Neuropeptide Y content in the hypothalamic paraventricular nucleus responds to fasting and refeeding in broiler chickens

To examine the neural mechanism by which hypothalamic neuropeptide Y (NPY) regulates energy homeostasis and feeding behavior in commercial broilers, we measured NPY content in several hypothalamic regions of birds that were fasted and then refed. After fasting for 48 and 72 h, body weight significantly decreased, and food intake significantly increased during the subsequent refeeding. The lost body weight was not restored to ad libitum feeding levels even after 3 days of refeeding. Plasma glucose concentration and body fat content significantly decreased and plasma non-esterified fatty acid (NEFA) concentration significantly increased after 48- and 72-h fasting. Refeeding for 24 h restored plasma metabolites and body fat content to pre-fasting levels. NPY content in the paraventricular nucleus (PVN) and infundibular nucleus significantly increased during fasting, and NPY content of the PVN was restored to pre-fasting levels after 24-h refeeding. However, there was no significant change in the NPY content of the lateral hypothalamic area during fasting or refeeding. The present results of changes in the hypothalamic NPY content during fasting and refeeding support the hypothesis that NPY plays a central role in regulation of energy homeostasis, with especially important effect on feeding behavior and body weight in broiler chickens.     

Distinctive iron requirement of tryptophan 5-monooxygenase: TPH1 requires dissociable ferrous iron

A peripheral type of tryptophan 5-monooxygenase (EC 1.14.16.4), TPH1, is very unstable in vitro, but the inactivation was reversible and full reactivation occurs upon anaerobic incubation with a high concentration of dithiothreitol (DTT, 15 mM). In this study, distinctive iron requirement of TPH1 was revealed through analysis of the enzyme's inactivation and activation by DTT. For this purpose, all the glasswares, plastics, Sephadex G-25 gels, and reagents including protein solutions had been treated with metal chelators, and apo-TPH was prepared by treatment with EDTA. Apo-TPH thus prepared exclusively required free Fe2+ for its catalytic activity; 10(-8) M was enough under the strict absence of Fe3+ but 10(-12) M was too low. No other metal ions including Fe3+ were effective. It appeared that Fe3+ bound to the enzyme with a higher affinity than Fe2+, resulting in the inactivation. Ascorbate, a non-thiol reducing agent, did not substitute DTT in the activation of TPH1, but enhanced the Fe2+-dependent activity of apo-TPH as effectively as DTT. Thus, the DTT-activation was essentially substituted by preparation of apo-TPH by the EDTA treatment and the assay of apo-TPH in the presence of Fe2+ and ascorbate. The activation of TPH1 by incubation with DTT was accompanied by exposure of 9 sulfhydryls out of the total 10 cysteine residues, but the cleavage of disulfide bonds seemed not to be crucial, even if it occurred. The effect of DTT was substituted by some other sulfhydryls whose structure was analogous to that of commonly used metal chelators. Based on these observations, the following dual roles of DTT are proposed: (1) in the activation of TPH, DTT removes inappropriate bound iron (Fe3+) as a chelator, keeping Fe3+ away from the enzyme's binding site which needs to bind Fe2+ for the catalytic activity, and (2) in both the activation and reaction processes, DTT prevents oxidation of Fe2+ to Fe3+ as a reducing agent.     

Efficacy of antibodies against a chimeric synthetic peptide encompassing epitopes of bonnet monkey (Macaca radiata) zona pellucida-1 and zona pellucida-3 glycoproteins to inhibit in vitro human sperm-egg binding

Immunocontraception achieved by immunization with zona pellucida (ZP) glycoproteins is invariably associated with ovarian dysfunction. Use of ZP glycoprotein-based synthetic peptides as immunogens has been proposed to overcome adverse side effects on ovaries. In the present study, a chimeric peptide encompassing the epitopes of bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1; amino acid residues 251-273) and ZP glycoprotein-3 (bmZP3; amino acid residues 324-347), separated by a tri-glycine spacer, was synthesized and conjugated to diphtheria toxoid (DT). Immunization of female BALB/cJ mice and bonnet monkeys with the chimeric peptide led to generation of antibodies that reacted with the chimeric peptide, individual bmZP1 & bmZP3 peptides, and also recombinant bmZP1 and bmZP3 proteins expressed by E. coli in an ELISA. Indirect immunofluorescence studies revealed that the immune serum also recognized human as well as bonnet monkey ZP. A significant inhibition of human sperm binding to ZP was observed with antibodies generated against the chimeric peptide in mice (P = 0.0001) as well as monkeys (P = 0.0002) in a hemizona assay (HZA). The inhibition efficacy was significantly higher than that observed by using antibodies against the individual bmZP1 and bmZP3 peptides. Interestingly, no ovarian pathology was observed in female bonnet monkeys immunized with the chimeric peptide. These studies have demonstrated that the chimeric peptide encompassing peptides of multiple ZP glycoproteins may be a promising candidate antigen for designing immunocontraceptive vaccines.     

Inhibition of preadipocyte differentiation by germacranolides from Calea urticifolia in 3T3-L1 cells

The effects of germacranolides isolated from Calea urticifolia on adipocytic differentiation of 3T3-L1 cells were examined. These germacranolides inhibited adipogenesis at a concentration of 1.25-5 microM. But no inhibitory activity against cell proliferation and no nonspecific binding activity to protein were observed. These results indicate that these germacranolides are the specific inhibitors of preadipocyte differentiation.     

Purification and characterization of a yeast carbonyl reductase for synthesis of optically active (R)-styrene oxide derivatives

Optically active styrene oxide derivatives are versatile chiral building blocks. Stereoselective reduction of phenacyl halide to chiral 2-halo-1-phenylethanol is the key reaction of the most economical synthetic route. Rhodotorula glutinis var. dairenensis IFO415 was discovered on screening as a potent microorganism reducing a phenacyl halide to the (R)-form of the corresponding alcohol. An NADPH-dependent carbonyl reductase was purified to homogeneity through four steps from this strain. The relative molecular mass of the enzyme was estimated to be 40,000 on gel filtration and 30,000 on SDS-polyacrylamide gel electrophoresis. This enzyme reduced a broad range of carbonyl compounds in addition to phenacyl halides. Some properties of the enzyme and preparation of a chiral styrene oxide using the crude enzyme are reported herein.     

Tryptophan at position 104 is involved in transforming light signal into changes of beta-sheet structure for the signaling state in the BLUF domain of AppA

AppA is a member of an FAD-based new class blue-light sensory protein known as sensor of blue light using FAD (BLUF) protein. The spectroscopic properties of an AppA BLUF domain (AppA126), in which the tryptophan residue at position 104 had been replaced with alanine (W104A), were characterized. The W104A mutant AppA126 showed a nearly normal absorption red shift in the FAD UV-visible absorption upon illumination; however, the light state relaxed to the dark state at a rate approximately 150 times faster than that of wild-type AppA126. Light-induced structural changes of FAD and apoprotein in the wild-type and mutant AppA126 were studied by means of light-induced Fourier transform infrared (FTIR) difference spectroscopy using AppA126, in which the apoprotein had been selectively labeled with 13C. The light-induced FTIR spectrum of the W104A mutant AppA126 revealed bands corresponding to a C4 = O stretch of the FAD isoalloxazine ring and structural changes of apoprotein, but with some alterations in the bands' features. Notably, however, prominent protein bands at 1,632(+)/1,619(-) cm(-1) caused by changes in the beta-sheet structure were eliminated by the mutation, indicating that Trp104 is responsible for transforming the light signal into a specific beta-sheet structure change in the apoprotein of the AppA BLUF domain in the signaling state.     

EBV-specific CD8+ T cell memory: relationships between epitope specificity, cell phenotype, and immediate effector function

EBV infection in humans induces CD8+ T cell memory to viral epitopes derived from both lytic and latent cycle Ags. We have analyzed the relationship between the phenotype and function of the memory pool of T cells specific for these Ags. Lytic epitope-specific populations were heterogeneous in terms of CD45RO/RA and CD28 expression, whereas latent epitope-specific populations were uniformly CD45RO+ and CD28+, consistent with the higher antigenic challenge from lytic epitopes driving some memory cells toward a CD45RA+, CD28- phenotype. However, both types of memory population showed immediate epitope-specific cytotoxicity and type 1 cytokine production in ex vivo assays. Cytotoxic function was not associated with preactivated T cells, as EBV-specific populations were negative for activation markers such as CD69 or CD38, nor could cytotoxic function be ascribed to CD27- or CD56+ subsets, as such cells were not detected in EBV-specific memory. Furthermore, cytotoxicity was not limited to CD45RA+ and/or CD28- fractions, but also was observed in CD45RO+, CD28+ populations in lytic and latent epitope-specific memory. Cytokine (IFN-gamma, TNF-alpha) responses, measured by intracytoplasmic staining after peptide stimulation, also were detectable in CD45RO+ and RA+ subsets as well as CD28+ and CD28- subsets. Of other markers that were heterogeneous in both lytic and latent epitope populations, CCR7 gave the best discrimination of functionality; thus, CCR7+ cells consistently failed to give an IFN-gamma or TNF-alpha response, whereas many CCR7- cells were responsive. Our data are consistent with effector functions having a broad distribution among phenotypically distinct subsets of "effector memory" cells that have lost the CCR7 marker.