The cannabinoid CB1 receptor is expressed in pancreatic delta-cells

Antagonists of cannabinoid CB1 receptor (CB1, CNR1) promote weight loss and decrease hyperglycemia in patients with type 2 diabetes. While the endocannabinoid system may modulate islet hormone secretion, the cell-type expressing CB1 receptor in islets has not been fully resolved. In this study, we verified receptor gene expression in rodent islets and cell lines and examined the distribution of CB1 receptor in mouse, rat, and human islets by confocal immunofluorescence (IF) microscopy. IF demonstrated CB1 receptor was present in β-cell lines, but co-localized solely with somatostatin in the islet δ-cells of Zucker rats, C57BL/6 mice, and humans; no CB1 receptor expression was observed in α-, β-, or pp-cells. Similarly, a rat somatostatinoma cell line, MSL-G2-Tu6, was found to express CB1 receptor. We also found monoacylglycerol lipase (MAGL) to be expressed in δ-cells and fatty acid amide hydrolase (FAAH) to be expressed in α-cells. The specific expression of CB1 in δ-cells suggests that the ECS may play a role in modulating islet hormone secretion. As there are some differences between our findings and previous reports, further studies, including detailed physiological studies of the effects of the ECS on islet function, are warranted.     

High-level production of recombinant human IFN-α2a with co-expression of tRNAArg(AGG/AGA) in high-cell-density cultures ofEscherichia coli

The co-expression of theargU gene in a double-vector expression system of recombinantEscherichia coli BL21(DE3)[pET-IFN2a+pAC-argU] significantly enhanced the production level of recombinant human interferon-α2a (rhIFN-α2a) in high cell density cultures compared to a recombinantE coli culture containing only the single expression vector, pET-IFN2a. The dry cell mass concentration increased to almost 100 g/L, and more than 4 g/L of rhIFN-α2a was accumulated in the culture broth. Evidently, the synthesis of rhIFN-α2a was strongly dependent on the pre-induction growth rate and more efficient at a higher specific growth rate. The additional supply of tRNA^Arg(AGG/AGA) enhanced the expression level of the rhIFN-α2a gene in the early stage of the post-induction phase, yet thereafter the specific production rate of rhIFN-α2a rapidly decreased due to severe segregational instability of plasmid vector pET-IFN2a. It would appear that the plasmid instability, which only occurred to pET-IFN2a in the double vector system, was related to the effect of translational stress due to the overexpression of rhIFN-α2a.     

Enhancement of recombinant erythropoietin production in CHO cells in an incubator without CO2 addition

The effect of low levels of carbon dioxide (CO₂) in the gas phase on the production of recombinant human erythropoietin (EPO)in CHO cells was explored. A T-flask culture in an incubator without CO₂ addition showed a slow cell growth initially followed by the cessation of growth, while other cultures incubated under 0.5–5% CO₂ concentrations grew normally at the same rate during the entire period of cultivation. Interestingly, the production of EPO in the culture incubated under no CO₂ supply was highest among the tested cultures. The cell specific secretion rate of EPO (q_EPO) of the culture under no CO₂ supply was about 3 times higher than that of the culture under 5% CO₂ supply. Western blot analysis and in vivo bioassay of EPO showed no apparent changes in EPO quality between the two cases of different CO₂ environments (air vs. 5% CO₂), suggesting robust glycosylation of EPO by CHO cells even under very reduced CO₂ environment. Various combinations of the two extreme cases, with 5% CO₂ supply (suitable for cell growth) and no CO₂ addition (better for EPO production), were made in order to maximize the volumetric productivity of EPO secretion (P_V) in CHO cells. The P_V of the cultures programmed with initial incubation under 5% CO₂ followed by no CO₂ supply was about 2 times superior to that of the culture incubated only under no CO₂ supply. The P_V of the culture under no CO₂ supply was slightly lower than that of culture grown under 5% CO₂. However, the q_EPO of the no CO₂ supply case was more than 5 times higher than that of the culture under 5% CO₂ supply. In conclusion, we have demonstrated that a simple programming of CO₂ supply to an incubator can enhance the production of EPO in CHO cells remarkably, without any apparent change of the EPO quality. This revised version was published online in August 2006 with corrections to the Cover Date.     

Isovaleric acid ameliorates ovariectomy-induced osteoporosis by inhibiting osteoclast differentiation

Osteoclasts (OCs) play important roles in bone remodelling and contribute to bone loss by increasing bone resorption activity. Excessively activated OCs cause diverse bone disorders including osteoporosis. Isovaleric acid (IVA), also known as 3-methylbutanoic acid is a 5-carbon branched-chain fatty acid (BCFA), which can be generated by bacterial fermentation of a leucine-rich diet. Here, we find that IVA suppresses differentiation of bone marrow-derived macrophages into OCs by RANKL. IVA inhibited the expression of OC-related genes. IVA-induced inhibitory effects on OC generation were attenuated by pertussis toxin but not by H89, suggesting a G_i-coupled receptor-dependent but protein kinase A-independent response. Moreover, IVA stimulates AMPK phosphorylation, and treatment with an AMPK inhibitor blocks IVA-induced inhibition of OC generation. In an ovariectomized mouse model, addition of IVA to the drinking water resulted in significant decrease of body weight gain and inhibited the expression of not only OC-related genes but also fusogenic genes in the bone tissue. IVA exposure also blocked bone destruction and OC generation in the bone tissue of ovariectomized mice. Collectively, the results demonstrate that IVA is a novel bioactive BCFA that inhibits OC differentiation, suggesting that IVA can be considered a useful material to control osteoclast-associated bone disorders, including osteoporosis.     

Purification and characterization of yolk protein-2 from the fall webworm,Hyphantria cunea drury

Yolk proteins (YP1, YP2, and YP3) of the fall webworm, Hyphantria cunea, are of relatively low molecular weight. Yolk protein-2 (YP2) was purified from gel slices and by KBr density gradient ultracentrifugation followed by ion exchange chromatography. YP2 is composed of one subunit with a molecular weight of 35.5 kDa. YP2 contains neutral lipids (diacylglycerol and triacylglycerol) and phospholipids (phosphatidylcholine and phosphatidylethanolamine). The neutral lipids are largely composed of lauric acid and palmitoleic acid. YP2 contains relatively large amounts of glutamic acid and aspartic acid but small amounts of tyrosine, phenylalanine, and methionine. YP2 is a vitellin (Vn) synthesized by the fat body. Vitellogenin-2 (Vg2), the precursor of YP2, is present in very small amounts in the hemolymph. Lipophorin and storage protein also are found in the ovary of H. cunea, and these proteins do not immunologically cross-react with YP2. YP2 is detected in first instar larvae but completely disappears during the second instar, indicating that YP2 is intensively utilized during postembryonic development. Anti-YP2 antibodies cross-react with ovarial extracts of Bombyx mori but not with those of insects from other orders such as Cletus schmidti (Hemiptera), Lucilia illustris (Diptera), Anechura japonica (Dermaptera), Periplaneta americana (Dictyoptera), and Ducetia japonica (Orthoptera). © 1995 Wiley-Liss, Inc.     

Imidazole dipeptides can quench toxic 4‐oxo‐2(E)‐nonenal: Molecular mechanism and mass spectrometric characterization of the reaction products

Imidazole dipeptides, such as carnosine (β‐alanyl‐l ‐histidine) and anserine (β‐alanyl‐N^π‐methyl‐l ‐histidine), are highly localized in excitable tissues, including skeletal muscle and nervous tissue, and play important roles such as scavenging reactive oxygen species and quenching reactive aldehydes. We have demonstrated several reactions between imidazole dipeptides (namely, carnosine, and anserine) and a lipid peroxide‐derived reactive aldehyde 4‐oxo‐2(E)‐nonenal. Seven carnosine adducts and two anserine adducts were characterized using liquid chromatography/electrospray ionization‐multiple‐stage mass spectrometry. Adduct formation occurred between imidazole dipeptides and 4‐oxo‐2(E)‐nonenal mainly through Michael addition, Schiff base formation, and/or Paal‐Knorr reaction. The reactions were much more complicated than the reaction with a similar lipid peroxide‐derived reactive aldehyde, 4‐hydroxy‐2(E)‐nonenal.     

Expression and characterization of glutamate decarboxylase from <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> HYE1 isolated from <Emphasis Type="Italic">kimchi</Emphasis>

A putative gene (gadlbhye1) encoding glutamate decarboxylase (GAD) was cloned from Lactobacillus brevis HYE1 isolated from kimchi, a traditional Korean fermented vegetable. The amino acid sequences of GADLbHYE1 showed 48% homology with the GadA family and 99% identity with the GadB family from L. brevis. The cloned GADLbHYE1 was functionally expressed in Escherichia coli using inducible expression vectors. The expressed recombinant GADLbHYE1 was successfully purified by Ni–NTA affinity chromatography, and had a molecular mass of 54 kDa with optimal hydrolysis activity at 55 °C and pH 4.0. Its thermal stability was determined to be higher than that of other GADs from L. brevis, based on its melting temperature (75.18 °C). Kinetic parameters including K_m and V_max values for GADLbHYE1 were 4.99 mmol/L and 0.224 mmol/L/min, respectively. In addition, the production of gamma-aminobutyric acid in E. coli BL21 harboring gadlbhye1/pET28a was increased by adding pyridoxine as a cheaper coenzyme.     

New Lignans from the Flower of Forsythia koreana and Their Suppression Effect on VCAM‐1 Expression in MOVAS Cells

Six lignans including two new lignans were obtained as the principal components of the Forsythia koreana flowers via silica gel (SiO₂), octadecyl SiO₂ (ODS) as well as Sephadex LH‐20 column chromatography. In addition to two new lignans, named koreanaside A ((7R,8S,7′R,8′S)‐7,7′‐diepoxy‐5′‐hydroxy‐3,3′‐dimethoxylignan 4‐O‐β‐d ‐glucopyranoside) and koreanaside B ((7R,8S,7′S,8′R)‐7,9′‐epoxy‐9,5′,7′‐trihydroxy‐3,3′‐dimethoxylignan 4‐O‐β‐d ‐glucopyranoside), four known lignans were identified to be (+)‐phylligenin, (?)‐epipinoresinol, pinoresinol, and tinosposide A. The structures and absolute configurations of koreanasides A and B were established by means of analysis of spectroscopic data (NMR, IR, FAB‐MS, and CD), whereas the structures of known lignans were identified by comparison their NMR and MS values with those in the reported literature. Their chemical structures including configuration were established by means of analysis of spectroscopic data (NMR, IR, FAB‐MS, and CD) but also comparison of their NMR and MS values with those in the reported literature. This is the first article for isolation of six lignans of F. koreana flowers. Koreanasides A and B showed high radical scavenging activity with oxygen radical absorbance capacity (ORAC) values of 0.97 ± 0.01 and 1.02 ± 0.01, respectively. Koreanaside A also prohibited expressing VCAM‐1 in MOVAS cells with 80.5% at 25 mg/mL.     

THERPA: A small molecule database related to prion protein regulation and prion diseases progression

Prion diseases are fatal neurodegenerative disorders that affect humans and animals. Although various small molecules have been evaluated for application in the treatment of prion diseases, none have been shown to be efficacious. Expanding our knowledge of these molecules is important for understanding of the complex mechanisms of prion diseases. To improve access to the scattered information on small molecules related to prion diseases, we built a database of therapeutic molecules associated with prion diseases (THERPA, therpa.pythonanywhere.com). THERPA includes 119 small molecules and their 283 relationships with prion diseases. THERPA is an interactive visual database and useful for improving search efficiency which can help researchers identify intrinsic small molecules that can be used for developing therapeutics for prion diseases.