A cDNA for a protein that interacts with the human immunodeficiency virus Tat transactivator

The human immunodeficiency virus (HIV) tat protein (Tat) is a positive regulator of virus gene expression and replication. Biotinylated Tat was used as a probe to screen a lambda gt11 fusion protein library, and a complementary DNA encoding a protein that interacts with Tat was cloned. Expression of this protein, designated TBP-1 (for Tat binding protein-1), was observed in a variety of cell lines, with expression being highest in human cells. TBP-1 was localized predominantly in the nucleus, which is consistent with the nuclear localization of Tat. In cotransfection experiments, expression of TBP-1 was able to specifically suppress Tat-mediated transactivation. The strategy described may be useful for direct identification and cloning of genes encoding proteins that associate with other proteins to modulate their activity in a positive or negative fashion.     

Adipsin and complement factor D activity: an immune-related defect in obesity

Adipsin is a serine protease that is secreted by adipocytes into the bloodstream; it is deficient in several animal models of obesity, representing a striking example of defective gene expression in this disorder. Recombinant mouse adipsin was purified and its biochemical and enzymatic properties were studied in order to elucidate the function of this protein. Activated adipsin has little or no proteolytic activity toward most substrates but has the same activity as human complement factor D, cleaving complement factor B when it is complexed with activated complement component C3. Like authentic factor D, adipsin can activate the alternative pathway of complement, resulting in red blood cell lysis. Decreased (58 to 80 percent) complement factor D activity, relative to lean controls, was observed as a common feature of several experimental models of obesity, including the ob/ob, db/db, and monosodium glutamate (MSG)-injected mouse and the fa/fa rat. These results suggest that adipsin and the alternative pathway of complement may play an unexpected but important role in the regulation of systemic energy balance in vivo.     

Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists

Blood lymphocyte numbers, essential for the development of efficient immune responses, are maintained by recirculation through secondary lymphoid organs. We show that lymphocyte trafficking is altered by the lysophospholipid sphingosine-1-phosphate (S1P) and by a phosphoryl metabolite of the immunosuppressive agent FTY720. Both species were high-affinity agonists of at least four of the five S1P receptors. These agonists produce lymphopenia in blood and thoracic duct lymph by sequestration of lymphocytes in lymph nodes, but not spleen. S1P receptor agonists induced emptying of lymphoid sinuses by retention of lymphocytes on the abluminal side of sinus-lining endothelium and inhibition of egress into lymph. Inhibition of lymphocyte recirculation by activation of S1P receptors may result in therapeutically useful immunosuppression.     

Isolation and identification of stilbenes in two varieties of Polygonum cuspidatum

The roots of two varieties of Polygonum cuspidatum (Hu Zhang and Mexican Bamboo) were analyzed for resveratrol and analogues. The roots of each variety were dried and ground into a powder. The powdered roots were then extracted with methanol and ethyl acetate. The ethyl acetate fraction of the Mexican Bamboo was then subjected to fractionation and purification using silica gel column chromatography and semipreparative HPLC. In addition to resveratrol (3,5,4'-trihydroxystilbene), three stilbene glucosides were identified by (1)H NMR, (13)C NMR, and MS. The stilbene glucosides were shown to be a piceatannol glucoside (3,5,3', 4'-tetrahydroxystilbene 4'-O-beta-D-glucopyranoside), resveratroloside (3,5,4'-trihydroxystilbene 4'-O-beta-D-glucopyranoside), and piceid (3,5,4'-trihydroxystilbene 3-O-beta-D-glucopyranoside). The levels of the piceatannol glucoside and piceid were twice as high in the Mexican Bamboo as compared to the Hu Zhang.     

Analysis of theaflavins and thearubigins from black tea extract by MALDI-TOF mass spectrometry

Black tea contains two major groups of pigments, theaflavins (TFs) and thearubigins (TRs). TFs contain a bis-flavan substituted 1,2-dihydroxy-3,4-benzotropolone moiety. Unlike the TFs, TRs have not yet been characterized. The chemical structure of the TRs remains a mystery. The present paper reports our effort to study the structure of TFs and TRs using delayed pulsed ion extraction of ions generated via the matrix-assisted laser desorption ionization (MALDI) technique, on line with a Linear time-of-flight (TOF) mass spectrometer. Spectra of standard TFs show not only pseudomolecular ions but also ions resulting from fragmentation. The analysis of MALDI-TOF spectra of black tea fractions shows the structure of some TRs, which are similar to those of TFs because the same loss of mass is observed.     

Determination of linuron in potatoes using capillary column gas chromatography/mass spectrometry

A convenient method for the determination of the N-methyl,N-methoxy-phenylurea herbicide (linuron) in potatoes has been developed. The herbicide is extracted from potatoes using a slightly modified Luke multiresidue procedure. The extract is analyzed directly by gas chromatography with cold on-column injection, using an ion trap mass spectrometer in the chemical ionization mode as the detector. Quantitation is performed using p-bromonitrobenzene as the internal standard. The limit of detection is 0.1 ppm. Recoveries of linuron in potatoes averaged 112 +/- 6% at the 0.5 ppm level, and 110 +/- 2% at the 0.2 ppm level. No linuron residues were found in 25 potato samples that were analyzed by this method. Two other N-methyl,N-methoxy-phenylurea herbicides, metobromuron and chlorbromuron, are also sufficiently stable to be determined by this method, but the N,N-dialkyl-phenylurea herbicides neburon, diuron, and monuron are too thermally unstable and degrade in the gas chromatograph.     

Identification of six phenylpropanoids from garlic skin as major antioxidants

The extract of garlic skins (peels) showed strong antioxidant activity, and some responsible constituents were isolated and identified. Garlic (Allium sativum L.) has been used as an herbal medicine, but there is no report on the health benefits of the skin or peel. In this study, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of garlic skin extract was evaluated. Using chromatographic techniques, the active constituents were isolated and subsequently identified. Analyses by high-performance liquid chromatography coupled with a photodiode array detector (HPLC-PDA) suggested that these compounds were phenylpropanoids, which had a characteristic absorbance at 300-320 nm. Liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance analyses allowed the chemical structures of the isolated constituents to be postulated. The proposed compounds were subsequently synthesized and compared with the constituents in the extract using HPLC-PDA and LC-MS. N-trans-Coumaroyloctopamine, N-trans-feruloyloctopamine, guaiacylglycerol-beta-ferulic acid ether, and guaiacylglycerol-beta-caffeic acid ether were identified as were trans-coumaric acid and trans-ferulic acid. Also, the antioxidant activities of these compounds were determined.