorial and modular protein (PCMP) family consisting of more than 200 genes in Arabidopsis. CRR2 functions in the intergenic processing of chloroplast RNA between rps7 and ndhB, which is possibly essential for ndhB translation. We have determined the function of a PCMP family member, indicating that the family is closely related to pentatrico-peptide PPR proteins involved in the maturation steps of organellar RNA.     

Synthesis of chondroitin sulfate E hexasaccharide in the repeating region by an effective elongation strategy toward longer chondroitin oligosaccharide

We synthesized chondroitin and its sulfate E hexasaccharides (1 and 2) composed of the trimer of the repeating disaccharide, beta-D-GalNAc(+/-4,6-di-O-SO(3)Na)-(1-->4)-beta-D-GlcA, by employing an efficient synthetic strategy for longer chondroitin oligosaccharide. Successful elongation with the beta-D-GalNAc-(1-->4)-beta-D-GlcA unit instead of the corresponding disaccharide possessing an azide group avoided problematic reduction of the multiple azide groups on the hexasaccharide.     

Liquid chromatography studies on the pharmacokinetics of phentermine and fenfluramine in brain and blood microdialysates after intraperitoneal administration to rats

A highly sensitive and simple HPLC method with fluorescence detection for the determination of phentermine (Phen), fenfluramine (Fen) and norfenfluramine (Norf, the active metabolite of Fen) in rat brain and blood microdialysates has been developed. The brain and blood microdialysates were directly subjected to derivatization with 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride (DIB-Cl) in the presence of carbonate buffer (0.1 M, pH 9.0) at room temperature. The chromatographic conditions consisted of an ODS column and mobile phase composition of acetonitrile and water (65:35, v/v) with flow rate set at 1.0 ml/min. The detection was performed at excitation and emission wavelengths of 325 and 430 nm, respectively. Under these conditions, the DIB-derivatives of Phen, Fen and Norf were well separated and showed good linearities in the studied ranges (5-2000 nM for Phen and 10-2000 nM for Norf and Fen) with correlation coefficients greater than 0.999. The obtained detection limits were less than 23 fmol on column (for the three compounds) in both brain and blood microdialysates at a signal-to-noise ratio of 3 (S/N=3). The intra- and the inter-assay precisions were lower than 10%. The method coupled with microdialysis was applied for a pharmacokinetic drug-drug interaction study of Phen and Fen following individual and combined intraperitoneal administration to rats. In addition, since the role of protein binding in drug interactions can be quite involved, the method was applied for the determination of total and free Phen and Fen in rat plasma and ultrafiltrate, respectively. The results showed that Fen and/or Norf significantly altered the pharmacokinetic parameters of Phen in both blood and brain but did not alter its protein binding. On the other hand, there was no significant difference in the pharmacokinetics of Fen when administered with Phen.     

A new alpha,beta,gamma,delta-unsaturated carboxylic acid and three new cyclic peroxides from the marine sponge,Monotria japonica,which selectively lyse starfish oocytes without affecting nuclear morphology

The marine sponge Monotria japonica contains cytolytic constituents, which have been fractionated to a new alpha,beta,gamma,delta-unsaturated carboxylic acid designated monotriajaponide A (1) and three new cyclic peroxides designated monotriajaponides B (2), C (3), and D (4), in addition to a known peroxide (5) and a known alpha,beta-unsaturated ester (6). The structures were determined on the basis of spectroscopic data. Compounds 1-5 lysed immature starfish (Asterina pectinifera) oocytes without affecting nuclear morphology at the minimum effective concentrations of 50, 6.3, 6.3, 6.3, and 13 microg/mL, respectively. On the other hand, compound 6, at the minimum effective concentration of 25 microg/mL, lysed both oocyte's plasma membrane and the nuclear envelope.     

A cysteine protease inhibitor prevents suspension-induced declines in bone weight and strength in rats

In this study, we examined the effects of a potent cysteine protease inhibitor, N-(L-3-trans-carboxyoxirane-2-cabonyl)-L-leucine-4-aminobutylamide (E-64a), on bone weight and strength in tail-suspended rats. We first administered a vehicle or 4 or 8 mg/rat of E-64a to rats fed with a low calcium diet for 7 wks to determine effective doses of E-64a on bone resorption in vivo. Femoral cathepsin K-like activity and serum hydroxyproline level in rats fed with a low calcium diet were significantly higher than those in rats fed with a standard diet. The intraperitoneal injection of 8 mg/rat of E-64a to rats decreased their serum calcium and hydroxyproline concentrations after 3 to 6 hrs in parallel with changes in femoral cathepsin K-like activity, while 4 mg/rat of E-64a had weaker effects on these parameters. Based on these results, we injected 8 mg/rat of E-64a to tail-suspended rats twice a day for 2 wks and compared the results with those of treatment with 1 mg/rat of etidronate, a bisphosphonate, twice a week. In tail-suspended rats, femoral weight and strength, assessed by three-point bending test, significantly decreased from Day 5 to 21, while femoral cathepsin K-like activity and serum calcium and hydroxyproline concentrations did not change. E-64a inhibited femoral cathepsin K-like activity in tail-suspended rats, but etidronate did not. E-64a as well as etidronate significantly prevented the suspension-induced declines in bone weight and strength. However, more frequent injection and higher doses were required for E-64a to exhibit significant efficacy of antiresorption, compared with those of etidronate. Our results suggest that a cysteine protease inhibitor could improve suspension-induced osteopenia by inhibiting cathepsin K-like activity in bone; however, it needs several improvements in the effect as a clinical drug.