[Study on reproductive toxicity of nano-cadmium sulfide with different particle sizes on male mice].

Objective: To investigate the reproductive toxicity of cadmium sulfide nanoparticles (Nano-CdS) with different particle sizes on male mice. Methods: In January 2019, 30 SPF grade male mice were randomly divided into a control group, an experimental group[CdS Ⅰ group (particle size approximately 5 nm), and a CdS Ⅱ group (particle size approximately 50 nm) ], with 10 mice in each group. The experimental group was orally gavaged with 100 mg/kg, once a day, while the control group was gavaged with an equal volume of physiological saline for 45 consecutive days. After 45 days, levels of cadmium accumulation in testis were determined directly by AAS, deformity and testicular histopathological changes were also observed. Serum testosterone levels were measured by enzyme-linked immunosorbentassay (ELISA), expression levels of P450scc, 17ß-HSD and P450c17 mRNA were determined by real-time PCR. P450c17 protein was determinated by Western Blot. Results: The histopathological results showed that the testes of the experimental group mice showed varying degrees of damage; Ultrastructural observation showed that the ultrastructure of mouse testicular cells in each experimental group showed varying degrees of mitochondrial expansion and disappearance of cristae, as well as irregular nuclear membranes. The degree of damage in CdS Ⅰ group was milder than that in CdS Ⅱ group. Compared with the control group, the cadmium content in the testes of the CdS Ⅰ and CdS Ⅱ groups significantly increased (P=0.001, 0.001), and the CdS Ⅱ group was higher than the CdS Ⅰ group (P=0.001). Compared with the control group, the levels of testosterone in the CdS Ⅰ and CdS Ⅱ groups decreased with statistical significance (P=0.001, 0.001). Real time fluorescence quantitative PCR results showed that compared with the control group, the experimental group's P450scc, 17ß-HSD. The expression levels of 17ß-HSD and P450c17 mRNA were significantly reduced, with statistically significant differences (P=0.001, 0.001, 0.001), and CdS Ⅱ group 17ß-HSD. The expression levels of 17ß-HSD and P450c17 mRNA were significantly lower than those of CdS Ⅰ group (P=0.001, 0.036). The Western Blot assay results showed that the expression levels of P450c17 protein in the testes of CdS Ⅰ and CdS Ⅱ groups of mice were significantly reduced, with statistical significance (P=0.001, 0.001) ; And the CdS Ⅱ group was significantly lower than the CdS Ⅰ group (P=0.001). According to Spearman correlation analysis, testosterone levels are correlated with P450scc, P450c17, 17ß-HSD mRNA. There is a highly positive correlation between 17ß-HSD mRNA levels, with statistically significant differences (r(s)=0.88, 0.80, 0.70, P=0.001, 0.001, 0.004) . Conclusion: Nano cadmium sulfide may induce reproductive toxicity by reducing the expression levels of key enzyme genes and enzyme protein activity in testosterone and its synthesis in mice, and the CdS Ⅱ group has a stronger toxic effect.

High efficiency and collimated terahertz pulse from ultra-short intense laser and cone target.

We propose a new, to the best of our knowledge, method to radiate a high-efficiency and collimated terahertz (THz) pulse from a relativistic femtosecond laser and cone target. Particle-in-cell simulations demonstrate that a THz source of 40 mJ, pointing at an angle of ∼20 ∘, can be generated from a laser pulse of 1.9 J by using a cone target whose open angle is 10 ∘. The peak power of the THz pulse is 1011 W. This method, which manipulates the divergence angle and the energy conversion efficiency of the THz source, should promote THz science into the extra strong region with a compact laser system.

Excretory/secretory proteins of adult Toxocara canis induce changes in the expression of proteins involved in the NOD1-RIP2-NF-κB pathway and modulate cytokine production in mouse macrophages.

Dog roundworm (Toxocara canis) is the major causative agent of toxocarosis, a parasitic disease of both veterinary and medical importance. Knowledge gaps in fundamental and applied aspects hinder the control of this important zoonotic disease. To have a better understanding of Toxocara infection and host immune responses, mouse macrophages were exposed to excretory/secretory (ES) proteins released by adult worms of T. canis in vitro. The messenger RNA transcription and protein expression of nucleotide-binding oligomerization domain-containing protein 1 (NOD1), receptor interacting protein 2 (RIP2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in macrophages were analysed using quantitative real-time PCR (qRT-PCR) and Western blot. The levels of tumour necrosis factor alpha (TNF-ɑ), interleukin-1 beta (IL-1ß) and IL-6 released by the stimulated macrophages were analysed using enzyme-linked immunosorbent assay. It was found that 20 µg/mL ES proteins of adult T. canis induced the expression of NOD1, RIP2 and NF-κB in mouse macrophages at both transcriptional and translational levels after 9 h of incubation in vitro. Incubation with 20 µg/mL ES proteins also modulated the production of pro-inflammatory cytokines TNF-ɑ, IL-1ß and IL-6 by the macrophages. Taken together, ES proteins of adult T. canis appeared to be able to affect the macrophage NOD1-RIP2-NF-κB signalling pathway, which might play a role in regulating the production of proinflammatory cytokines. Further investigation of these aspects should lead to a better understanding of immune recognition of and modulation by Toxocara canis in host animals.

[Investigation and research on ticks carrying spotted fever group rickettsia in the border area of Tumen River Basin].

Objective: To understand the situation and genotype distribution of spotted fever group rickettsia (SFGR) in the border area of Tumen River Basin in free ticks in Yanbian Korean Autonomous Prefecture (Yanbian Prefecture), Jilin Province. Methods: From April to September, 2017, ticks were collected using flagging method from Hunchun, Tumen, Helong and Longjing cities in the Tumen River basin of Yanbian Prefecture. Outer membrane protein A (ompA) was detected by Polymerase Chain Reaction (PCR), then, the species were identified by gene sequencing and analyzed systematically. The positive rate of pools and MIR(minimum infection rate per 100 ticks,MIR) of SFGR were calculated, and the difference of positive rate of pools among ticks with different characteristics was compared by Chi-square test. Results: A total of 3 079 ticks were collected and divided into 536 pools. The positive rate of pools of SFGR nucleic acid was 39.7% (213 pools). The MIR of SFGR was 6.9%.The positive rate of pools of SFGR in Dermacentor silvarum, Haemaphysalis concinna, Haemaphysalis japonica, Haemaphysalis longicornis and Ixodes persulcatus were 80.4% (41/51), 14.0% (25/179), 20.2% (18/89), 78.9% (101/128) and 25.9% (21/81), and the difference was statistically significant (P<0.001). There was statistical difference in the positive rate of pools of SFGR in developmental stages of ticks (P<0.001); the positive rate of pools of female adults, male adults, nymph and larvae were 36.4% (95/261), 34.2% (67/196), 56.3% (40/71) and 7/8, and the MIR was 7.9%, 7.7%, 4.9% and 3.5%. The five genotype was detected which was Candidatus Rickettsia longicornii, Rickettsia raoultii, Rickettsia heilongjiangensis, Candidatus Rickettsia tarasevichiae,Rickettsia monacensis and have 98%-100% homology with known gene sequences. Candidatus Rickettsia longicornii, Rickettsia raoultii, Rickettsia heilongjiangensis and Candidatus Rickettsia tarasevichiae showed close evolutionary relationship with known specie (have 98%-100% homology with known gene sequences); Rickettsia monacensis showed Far from evolutionary relationship with known species (have 98% homology with known gene sequences). Conclusion: SFGR infection of ticks is common in the border areas of the Tumen River Basin. There was high diversity in SFGR species and tick species in the areas surveyed.

Construction of a recombinant baculovirus expressing swine hepatitis E Virus ORF2 and preliminary research on its immune effect.

In the swine hepatitis E virus (HEV), open reading frame 2 (ORF2) is rich in antigenic determinants and neutralizing epitopes that could induce immune protection. We chose the Bac-to-Bac® Baculovirus Expression System to express fragments containing the critical neutralizing antigenic sites within the HEV ORF2 protein of pigs to obtain a recombinant baculovirus. The fragment of swine HEV ORF2 region (1198-1881bp) was cloned into vector pFastBacTM. A recombinant baculovirus, rBacmid-ORF2, was obtained after transposition and transfection. The molecular mass of the recombinant protein was 26 kDa. Mice were immunized by the intraperitoneal and oral routes with cell lysates of recombinant baculovirus rBacmid-ORF2. Serum and feces of the mice were collected separately at 0, 14, 28, and 42 d after immunization and the antibody levels of IgG and secretory IgA against swine HEV were determined using an enzyme-linked immunosorbent assay. The results suggested that rBacmid-ORF2 induced antibodies of the humoral and mucosal immune responses in mice and that the oral route was significantly superior to the intraperitoneal route. This is the first study to demonstrate that that recombinant baculovirus swine HEV ORF2 could induce humoral and mucosal immune responses in mice.

Anti-tumor activity and immunogenicity of a mutated staphylococcal enterotoxin C2.

In this study, a novel SEC2 mutant with lower toxic activity, named 2M-118 (H118A/T20L/G22E), was engineered by site-directed mutagenesis of structural domains that are responsible for MHC class II molecule binding and TCR binding, respectively. Stimulating activity on murine splenocytes, anti-tumor effect and immunogenicity of 2M-118 were investigated in BALB/c mice. 2M-118 not only remained splenocyte stimulation activity, but also effectively inhibited the growth of S180 sarcoma in the BALB/c mice. Even though antibodies to 2M-118 could be induced after repeated administration, the action of 2M-118 was hardly neutralized or cross neutralized. Like other superantigens, immunosuppression could happen when 2M-118 was given at a greater dose. In conclusion, 2M-118 is a promising anti-tumor drug candidate for its acceptable toxicity and satisfying anti-tumour efficacy.

Effects of isomaltooligosaccharide and Bacillus supplementation on sow performance, serum metabolites, and serum and placental oxidative status.

This study investigated the effects of isomaltooligosaccharide (IMO) and Bacillus supplementation on sow performance, serum metabolites, and serum and placental oxidative status. Multiparous gestating sows (n = 130) with similar body conditions were randomly allocated to five groups (n = 26) receiving a basal diet (CON group) or a basal diet supplemented with 0.5% IMO (IMO group); 0.5% IMO and 0.02% Bacillus subtilis (IMO + S group); 0.5% IMO and 0.02% Bacillus licheniformis (IMO + L group); or 0.5% IMO, 0.02% Bacillus subtilis, and 0.02% Bacillus licheniformis (IMO + S+L group). There were no significant differences in the litter sizes among all dietary groups. The average piglet birth weight was improved in all treatment groups, and the placental efficiency was greater in the IMO + S and IMO + S+L groups than in the CON group (P < 0.05). The IMO + S+L group had increased the low-density lipoprotein cholesterol and reduced the total cholesterol in umbilical venous serum (P <  0.05). Additionally, the malondialdehyde concentrations were greater in umbilical venous serum of piglets in all treatment groups relative to that in the CON piglets (P <  0.05). The placental total antioxidant capacity was increased in the IMO+L and IMO+S+L groups (P <  0.05). Furthermore, the growth hormone concentration in umbilical venous serum was greater (P <  0.05) in all treatment groups. Overall, IMO and Bacillus supplementation during late gestation resulted in a changed metabolism of sows, improved the placental antioxidant capacity, and increased the growth hormone concentrations in umbilical venous serum, which ultimately improved the piglet birth weight and placental efficiency.

Effects of dietary supplementation with enzymatically treated Artemisia annua on growth performance, intestinal morphology, digestive enzyme activities, immunity, and antioxidant capacity of heat-stressed broilers.

The gastrointestinal tract is considered as one of the main target organs affected by heat stress. Phytogenic feed additives containing phenolics and flavonoids can improve the resistance of broilers to heat stress. This study was conducted to investigate the effects of dietary supplementation with enzymatically treated Artemisia annua (EA) on growth performance, intestinal morphology, digestive enzyme activities, immunity and antioxidant capacity of broilers challenged with heat stress. One hundred and forty-four 21-day-old male Arbor Acres broilers were randomly distributed into 3 treatments: 1) non-challenged control (CON); 2) heat-stress-challenged control (HS); and 3) heat-stress-challenged group + 1 g EA/kg diet (HS-EA). From 22 to 41 d, broilers in the CON group were housed at 22 ± 1°C, the HS and HS-EA groups, in which broilers were raised at 34 ± 1°C for 8 h (0900-1700 h) and the temperature for the rest time was the same as that of the CON group. The EA supplementation alleviated the compromised body weight gain and intestinal morphology impairment caused by heat stress challenge (P < 0.05). The EA attenuated heat-stress-induced decreased intestinal lipase, trypsin and total superoxide dismutase activities, and reduced intestinal secretory immunoglobulin A (SIgA) and IgG concentrations (P < 0.05). The EA inclusion prevented the elevation of intestinal malondialdehyde content and reduction of intestinal glutathione concentration induced by heat stress challenge (P < 0.05). The intestinal mRNA abundances of nuclear factor erythroid 2-related factor 2, heme oxygenase 1, glutathione peroxidase, gamma-glutamyl cysteine ligase larger catalytic subunit and gamma-glutamyl cysteine ligase smaller modulator subunit in heat-stressed broilers were increased in response to dietary EA treatment (P < 0.05). In conclusion, dietary supplementation of 1 g/kg EA could alleviate heat-stress-induced compromised growth performance and intestinal damage of broilers.

Evaluation of enzymatically treated Artemisia annua L. on growth performance, meat quality, and oxidative stability of breast and thigh muscles in broilers.

An experiment was conducted to evaluate the effects of including enzymatically treated Artemisia annua L. (EA) in broiler diets on growth performance, meat quality, and oxidative stability of breast and thigh muscles. A total of 256 one-d-old Arbor Acres broiler chicks were randomly allotted into four groups with eight replicates of eight birds each. Broilers in the four groups were offered basal diet supplemented with 0.0, 0.5, 1.0, and 1.5 g/kg EA during the 42-d experiment, respectively. The ADG, ADFI, and feed/gain ratio (F:G) were measured at 42 d of age. Breast and thigh muscle samples from eight birds per treatment were obtained at 42 d to determine meat quality, free radical scavenging activity, and lipid peroxidation. All treatment groups had similar ADG, ADFI, and F:G during the 42 d experiment (P > 0.05). Drip loss at 24 h and shearing force of breast muscle were linearly (P < 0.05) and quadratically (P < 0.05) decreased by EA addition. The drip loss at 24 h and 48 h, cooking loss and shearing force of thigh muscle followed the same fashion. The supplementation of EA quadratically increased 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) (P = 0.004) and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) (P = 0.035) free radical scavenging activities in breast muscle, and linearly (P < 0.05) and quadratically (P < 0.05) increased ABTS and DPPH scavenging activities of thigh muscle. Increasing levels of EA linearly (P < 0.05) or quadratically (P < 0.05) or both decreased the malondialdehyde (MDA) concentrations in breast and thigh muscle samples during 15 d of storage at 4°C. The results indicated that EA supplementation improved meat quality and oxidative stability of breast and thigh muscles in broilers. The inclusion level of 1.0 g/kg EA in broiler diet was recommended.

Effects of dietary vitamin E type on the growth performance and antioxidant capacity in cyclophosphamide immunosuppressed broilers.

Reactive oxygen species and free radicals play multiple roles in some immune-pathological events. Vitamin E, as a very potent antioxidant, perhaps deceases the potentially negative effects of such oxidative stress to prevent immune-pathological damage to broilers. Therefore, the current study investigated the effects of dietary natural (D-α-tocopherol) and synthetic (DL-α-tocopherol acetate) vitamin E on the growth performance and antioxidant capacity in cyclophosphamide (CY) immunosuppressed broilers. 192 one-day-old male Arbor Acre broilers were randomly distributed into 4 groups: 1) non-CY-challenged control; 2) CY-challenged control; 3) CY-challenged group+20 IU DL-α-tocopherol acetate per kg feed; and 4) CY-challenged group+20 IU D-α-tocopherol per kg feed. The maize-soybean basal diet in the control group contained α-tocopherol (7.12 mg/kg). Broilers were intramuscularly injected with 80 mg/kg body weight of CY or sterile saline at 16, 17, and 18 d of age. CY decreased (P < 0.05) the average daily gain and average daily feed intake, but vitamin E did not alter the growth performance of broilers before or after CY injection (P > 0.05). The decreased absolute weight of the spleen, thymus and bursa, serum interleukin 2 (IL-2), and interleukin 6 (IL-2) concentrations in CY-treated broilers were alleviated by vitamin E (P < 0.05) . The decreased relative weight (g/kg body weight) of the bursa in the CY-treated broilers was increased by natural vitamin E (P < 0.05). The CY-induced increases in malondialdehyde (MDA) content and decreases in total antioxidant capacity (T-AOC), glutathione, vitamin C, and α-tocopherol levels, and total superoxide dismutase and glutathione peroxidase activities in both serum and the liver were attenuated by vitamin E (P < 0.05). Additionally, natural vitamin E increased α-tocopherol and T-AOC levels and decreased MDA content in the liver of CY-treated broilers (P < 0.05) when compared to the synthetic form. In summary, both synthetic and natural vitamin E supplementation improved lymphoid organ weights, serum IL-2 and IL-6 levels, and antioxidant capacity of immunosuppressed broilers induced by CY. Especially, natural vitamin E was superior to the synthetic form and enhanced α-tocopherol and T-AOC levels, reduced MDA concentration in the liver, and alleviated the immune damage of the bursa.

Effects of Oridonin on growth performance and oxidative stress in broilers challenged with lipopolysaccharide.

This study was conducted to investigate the effects of oridonin (ORI) on growth performance and antioxidant capacity in broiler chickens that were repeatedly challenged with lipopolysaccharide (LPS). A total of 384 one-day-old male Arbor Acre broiler chickens were randomly assigned to 8 treatments with 6 replicate cages per treatment and 8 birds per replicate. There were 4 dietary treatments: the control group (birds fed the basal diet), the ORI 50 group, the ORI 80 group, and the ORI 100 group (the basal diet supplemented with 50, 80, and 100 mg/kg oridonin, respectively). Broilers were intraperitoneally injected with either 250 µg/kg BW LPS or an equivalent amount of sterile saline at 16, 18, and 20 d of age. LPS decreased the average daily weight gain (ADG), the average daily feed intake (ADFI), and the feed conversion ratio (FCR) of broiler chickens (P < 0.05); oridonin supplementation had no effects on performance whether before or after LPS injection (P > 0.05). LPS stimulation increased the relative weight of the spleen and bursa (P < 0.05); oridonin inclusion markedly attenuated the increased spleen index (P < 0.05). Additionally, the LPS-induced increases in the concentrations of malondialdehyde (MDA) and decreases in activities of total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC) and catalase (CAT) were dramatically attenuated by oridonin in both the serum and liver (P < 0.05). Furthermore, LPS down-regulated the mRNA expressions of nuclear factor erythroid 2-related factor 2 (Nrf2), copper and zinc superoxide dismutase (Cu/Zn-SOD), manganese superoxide dismutase (Mn-SOD), glutathione peroxidase (GPx1), and CAT in the liver (P < 0.05), However, oridonin inclusion increased the liver mRNA expression levels of Nrf2, Cu/Zn-SOD, Mn-SOD, CAT, and GPx1 (P < 0.05). It was concluded that the dietary oridonin supplementation at an optimum dose of 100 mg/kg improves the antioxidant capacity in broilers, as evidenced by the decrease in MDA and the increase in total SOD activities and mRNA expression levels of the liver antioxidant genes, although the effects on growth performance was negligible.

Effects of cello-oligosaccharide on intestinal microbiota and epithelial barrier function of weanling pigs.

A total of 144 piglets (Duroc × Landrace × Yorkshire; average initial weight of 6.13 kg weaned at 21 ± 1 d age) were allotted to 4 treatments for 2 wk, each of which had 6 pens with 6 pigs per pen. After the feeding experiment, 6 pigs per treatment were slaughtered to investigate the effects of cello-oligosaccharide (COS) on intestinal microbiota and epithelial barrier function. The COS was added to the basal diet at 0, 1.5, 3.0, and 4.5 g/kg diet at the expense of corn, respectively. Plasma -lactate, diamine oxidase (DAO), and the Ussing chamber technique were used to determine the intestinal barrier function. 16S rRNA-based methods were used for intestinal microbiota analysis. The results showed that incremental levels of COS had no effect ( > 0.05) on growth performance. Incremental levels of COS increased lactobacilli in jejunal and colonic contents ( < 0.05); decreased in jejunal contents ( < 0.05) and and in colonic contents ( < 0.05); reduced plasma DAO (linear, = 0.013, and quadratic, = 0.037); increased jejunal mucosa DAO (linear, = 0.003, and quadratic, = 0.008); decreased fluorescein isothiocyanate dextran 4 kDa flux of jejunum and colon ( < 0.05); and increased transepithelial electrical resistance (TER) in colon ( < 0.05), claudin-1 protein expression in jejunal mucosa (linear, = 0.001, and quadratic, = 0.003), and protein expressions of claudin-1 and zonula occludens-1 (ZO-1) in colonic mucosa linearly ( = 0.001 and = 0.001, respectively) and quadratically ( = 0.001 and = 0.002, respectively). The results indicated that the improved microbial ecosystem in the presence of COS might contribute to improvement in intestinal barrier function and tight junction proteins. Results also showed that the appropriate dietary COS supplementation level was 3.0 g/kg in weaned pig diets under our trial conditions.

Diosmectite-zinc oxide composite improves intestinal barrier restoration and modulates TGF-ß1, ERK1/2, and Akt in piglets after acetic acid challenge.

The present study evaluated the beneficial effect of diosmectite-zinc oxide composite (DS-ZnO) on improving intestinal barrier restoration in piglets after acetic acid challenge and explored the underlying mechanisms. Twenty-four 35-d-old piglets (Duroc × Landrace × Yorkshire), with an average weight of 8.1 kg, were allocated to 4 treatment groups. On d 1 of the trial, colitis was induced via intrarectal injection of acetic acid (10 mL of 10% acetic acid [ACA] solution for ACA, DS-ZnO, and mixture of diosmectite [DS] and ZnO [DS+ZnO] groups) and the control group was infused with saline. Twenty-four hours after challenged, piglets were fed with the following diets: 1) control group (basal diet), 2) ACA group (basal diet), 3) DS-ZnO group (basal diet supplemented with DS-ZnO), and 4) DS+ZnO group (mixture of 1.5 g diosmectite [DS]/kg and 500 mg Zn/kg from ZnO [equal amount of DS and ZnO in the DS-ZnO treatment group]). On d 8 of the trial, piglets were sacrificed. The results showed that DS-ZnO supplementation improved (P < 0.05) ADG, ADFI, and transepithelial electrical resistance and decreased (P < 0.05) fecal scores, crypt depth, and fluorescein isothiocyanate-dextran 4 kDa (FD4) influx as compared with ACA group. Moreover, DS-ZnO increased (P < 0.05) occludin, claudin-1, and zonula occluden-1 expressions; reduced (P < 0.05) caspase-9 and caspase-3 activity and Bax expression; and improved (P < 0.05) Bcl2, XIAP, and PCNA expression. Diosmectite-zinc oxide composite supplementation also increased (P < 0.05) TGF-ß1 expression and ERK1/2 and Akt activation. These results suggest that DS-ZnO attenuates the acetic acid-induced colitis by improving mucosa barrier restoration, inhibiting apoptosis, and improving intestinal epithelial cells proliferation and modulation of TGF-ß1 and ERK1/2 and Akt signaling pathway.

Germ cell-specific Atg7 knockout results in primary ovarian insufficiency in female mice.

Primary ovarian insufficiency (POI) is a common cause of infertility in around 1-2% of women aged <40 years. However, the mechanisms that cause POI are still poorly understood. Here we showed that germ cell-specific knockout of an essential autophagy induction gene Atg7 led to subfertility in female mice. The subfertility of Atg7 deletion females was caused by severe ovarian follicle loss, which is very similar to human POI patients. Further investigation revealed that germ cell-specific Atg7 knockout resulted in germ cell over-loss at the neonatal transition period. In addition, our in vitro studies also demonstrated that autophagy could protect oocytes from over-loss by apoptosis in neonatal ovaries under the starvation condition. Taken together, our results uncover a new role for autophagy in the regulation of ovarian primordial follicle reservation and hint that autophagy-related genes might be potential pathogenic genes to POI of women.

Absence of a conserved proline and presence of a conserved tyrosine in the CB2 cannabinoid receptor are crucial for its function.

A majority (84%) of G protein-coupled receptors have a proline (P5.50) in the middle of the fifth transmembrane domain. However, one of the unique structural features of cannabinoid receptors is the replacement of the conserved P5.50 by a leucine (L5.50). It has been shown that a conserved tyrosine (Y5.58), located at the cytoplasmic side of P5.50, is crucial for the signal transduction of several G protein-coupled receptors. We proposed that the replacement of P5.50 by L5.50 and the presence of the conserved Y5.58 in this context are important for the function of CB2. Mutating L5.50 to a proline abolished ligand binding, whereas mutating Y5.58 to an alanine resulted in a rightward shift of the competition binding curves. Both of these mutations led to a complete loss of the ability of cannabinoid agonists to inhibit forskolin-stimulated cAMP accumulation.

CB1 cannabinoid receptor-mediated tyrosine phosphorylation of focal adhesion kinase-related non-kinase.

The effect of cannabinoid on the tyrosine phosphorylation of focal adhesion kinase (FAK) and focal adhesion kinase-related non-kinase (FRNK) was investigated in differentiated mouse neuroblastoma N1E-115 cells. HU-210, a potent cannabinoid agonist, elicited a time-dependent enhancement of tyrosine phosphorylation of FRNK, but not FAK. Pretreatment of cells with antisense oligodeoxynucleotide targeting CB1 cannabinoid receptor abolished HU-210-induced FRNK tyrosine phosphorylation. In addition, pretreatment of cells with 8-Br-cAMP also blocked HU-210-induced FRNK tyrosine phosphorylation. These data demonstrated that HU-210 induces FRNK tyrosine phosphorylation by activating G(i)-coupled CB1 cannabinoid receptor in N1E-115 cells. This newly discovered, cannabinoid-induced FRNK tyrosine phosphorylation might be a novel mechanism for cannabinoid-induced functional changes.

Functional roles of the tyrosine within the NP(X)(n)Y motif and the cysteines in the C-terminal juxtamembrane region of the CB2 cannabinoid receptor.

In G protein-coupled receptors, a NP(X)(n)Y motif in the seventh transmembrane domain and cysteine residues in the C-terminal juxtamembrane region are conserved. In the current study, the roles of Y299 within the NPVIY motif and C313 and C320 in the C-terminal juxtamembrane region of the human CB2 cannabinoid receptor were investigated by site-directed mutagenesis. Replacing Y299 with alanine resulted in a complete loss of ligand binding and a severe impairment of cannabinoid-induced inhibition of forskolin-stimulated cAMP accumulation. The C313A and C320A mutations markedly reduced functional coupling to adenylate cyclase, but had no effect on ligand binding and agonist-induced receptor desensitization.

Molecular cloning of a novel candidate G protein-coupled receptor from rat brain.

A PCR cloning strategy using primers designed from sequences selectively conserved among a cannabinoid receptor and two orphan receptors, was used to isolate novel G protein-coupled receptors. rCNL3, a 1.75 kb cDNA encoding a 363 amino acid protein, was isolated from a rat cerebral cortex library. Sequence analysis showed that rCNL3 possesses a number of structural characteristics of G protein-coupled receptors and has 61% amino acid identity (from transmembrane region one through the carboxyl-terminus) with two other candidate G protein-coupled receptors. Therefore, these three receptors may comprise a receptor subfamily with identical or closely related endogenous ligands. Northern and in situ hybridization experiments demonstrated that rCNL3 mRNA is expressed in the rat brain, with a prominent distribution in striatum.

Effects of D3.49A, R3.50A, and A6.34E mutations on ligand binding and activation of the cannabinoid-2 (CB2) receptor.

In several G protein-coupled receptors (GPCRs), the Asp-Arg-Tyr (DRY) motif at the bottom of third transmembrane domain and the amino acid at position 6.34 in the sixth transmembrane domain have been shown to play important roles in signal transduction. In this study, we propose that in the cannabinoid-2 (CB2) receptor, R3.50 in the DRY motif may be crucial for interacting with G proteins, and D3.49 and A6.34 may be important for constraining the receptor in an inactive conformation. To test our hypothesis, R3.50A, D3.49A, and A6.34E mutations of the human CB2 receptor were made by site-directed mutagenesis. These mutant receptors were stably transfected into human embryonic 293 cells, and their ligand binding and signal transduction properties were analyzed. Similar to other GPCRs, R3.50 of the CB2 receptor is crucial for signal transduction. Unlike other GPCRs, D3.49 and A6.34 of the CB2 receptor do not seem to be important for keeping the receptor in an inactive state. Furthermore, D3.49A and A6.34E mutations abolished ligand binding, and all three mutations abolished constitutive activity of the wild-type CB2 receptor.

Long circulating liposomes encapsulating organophosphorus acid anhydrolase in diisopropylfluorophosphate antagonism.

These studies are focused on antagonizing organophosphorous (OP) intoxications by a new conceptual approach using recombinant enzymes encapsulated within sterically stabilized liposomes to enhance diisopropylfluorophosphate (DFP) degradation. The OP hydrolyzing enzyme, organophosphorous acid anhydrolase (OPAA), encapsulated within the liposomes, was employed either alone or in combination with pralidoxime (2-PAM) and/or atropine. The recombinant OPAA enzyme, from the ALTEROMONAS: strain JD6, has high substrate specificity toward a wide range of OP compounds, e.g., DFP, soman, and sarin. The rate of DFP hydrolysis by liposomes containing OPAA (SL)* was measured by determining the changes in fluoride-ion concentration using a fluoride ion-selective electrode. This enzyme carrier system serves as a biodegradable protective environment for the OP-metabolizing enzyme (OPAA), resulting in an enhanced antidotal protection against the lethal effects of DFP. Free OPAA alone showed some antidotal protection; however, the protection with 2-PAM and/or atropine was greatly enhanced when combined with (SL)*.