Increase of pancreatic somatostatin concentration in early phases of streptozotocin diabetes in rats

A small yet significant increase of immunoassayable pancreatic somatostatin concentration (0.107 +/- 0.005 vs. 0.156 +/- 0.017 microgram/g at 24 hr, p less than 0.05) was found in rats, 24 hr as well as 7 days after treatment with a diabetogenic dose of streptozotocin (65 mg/kg BW). These animals were characterized by marked decreases of insulin in the pancreas without any significant changes in pancreatic glucagon concentration. These results suggest that an abrupt deprivation of insulin from islets results in an elevation of pancreatic somatostatin concentration, and that glucagon in the pancreas plays a minor role in determining pancreatic somatostatin concentration in rats with insulin-deprived diabetes of short duration.     

Plasma anti-Müllerian hormone as a biomarker for bovine granulosa-theca cell tumors: Comparison with immunoreactive inhibin and ovarian steroid concentrations

Granulosa-theca cell tumors (GTCTs) are the most frequently reported ovarian tumors in cattle. Clinically, GTCTs could be confused with other ovarian abnormalities; therefore, the only definitive diagnosis for such tumors is histopathology of a biopsy from the affected ovary. However, this is an invasive technique and unsuitable for farm conditions. As a result, the key aim of this study was to evaluate the diagnostic value of anti-Müllerian hormone (AMH), a glycoprotein hormone that is synthesized exclusively by ovarian granulosa cells, as a sensitive noninvasive biomarker for diagnosing GTCTs in cattle. To achieve this aim, we conducted two experiments. In experiment 1, four clinically healthy Japanese Black cows had blood samples taken daily over one estrous cycle to characterize their AMH profiles throughout the estrous cycle. Additionally, single blood samples were collected from 21 cyclic cows to clarify the physiological range of AMH. In experiment 2, cows with histologically confirmed GTCT (GTCT group, n = 9) and cows affected with cystic ovarian disease (COD group, n = 8) had one blood sample taken before extraction of the tumorous ovary or therapeutic treatment for the COD. Blood samples (n = 105) from cyclic cows (n = 25) in experiment 1 were assigned as a physiologically cyclic group (PC group). Plasma AMH, immunoreactive inhibin (ir-INH), estradiol-17β (E₂), testosterone (T), and progesterone (P₄) concentrations were assayed in all samples. In experiment 1, the mean plasma AMH concentration was 0.09 ± 0.003 ng/mL and did not show substantial fluctuation throughout the estrous cycle. In experiment 2, plasma AMH, ir-INH, and E₂ concentrations were significantly elevated in the GTCT group in comparison with the PC group; among these parameters, only the AMH concentrations were significantly higher in the GTCT group than in the COD group. The area under the receiver operating characteristic curve of AMH for diagnosis of GTCT was 0.99 and was significantly higher than that of ir-INH (P < 0.001) and E₂ (P < 0.01). Moreover, the AMH at a cutoff point of ≥0.36 ng/mL had the highest diagnostic accuracy (99.2%), sensitivity (100%), and specificity (99.1%) compared with the other tested parameters. In conclusion, plasma AMH concentration is probably a more reliable and sensitive biomarker for bovine GTCTs than the concentrations of ir-INH or ovarian steroids.     

Ethylene inhibitors enhance in vitro root formation on faba bean shoots regenerated on medium containing thidiazuron

The possible involvement of ethylene in in vitrorooting of faba bean (Vicia faba L.) shootsregenerated on medium containing thidiazuron wasinvestigated. The effects of the ethylene precursor1-aminocyclopropane-1-carboxylic acid (ACC) and threeethylene inhibitors, silver nitrate (AgNO₃),acetyl salicylic acid (ASA) and cobalt chloride(CoCl₂) on root formation were tested in vitrousing TDZ-induced shoots of faba bean accession 760.ACC inhibited root formation. In contrast, ethyleneinhibitors promoted root formation, AgNO₃ at theappropriate concentrations enhanced root emergence andincreased root number per shoot, root growth rate, androot length. Both CoCl₂ and ASA at theappropriate concentrations increased rootingefficiency. These promotive effects may result from areduction in ethylene concentration or inhibition ofethylene action. The results offer a new approach toimprove the rooting efficiency of TDZ-induced shootsof faba bean and possibly of other plant species.     

Heat Shock Cognate Protein 71-Associated Peptides Function as an Epitope for Toxoplasma gondii-Specific CD4+ CTL

HLA-DR-restricted CD4⁺ cytotoxic T-lymphocyte (CTL) lines specific for Toxoplasma gondii (T. gondii)-infected melanoma cells have been established from peripheral blood lymphocytes (PBLs) of a patient with chronic toxoplasmosis. The role of heat shock cognate protein (HSC) 71 in antigen (Ag) processing and presentation of T. gondii-infected melanoma cells to these CD4⁺ CTL lines was investigated. A human melanoma cell line (P36) pulsed with T. gondii-infected P36 cell-derived HSC71 was lysed by a T. gondii-specific CD4⁺ CTL line (Tx-HSC-1). The Tx-HSC-1 also killed T. gondii-infected P36 cells. The lytic activity of Tx-HSC-1 against P36 cells pulsed with T. gondii-infected P36 cell-derived HSC71 was inhibited by monoclonal antibodies (mAbs) against HSC71. Anti-human leukocyte antigen (HLA)-DR mAb also partially blocked the lytic activity, whereas anti-HLA-A,B,C mAb did not block the lytic activity. In addition, a flow cytometric analysis with these specific mAbs against HSC71 showed HSC71 to be expressed on the cell surface of T. gondii-infected P36 cells as well as uninfected P36 cells. These data indicate that HSC71 molecules are expressed on human melanoma cell line P36, and that HSC71 may play a potential role in Ag presentation and processing of T. gondii-infected P36 cells to CD4⁺ CTL.     

Effects of sulfur amino acids, <Emphasis Type="SmallCaps">l</Emphasis>-methionine, <Emphasis Type="SmallCaps">l</Emphasis>-cystine and <Emphasis Type="SmallCaps">l</Emphasis>-cysteine on lipoprotein lipase and hormone-sensitive lipase in differentiated mouse 3T3-L1 adipocytes

Rats subcutaneously implanted with AH109A hepatoma cells show hyperlipidemia with high concentrations of serum triglyceride and nonesterified fatty acid, suppression of lipoprotein lipase (LPL), and elevation of hormone-sensitive lipase (HSL) activities during the growth of the hepatoma. Supplementation of the diet with sulfur amino acids such as l-methionine (Met) and l-cystine (Cys) improved hyperlipidemia by restoring LPL and HSL activities. In the present study, we have attempted to examine the effects of sulfur amino acids on the activity and mRNA level of LPL and the activity of HSL using 3T3-L1 cells, which are known to differentiate to adipocytes. The adipocytes were incubated with various concentrations of Met, Cys or l-cysteine (CysH) in the absence or presence of tumor necrosis factor-α (TNF-α). LPL activity was suppressed by TNF-α. In the absence of TNF-α, Met, Cys and CysH did not change the LPL activity. In the presence of TNF-α, Met and Cys significantly increased the LPL activity, and Met also enhanced the LPL mRNA level. HSL activity was also suppressed by TNF-α. In the absence of TNF-α, Met enhanced the HSL activity. In the presence of TNF-α, Met, Cys and CysH suppressed the HSL activity. Sulfur amino acids such as Met, Cys and CysH affected the LPL activity, mRNA level, and HSL activity in 3T3-L1 adipocytes. Some of these effects of sulfur amino acids were different between LPL and HSL, between the absence and the presence of TNF-α, and between 3T3-L1 adipocytes and the adipose tissue from rats.     

A delicate interplay of structure, dynamics, and thermodynamics for function: a high pressure NMR study of outer surface protein A

Outer surface protein A (OspA) is a crucial protein in the infection of Borrelia burgdorferi causing Lyme disease. We studied conformational fluctuations of OspA with high-pressure ¹⁵N/¹H two-dimensional NMR along with high-pressure fluorescence spectroscopy. We found evidence within folded, native OspA for rapid local fluctuations of the polypeptide backbone in the nonglobular single layer β-sheet connecting the N- and C-terminal domains with τ << ms, which may give the two domains certain independence in mobility and thermodynamic stability. Furthermore, we found that folded, native OspA is in equilibrium (τ >> ms) with a minor conformer I, which is almost fully disordered and hydrated for the entire C-terminal part of the polypeptide chain from β8 to the C-terminus. Conformer I is characterized with ΔG⁰ = 32 ± 9 kJ/mol and ΔV⁰ = −140 ± 40 mL/mol, populating only ∼0.001% at 40°C at 0.1 MPa, pH 5.9. Because in the folded conformer the receptor binding epitope of OspA is buried in the C-terminal domain, its transition into conformer I under in vivo conditions may be critical for the infection of B. burgdorferi. The formation and stability of the peculiar conformer I are apparently supported by a large packing defect or cavity located in the C-terminal domain.     

Chemical analysis of late stages of pheomelanogenesis: conversion of dihydrobenzothiazine to a benzothiazole structure

Pheomelanogenesis is a complex pathway that starts with the oxidation of tyrosine (or DOPA, 3,4‐dihydroxyphenylalanine) by tyrosinase in the presence of cysteine, which results in the production of 5‐S‐cysteinyldopa and its isomers. Beyond that step, relatively little has been clarified except for a possible intermediate produced, dihydro‐1,4‐benzothiazine‐3‐carboxylic acid (DHBTCA). We therefore carried out a detailed study on the course of pheomelanogenesis using DOPA and cysteine and the physiological enzyme tyrosinase. To elucidate the later stages of pheomelanogenesis, chemical degradative methods of reductive hydrolysis with hydroiodic acid and alkaline peroxide oxidation were applied. The results show that: (1) DHBTCA accumulates after the disappearance of the cysteinyldopa isomers, (2) DHBTCA is then oxidized by a redox exchange with dopaquinone to form ortho‐quinonimine, which leads to the production of pheomelanin with a benzothiazine moiety, and (3) the benzothiazine moiety gradually degrades to form a benzothiazole moiety. This latter process is consistent with the much higher ratio of benzothiazole‐derived units in human red hair than in mouse yellow hair. These findings may be relevant to the (photo)toxic effects of pheomelanin.     

Linking temperature sensitivity of soil organic matter decomposition to its molecular structure,accessibility, and microbial physiology

Temperature sensitivity of soil organic matter (SOM) decomposition may have a significant impact on global warming. Enzyme‐kinetic hypothesis suggests that decomposition of low‐quality substrate (recalcitrant molecular structure) requires higher activation energy and thus has greater temperature sensitivity than that of high‐quality, labile substrate. Supporting evidence, however, relies largely on indirect indices of substrate quality. Furthermore, the enzyme‐substrate reactions that drive decomposition may be regulated by microbial physiology and/or constrained by protective effects of soil mineral matrix. We thus tested the kinetic hypothesis by directly assessing the carbon molecular structure of low‐density fraction (LF) which represents readily accessible, mineral‐free SOM pool. Using five mineral soil samples of contrasting SOM concentrations, we conducted 30‐days incubations (15, 25, and 35 °C) to measure microbial respiration and quantified easily soluble C as well as microbial biomass C pools before and after the incubations. Carbon structure of LFs (<1.6 and 1.6–1.8 g cm^?3) and bulk soil was measured by solid‐state ¹³C‐NMR. Decomposition Q₁₀ was significantly correlated with the abundance of aromatic plus alkyl‐C relative to O‐alkyl‐C groups in LFs but not in bulk soil fraction or with the indirect C quality indices based on microbial respiration or biomass. The warming did not significantly change the concentration of biomass C or the three types of soluble C despite two‐ to three‐fold increase in respiration. Thus, enhanced microbial maintenance respiration (reduced C‐use efficiency) especially in the soils rich in recalcitrant LF might lead to the apparent equilibrium between SOM solubilization and microbial C uptake. Our results showed physical fractionation coupled with direct assessment of molecular structure as an effective approach and supported the enzyme‐kinetic interpretation of widely observed C quality‐temperature relationship for short‐term decomposition. Factors controlling long‐term decomposition Q₁₀ are more complex due to protective effect of mineral matrix and thus remain as a central question.