Optimization of reverse chemical ecology method: false positive binding of Aenasius bambawalei odorant binding protein 1 caused by uncertain binding mechanism.

Odorant binding proteins (OBPs) are considered as the core molecular targets in reverse chemical ecology, which is a convenient and efficient method by which to screen potential semiochemicals. Herein, we identified a classic OBP, AbamOBP1 from Aenasius bambawalei, which showed high mRNA expression in male antennae. Fluorescence competitive binding assay (FCBA) results demonstrated that AbamOBP1 has higher binding affinity with ligands at acid pH, suggesting the physiologically inconsistent binding affinity of this protein. Amongst the four compounds with the highest binding affinities at acid pH, 2, 4, 4-trimethyl-2-pentene and 1-octen-3-one were shown to have attractant activity for male adults, whereas (-)-limonene and an analogue of 1-octen-3-ol exhibited nonbehavioural activity. Further homology modelling and fluorescence quenching experiments demonstrated that the stoichiometry of the binding of this protein to these ligands was not 1: 1, suggesting that the results of FCBA were false. In contrast, the apparent association constants (Ka) of fluorescence quenching experiments seemed to be more reliable, because 2, 4, 4-trimethyl-2-pentene and 1-octen-3-one had observably higher Ka than (-)-limonene and 1-octen-3-ol at neutral pH. Based on the characteristics of different OBPs, various approaches should be applied to study their binding affinities with ligands, which could modify and complement the results of FCBA and contribute to the application of reverse chemical ecology.

Supplementation dietary zinc levels on growth performance, carcass traits, and intramuscular fat deposition in weaned piglets.

This study was conducted to estimate dietary zinc (Zn) levels on growth performance, carcass traits, and intramuscular fat (IMF) deposition in weaned piglets. Sixty piglets were randomly divided into five groups, as follows: control (basal diet), Zn250, Zn380, Zn570, and Zn760 with supplementation of 250, 380, 570, and 760 mg Zn/kg of the basal diet, respectively. The final weight, average daily gain (ADG), gain/feed (G/F), lean meat percentage, fat meat percentage, lean eye area, backfat thickness, and IMF content were dose-dependently increased in all groups of Zn treatment. The serum total triglycerides (TG) and free fatty acid (FFA) were significantly higher in all Zn treatments than in the control. The enzyme activities of lipoprotein lipase (LPL), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC) were markedly higher, while enzyme activities of hormone-sensitive lipase (HSL) and carnitine palmitoyltransferase-1 (CPT-1) were significantly lower in all Zn treatments than in the control. The messenger RNA (mRNA) levels of sterol regulatory element-binding protein 1 (SREBP-1), stearoyl-CoA desaturase (SCD), FAS, ACC, peroxisome proliferator-activated receptor γ (PPARγ), LPL, and adipocyte fatty acid-binding protein (A-FABP) were significantly higher, while the mRNA levels of CPT-1 and HSL were significantly lower in all Zn treatments compared with the control. These results indicated that high levels of Zn increased IMF accumulation by up-regulating intramuscular lipogenic and fatty acid transport gene expression and enzyme activities while down-regulating lipolytic gene expression and enzyme activities.

[MK-801 attenuates NOS expression and NO content in the spinal cord of rats with inflammatory pain].

Using NADPH-d histochemistry, the effect of NMDA receptor antagonist MK-801 on the expression of nitric oxide synthase (NOS) in the dorsal horn of the spinal cord was investigated during inflammatory pain and hyperalgesia induced by injection of formalin into the right hind paw. The course of the change in the nitric oxide (NO) content of the lumbar intumescence was observed by measuring the ratio of nitrate/nitrite (NO3T/NO2T) and also the end product of NO. The results showed that the NOS expression and NO contents significantly increased 24 h after formalin injection, which were substantially inhibited when MK-801 was intrathecally injected 15 min prior to formalin injection or 12 h after formalin injection. The results suggest that the increases in the expression of NOS and NO contents in the dorsal horn of the spinal cord are mediated by activation of NMDA receptors during pain and hyperalgesia after formalin injection.

[Increase in amount and affinity of adenosine receptor in rat hippocampal cellular membranes induced by cerebral ischemic preconditioning and its protective effects on the neurons].

To determine the role of adenosine (ADO) receptor in the induction of brain ischemic tolerance, changes in amount and affinity of ADO receptor in rat hippocampal cellular membranes after transient ischemia were investigated using radioligand binding method. Ischemia for 6 min resulted in an apparent delayed neuron death (DND) in the hippocampus, while ischemia for 3 min did not cause DND. Preconditioning ischemia for 3 min could apparently decrease DND caused by ischemia for 6 min after the preconditioning at an interval of 1 d reperfusion. In correspondence with the histological changes, ischemia for 3 min caused an increase in amount and affinity of ADO receptor at 1 or 3 days after reperfusion (P<0.05 vs sham), while ischemia for 6 min caused a decrease in the amount and an increase in the affinity (P<0.05 vs sham). Compared with the rats suffering from ischemia for 6 min followed by reperfusion for 4 h and 1 or 3 d, the amount and affinity of ADO receptor increased in rats with preconditioning ischemia (3 min) 1 d before the ischemia for 6 min. The above results showed that cerebral ischemic preconditioning increased the amount and affinity of ADO receptor in hippocampal cellular membranes, and resisted the down-regulating of ADO receptor caused by severe ischemia, suggesting an important role of the increase in amount and affinity of ADO receptor in the induction of brain ischemic tolerance.

Promotion of pulmonary fibroblast proliferation and apoptosis by sodium nitroprusside.

The effects of nitric oxide (NO) donor sodium nitroprusside (SNP) on the proliferation and apoptosis and on Bcl-2, Bax and p53 proteins of pulmonary fibroblasts were investigated by using MTT cleavage assay, agarose gel electrophoresis and flow cytometric analysis. The results showed increases in the optical density (550 nm) of MTT cleavage assay, the number of cells and the proliferation index (PI), in comparison with the control. The number of apoptotic cells was also increased, though the percentage of apoptotic cells was too low to reveal oligonucleosomal fragmentation of characteristic ladder pattern, which is associated with apoptosis. In the meantime, the level of Bcl-2 decreased and that of Bax increased, while the p53 remained unchanged. These results suggest that exogenous NO has a dual effect on proliferation and apoptosis; and the action of NO on pulmonary fibroblasts is mainly proliferative. Down regulation of Bcl-2 and up regulation of Bax are implicated in the molecular mechanisms of this action.

Multiple forms of prostate-specific antigen in serum: differences in immunorecognition by monoclonal and polyclonal assays.

Prostate-specific antigen (PSA) in serum is primarily complexed with alpha 1-antichymotrypsin (alpha 1-ACT). However, 12-15% of prostate cancer (PCa) patients present with the predominant form being uncomplexed (free) PSA (Lilja et al., Clin Chem 1991;37:1618-24). We report that commercial immunoassays demonstrate variations in reactivity, especially to the uncomplexed form. We fractionated and analyzed commercial controls, PSA complexes prepared in vitro, and sera from patients with PCa or benign prostatic hyperplasia, using molecular sieve chromatography and Hybritech Tandem-R, Abbott IMx, and Ciba Corning ACS PSA assays. Peak integration of PCa samples demonstrated ACS:Tandem-R ratios of 1-1.3 for PSA/alpha 1-ACT complex. In contrast, ratios of uncomplexed peaks ranged from 2 to 4, suggesting a greater reactivity of the uncomplexed form in the ACS PSA assay. Discrepancies between assays, when PSA was measured in unfractionated sera, correlated directly with the percentage of the uncomplexed form. In controls, fractionation revealed the presence of uncomplexed PSA only, with ratios of ACS:Tandem-R and IMx:Tandem-R of 3:1 and 1.8:1, respectively. Immunoblots of PCa sera detected uncomplexed PSA (approximately 30 kDa) and PSA complexes of approximately 95 kDa (PSA/alpha 1-ACT) and > 200 kDa, indicative of alpha 2-macroglobulin. Maximal recognition of all forms of PSA may be important for early detection of disease progression.

Direct evidence for translational regulation by leader RNA and Tat protein of human immunodeficiency virus type 1.

Translational effects of the RNA leader and Tat protein of human immunodeficiency virus type 1 (HIV-1) were investigated in rabbit reticulocyte lysate. Hybrid RNA species with natural or mutated HIV-1 leader fused to human interferon- gamma mRNA were produced in vitro from recombinant plasmids. HIV-1 leader RNA was found to inhibit translation through two mechanisms. A 3-fold trans-inhibition of translation was demonstrated by mixing hybrid HIV-1 leader RNA with indicator interferon mRNA. By comparison, HIV-1 leader caused a 50-fold cis-inhibition in lysate in which two trans-inhibitory factors, double-stranded RNA-dependent protein kinase and (2'-5')oligoadenylate synthetase, were suppressed. In contrast, purified HIV-1 Tat protein produced in Escherichia coli enhanced by 4-fold translation from HIV-1 leader-interferon mRNA but not from interferon mRNA lacking HIV sequences or from total poly(A)+ RNA. Translation of mRNA containing either a single base substitution in the loop of the "trans-acting responsive" sequence (TAR) or an alternative stem-loop in TAR was nevertheless stimulated by Tat. The enhancement of translation by Tat was largely due to relief of cis-inhibition, since the effect was found even in lysate in which double-stranded RNA-dependent protein kinase was inhibited with 2-aminopurine. These results suggest that translation is an important level of control in the replication cycle of HIV-1.

[The discovery and study on invasive strain of Escherichia coli O121].

A strain of Enteroinvasive Escherichia coli was isolated from the stool with blood and mucus of a child suffering from acute diarrhea. The strain shows the following characteristics: rapid fermentation of glucose (with gas), no fermentation of lactose, beta-galactosidase reaction positive, growth in acetate media, lysine decarboxylase negative, non-motility causing keratoconjunctivitis in guinea pigs and invading into epithelial cells, with a plasmid of 140 Md, Serotype is O121:H- which is a new serotype of Enteroinvasive Escherichia coli.