[Deletion of Aldh4a1 Leads to Impaired Sperm Maturation in Mice].

ALDH4A1, a member of the aldehyde dehydrogenase superfamily, is a key enzyme in the mitochondrial proline metabolism pathway. Recent studies have shown that mutations in aldh4a1 lead to reduced fertility and reproductive premature aging of male nematodes. However, the effect of ALDH4A1 on fertility of male mice has not been studied. In this study, we used CRISPR-Cas9 technology to construct a knockout mouse model of Aldh4a1 for the first time to explore the effect of this gene on the reproduction of male mice. The results showed that compared with WT male mice, Aldh4a1^(-/-) male mice were fertile, had normal spermatogenesis but defect in sperm maturation in the epididymis documented by impaired motility, increased morphological abnormalities and increased spontaneous acrosome reaction. In addition, transmission electron microscopy showed vacuoles in the sperm mitochondria, and fracture in the neck of sperms and vacuoles in these mice. These results revealed that ALDH4A1 plays a vital role in the structure of sperm flagellum and the process of sperm maturation in mice.

[Effect of cigarette smoke exposure on the expression of CCR7 and levels of Th1/Th2 cytokines in asthmatic rats].

Objective: To investigate the effect of cigarette smoke exposure on the expression of CC Chemokine receptor 7 (CCR7) and levels of Th1/Th2 cytokines in asthmatic rats. Methods: Forty Wistar rats were randomly divided into four groups: control group, asthma group, smoke exposure group, asthma-smoke exposure group. The asthma group were sensitized with ovalbumin (OVA) and Aluminum hydroxide at day 1, 8 and challenged with OVA at day 15 by atomization for 8 weeks.While control group was sensitized and challenged with normal saline instead of OVA.The smoke exposure group was sensitized and challenged with normal saline instead of OVA followed passive smoking for 8 weeks. The asthma-smoke exposure group was challenged with OVA followed passive smoking. The pathological changes of different groups were observed by HE-staining. CCR7 was semiquantitatively analyzed in lungs by immunohistochemistry.The concentration of CC chemokine ligand (CCL)19, CCL21, interferon (IFN)-γ and interleukin (IL)-4 in peripheral blood and CCL19 and CCL21 in bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent (ELESA) assay. Results: In asthma group, smoke exposure group and asthma-smoke exposure group, the various degrees of inflammatory reaction appeared in lung tissue and the asthma-smoke exposure group was with the most significant reaction. In the lung tissues of the rats from asthma group, smoke exposure group and asthma-smoke exposure group, the average optical density (AOD) of CCR7 were significantly higher than those in control group (0.350±0.023, 0.252±0.022, 0.400±0.029 vs 0.180±0.020, all P<0.01). The AOD of CCR7 of asthma-smoke exposure group was much higher than both that in asthma group and in smoke exposure group (both P<0.01). In asthma group, smoke exposure group and asthma-smoke exposure group, the concentrations of both CCL19 and CCL21 in peripheral blood and BALF were significantly higher than that in control group (all P<0.01). The concentrations of both CCL19 and CCL21 in peripheral blood and BALF of asthma-smoke exposure group were significantly higher than the results in asthma group and in smoke exposure group (all P<0.01). The concentrations of IFN-γ in peripheral blood of asthma group and asthma-smoke exposure group were lower than those in control group [(33±3), (17±3) vs (70±4) pg/ml], but asthma-smoke exposure group was much lower than the results in asthma group (all P<0.01). The concentration of IFN-γ in peripheral blood of smoke exposure group[(100±5)pg/ml]was higher than that in control group and asthma-smoke exposure group (both P<0.01). In asthma group, smoke exposure group, asthma-smoke exposure group, the concentrations of IL-4 in peripheral blood were significantly higher than those in control group [(54±4), (42±4), (76±4) vs (30±4) pg/ml, all P<0.01]. The concentrations of IL-4 in peripheral blood of asthma-smoke exposure group was significantly higher than those in asthma group and in smoke exposure group (both P<0.01). Conclusion: Cigarette smoke could enhance the expression of CCR7 and its ligand, and it can also result in exacerbations of asthma by reducing the expression level of IFN-γ (the representative of Th1 cytokine) and increasing the expression level of IL-4 (the representative of Th2 cytokine).

HPV16 infection promotes an M2 macrophage phenotype to promote the invasion and metastasis of esophageal squamous cell carcinoma.

OBJECTIVES: High-risk human papillomavirus (HR-HPV) is an important risk factor for esophageal cancer. Macrophages constitute a crucial immune medium for regulating HPV-related tumors; however, the specific regulatory mechanisms remain unknown. Therefore, the purpose of our current study was to investigate the mechanism by which HPV16E6 regulates macrophages to promote the invasion and metastasis of esophageal cancer. METHODS: HPV16E6 infection was detected by polymerase chain reaction. Immunohistochemistry was used to verify the distribution of tumor-associated macrophages (TAMs) and MMP-9 expression in esophageal squamous cell carcinoma tissues (ESCCs), and cancer adjacent normal tissues (CANs) from Kazakh patients. ESCC cells were transfected with a plasmid over-expressing HPV16E6 and non-contact cocultured with macrophages. RESULTS: The infection rate of HPV16E6 in Kazakh ESCCs was clearly higher than that in CANs (P < 0.05). The density of CD163-positive TAMs was significantly positively correlated with HPV16E6 infection in ESCCs (P < 0.05). After coculturing macrophages and EC9706 cells transfected with the HPV16E6 plasmid, the phenotype of macrophages transformed into M2 macrophages. The migration and invasion ability of ESCC cells were higher in the HPV16E6-transfected and coculture group than in the HPV16E6 empty vector-transfected and non-cocultured HPV16E6-transfected groups (all P < 0.05). The density of M2-like TAMs in ESCCs was positively correlated with the level of MMP-9 expression. MMP-9 expression in the HPV16E6-ESCC coculture macrophages group was substantially higher than that in controls (all P < 0.05). CONCLUSIONS: HPV16 infection mediates tumor-associated macrophages to promote ESCC invasion and migration.

Nitric oxide is involved in phosphorus deficiency-induced cluster-root development and citrate exudation in white lupin.

*White lupin (Lupinus albus) forms specialized cluster roots characterized by exudation of organic anions under phosphorus (P) deficiency. Here, the role of nitric oxide (NO) in P deficiency-induced cluster-root formation and citrate exudation was evaluated. *White lupin plants were treated with the NO donor sodium nitroprusside (SNP) and scavenger or inhibitor of NO synthase under conditions of P deficiency (0 muM) or P sufficiency (50 muM). *Phosphorus deficiency enhanced NO production in primary and lateral root tips, with a greater increase in cluster roots than in noncluster roots. NO concentrations decreased with cluster root development from the pre-emergent stage, through the juvenile stage, to the mature stage. The P deficiency-induced increase in NO production was inhibited by antagonists of NO synthase and xanthine oxidoreductase, suggesting the involvement of these enzymes in NO production. SNP markedly increased the number of cluster roots. Citrate exudation from different root segments in P-deficient roots was positively correlated with endogenous root NO concentrations. *These findings demonstrate differential patterns of NO production in white lupin, depending on root zone, developmental stage and P nutritional status. NO appears to play a regulatory role in the formation of cluster roots and citrate exudation in white lupin under conditions of P deficiency.

Nociception, enkephalin content and dipeptidyl carboxypeptidase activity in brain of mice treated with exopeptidase inhibitors.

Thiorphan (60 micrograms intracerebrally) increased the met5-enkephalin content of mouse striatum by 30% in 30 min. This increase was no longer evident at 1 hr. If the dipeptidyl carboxypeptidase, inhibited by thiorphan, were located extraneuronally as suggested by De La Baume, Patey and Schwartz (1981), the met5-enkephalin accumulation represents the rate at which the pentapeptide is released extraneuronally. The increase in met5-enkephalin content was accompanied by an inhibition, greater than 80%, of the dipeptidyl carboxypeptidase that degrades striatal met5-enkephalin. Such an inhibition lasted longer then 2 hr. Thiorphan, given to mice intracerebrally, prolonged the latency time to jump off a 54 degree plate. The effects of thiorphan on brain met5-enkephalin content and hot plate latencies were significantly potentiated by bestatin, which inhibits aminopeptidase B and leucine aminopeptidase.

Dynorphin receptor in the blood vessel.

Using (3H)etorphine, (3H)E, in binding studies, the KD and Bmax for rabbit mesentery and aorta membrane preparations were 0.61 nM and 0.17 fmol/mg tissue respectively, while it was 0.30 nM and 12 fmol/mg tissue in the brain. The IC50 of dynorphin (1-13) (D1-13) for displacing (3H)E binding in the blood vessel was 20 +/- 2.8 nM (S,E,M,), while PLO17, D-ala2-D-leu5-enkephalin (DADLE) and met5-enkephalin-arg6-phe7 showed very weak inhibition (IC50 greater than 1000nM) though they displaced (3H)E binding very well in the brain. In vitro study showed that D1-13 inhibited electric field stimulation induced vasoconstriction with an IC50 of 53 +/- 12 (rabbit ear artery) and 510 +/- 120 (dog mesenteric artery)nM. Such effect was partially reversed by 1 microM of naloxone. D-ala2-met5-enkephalin and metorphamide displayed much weaker inhibition and DADLE was completely ineffective at doses up to 1 microM. D1-13 did not antagonize noradrenaline (NA) induced vasoconstriction, while phentolamine could abolish vasoconstriction induced either by stimulation or by NA. The result suggests that D1-13 acts presynaptically on neuronal kappa receptor in the blood vessel and inhibits NA release, thus causes vasodilation.

Lower CSF level of dynorphin(1-8) immunoreactivity in schizophrenic patients.

Using a highly specific and sensitive radioimmunoassay for dynorphin(1-8) (D 1-8), and a singly blind test design, we measured D(1-8) immunoreactivity (ir D 1-8) in CSF of 35 first break cases of acute schizophrenic patients. All patients were free of psychotropic medication for at least one week before the study. Another 31 neurological patients suffered from cervical arthrosis, tumor, myelopathy etc. were studied as controls. The ir D(1-8) in CSF of schizophrenic patients were significantly lower than that of controls (91.8 +/- 5.6, means +/- S.E.M., and 131.9 +/- 6.8 fmol/ml CSF respectively, p less than 0.001).

The analgesic and respiratory depressant actions of metorphamide in mice and rabbits.

Metorphamide (MET) elicited a potent, dose-dependent analgesia and respiratory depression in mice and rabbits. MET induced-analgesia was naloxone reversible and potentiated by bestatin. Naloxonazine, a relatively selective mu 1 blocker, at certain dosage (50 micrograms per rabbit, icv), could abolish the analgesia but not the respiratory inhibition produced by MET. Our result indicates that mu 1 receptors mediate the MET induced-analgesia but not its respiratory effect. Since MET is a mu- and kappa-ligand with very low delta activity, the MET induced respiratory depression may be mediated by mu 2 or kappa binding sites.

Ontogeny of opioid pharmacology and receptors: high and low affinity site differences.

The development of the high affinity binding of a variety of opiates and enkephalins is distinct from low affinity binding. During the first 2 weeks after birth, low affinity binding in both brain and spinal cord remains relatively constant while high affinity binding increases up to 3-fold. Differences in the development of analgesic and respiratory sensitivity to opiates are also found. Whereas morphine, beta-endorphin and D-Ala2-Met5-enkephalin-amide depress respiratory rates in 2-day old rats at doses equal to or lower than those active in 14-day old rats, morphine's analgesic ED50 is 40-fold greater in 2-day old than in 14-day old rats. Similarly, beta-endorphin and D-Ala2-Met5-enkephalinamide are analgesic in 14-day old rats but not 7-day old rats. The small effect of spinal transections on morphine analgesia in 14-day old rats suggests that the change in analgesic sensitivity is at a segmental spinal level and not a result of descending pathways. These results suggest an interesting correlation between high affinity binding and analgesia and between low affinity binding and respiratory effects.

Met5-enkephalin-arg6-phe7 and its receptor in lung.

The presence of met5-enkephalin-arg6-phe7 (YGGFMRF) and opiate receptors in rat, guinea pig and human lung was investigated with specific and sensitive radio immuno- and radio-receptor assays. 1) High and low molecular weight YGGFMRF-like immunoreactivity were detected in lung extracts using Bio-Gel P-2 column chromatography followed by radioimmunoassay. Using HPLC, we determined that the low molecular weight YGGFMRF-like immunoreactivity is authentic YGGFMRF. 2) The contents of YGGFMRF were 0.68 +/- 0.08, 0.76 +/- 0.12 and 0.63 pmol/mg protein in lung of rat, guinea pig and human, respectively. In the lung of these three species, the content of YGGFMRF is much greater than that of met5-enkephalin. 3) 47 mM KCl released YGGFMRF from rat lung slices in a Ca++ dependent manner. 4) Rat lung membranes were shown to bind [3H]-etorphine in a saturable manner. There are two populations of binding sites with a Kd = 0.6 and 7.1 nM and a Bmax = 7.8 and 28.5 fmol/mg protein, respectively. This binding could be displaced by YGGFMRF with high affinity, the other endogenous opioid peptides were poor displacers. From these results, we infer that YGGFMRF might be a putative neurotransmitter or neuromodulator, its role in the regulation of lung function can now be investigated.

Dextrorphan: an antagonist for phencyclidine receptors.

Radio-binding assay, bioassay and HPLC detection were used to observe the antagonistic effects of dextrorphan on PCP's actions. Dextrorphan displayed high affinity to PCP receptor in the rabbit mesenteric blood vessels. It had weak PCP-like bioactivity, but could antagonize PCP's action dose-dependently in vitro study with the rabbit ear artery preparation and shifted the dose-response curve of PCP to the right. After PCP administration, the content of norepinephrine in the vascular bath medium was increased, which was reversed by dextrorphan. Thus suggests that dextrorphan is an antagonist with very mild agonistic action for PCP receptors.

[Mechanism of dynorphin inhibition on vasoconstriction in vitro].

Bioassay and HPLC detection were used to analyze the mechanism of inhibition of stimulation-induced vasoconstriction by dynorphin 1-13 (D1-13). Bioassay showed that D1-13 inhibited the contraction of rabbit ear artery and mesenteric artery induced by electrical field stimulation with IC50s of 8.5 +/- 1.2 x 10(-8) mol/L (n = 4) and 5.02 +/- 1.3 x 10(-7) mol/L (n = 5), respectively. D1-13 was ineffective in rabbit femoral artery at a concentration even larger than 10(-6) mol/L. D1-13 did not alter the basal tension of the blood vessel, nor the vasoconstriction induced by adding norepinephrine (NE) into the bath medium, and both constriction were markedly inhibited by phentolamine, an alpha-adrenoceptor blocker. With HPLC detection, the contents of NE in the bath medium were significantly reduced by D1-13 (5 x 10(-7) mol/L) from 340.56 +/- 73.13 pg/ml to 76.91 +/- 10.26 pg/ml as compared with control group (P less than 0.05). The effect could be completely reversed by naloxone at a concentration of 10(-6) mol/L (P less than 0.05). The results suggest that D1-13 reduces stimulation-induced vasoconstriction probably through a presynaptic inhibition of NE release from the nerve terminals.

Roles of opioid peptides of PAG in analogous electro-acupuncture inhibition of experimental arrhythmia: analyzed by specific antisera microinjection.

Using specific antisera microinjected into periaqueductal grey (PAG), we further studied the mechanism of the analogous electroacupuncture, deep peroneal nerve stimulation (DPNS), inhibition on the experimental arrhythmia, hypothalamic stimulation-induced ventricular extrasystoles (HVE). Neither normal serum nor anti-leu-enkephalin antiserum interfered with DPNS inhibition; anti-dynorphin A (1-8) tended to attenuate the inhibition; and anti-beta-endorphin antiserum blocked it. The results provided further evidence for the hypothesis that beta-endorphin in PAG plays an important role in the neural circuit of DPNS inhibition on HVE.

The effect of acupuncture on blood pressure: the interrelation of sympathetic activity and endogenous opioid peptides.

Experimental hypertension was produced by intravenous infusion of norepinephrine in 13 conscious dogs. Electroacupuncture at the dogs' "Tsu San-Li" (St. 36) points showed a significant decrease in blood pressure, while the heart rate was not affected. The depressor effect was naloxone (0.2 mg/kg iv) reversible, and accompanied by an increased blood flow at the mesenteric artery, so it is suggested that the depressor effect was due to inhibition of the sympathetic vasoconstrictor tone. This inhibition was mediated by endogenous opioid peptides released by acupuncture. The location of this mediation was further analyzed. The central mechanism was evidenced by the ineffectiveness of acupuncture in reducing blood pressure in anesthetized dogs. On the other hand, the demonstration of opiate receptors in the blood vessels by radio-receptor assay provided evidence of peripheral mediation of blood pressure by opioids. The blood vessel might be a target organ for the plasma opioids, which can also be increased by acupuncture.

Inhibitory effect of analogous electro-acupuncture on experimental arrhythmia.

Rabbits were used to study the inhibitory effect of analogous electro-acupuncture on the experimental arrhythmias, i.e. hypothalamic stimulation-induced ventricular extrasystole (HVE). The result revealed that HVE, which was due to an increased cardiac sympathetic activity, could be inhibited by deep peroneal nerve stimulation with an electrical current of low frequency and low intensity. Such effect is related to endogenous opioid peptides and serotonin in the arcuatus area, the periaqueductal gray and and the medial medulla. The acupuncture correction of arrhythmias may have the same mechanism.