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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, 17β-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, 17β-HSD. The expression levels of 17β-HSD and P450c17 mRNA were significantly reduced, with statistically significant differences ( P =0.001, 0.001, 0.001), and CdS Ⅱ group 17β-HSD. The expression levels of 17β-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, 17β-HSD mRNA. There is a highly positive correlation between 17β-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.

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 10 11 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.

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-1β) 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-1β 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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (Copyright© by the Polish Academy of Sciences.)

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., (© 2017 Poultry Science Association Inc.)

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., (© 2016 Poultry Science Association Inc.)

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., (© 2016 Poultry Science Association Inc.)

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., (© Crown copyright 2016.)

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-β1 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-β1 and ERK1/2 and Akt signaling pathway.

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.

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.

Interferon-γ (IFN-γ), a pleiotropic lymphokine, has important regulatory effects on many cell types. Although IFN-γ is essential for the initiation of uterine vascular modifications and maintenance of decidual integrity, IFN-γ administration can also cause pregnancy failure in many species. However, little is known about the effector mechanisms involved. In this study, using an IFN-γ-induced abortion mouse model, we reported that no Dolichos biflorus agglutinin lectin-positive uterine natural killer (uNK) cells were observed in the uteri from IFN-γ-induced abortion mice. By contrast, the percentage of CD3(-)CD49b(+) NK cells in the uterus and blood from a foetal resorption group was significantly higher than that of the control group. Similarly, significantly upregulated expression of CD49b (a pan-NK cell marker), CX3CL1 and CX3CR1 (CX3CL1 receptor) was detected in the uteri of IFN-γ-induced abortion mice. Using isolated uterine stromal cells, we showed that upregulated expression of CX3CL1 by IFN-γ was dependent on a Janus family kinase 2-signal transducers and activators of transcription 1 (JAK2-STAT1) pathway. We further demonstrated the chemotactic activity of CX3CL1 in uterine stromal cell conditioned medium on primary splenic NK cells. Finally, we observed increased recruitment of CD49b(+) NK cells into the endometrium after exogenous CX3CL1 administration. Collectively, our findings indicate that IFN-γ can significantly increase uterine CX3CL1 expression via activation of the JAK2-STAT1 pathway, thus inducing CD49b(+) NK cell uterine homing, and eventually provoke foetal loss. Thus, we provide a new line of evidence correlating the deleterious effects of IFN-γ on pregnancy with the aberrant regulation of CX3CL1 and CD49b(+) NK cells.

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.

A sensitive chemiluminescence method, based on the enhancive effect of phenobarbital on the chemiluminescence reaction between luminol and dissolved oxygen in a flow injection system, was proposed for the determination of phenobarbital. The chemiluminescence intensity responded to the concentration of phenobarbital linearly ranging from 0.05 to 10 ng x ml(-1) with the detection limit of 0.02 ng x ml(-1) (3 sigma). At a flow rate of 2.0 ml x min(-1), a complete determination of phenobarbital, including sampling and washing, could be accomplished in 0.5 min, offering the sampling efficiency of 120 h(-1) accordingly. The method was applied successfully in an assay of PB for pharmaceutical preparations, human urine and serum without any pretreatment with recovery from 95.7 to 106.7% and RSDs of less than 3.0%.

A convenient method for fetal murine premeiotic germ cells to develop into oocytes in vitro has been established. Fetal ovaries from mice, collected 12.5 d postcoitus (dpc), were organ-cultured in vitro using a medium for organ growth, and the developmental potential regarding oocyte formation was determined. After 28 d of culture, premeiotic female germ cells developed into oocytes with a mean (+/-SD) diameter of 73.3+/-7.7 microm. However, follicles developed in vitro versus in vivo had fewer granulosa cells (32+/-2.6 vs. 142+/-9.5, respectively; P<0.01), and the ovaries had less mRNA for Cx37 and Cx43 (P<0.01). Oocytes in the first meiotic division phase were isolated from cultured ovaries or after hormone treatments. After exposure to okadaic acid at a final concentration of 1 microM, oocytes derived from premeiotic fetal female germ cells were able to undergo germinal vesicle breakdown but failed to complete the first meiotic division. Furthermore, the intracellular content of GSH in oocytes cultured in vitro was lower than that of oocytes matured in vivo (P<0.01). In conclusion, premeiotic germ cells derived from murine fetuses as early as 12.5 dpc were able to differentiate into germinal vesicle-stage oocytes but were unable to complete meiosis I in vitro.

The 24-h median lethal concentrations of pentachlorophenol to Chironomus plumousus, Tubifex sinicus and Galba pervia were 0.302, 0.977 and 0.293 mg/L, respectively. Bioconcentration factors of C. plumousus, T. sinicus and G. pervia to pentachlorophenol were 108, 367 and 85 at 0.02 mg/L pentachlorophenol, respectively. As pentachlorophenol concentration increased, the G. pervia egg hatching rates became lower, and the total hatched time became longer. Pentachlorophenol teratogenesis was demonstrated by observing the deformation of C. plumousus larvae mentum.

One new phenylethanoid glycoside, cistansinenside A and one new oligosaccharide, cistansinensose A1/A2, were isolated from the stems of Cistanche sinensis, together with six known compounds. The structures of the new compounds were elucidated on the basis of spectral data.

Cordyceps is an expensive traditional Chinese medicine, which has anti-tumor activity and significant effects on the immune system. In Southeast Asia, Cordyceps is commonly sold in capsule form as a health food product. Most of these products are derived from cultured Cordyceps mycelia. Because of the price difference, some manufacturers claim their products are from natural Cordyceps. In order to distinguish among various types of Cordyceps in the market, the profiles of water-soluble constituents derived from different sources of Cordyceps were determined by capillary electrophoresis (CE). Both natural and cultured Cordyceps showed three peak clusters migrated at 5-7, 9-11 and 12-13 min, and the height and resolution of these peak clusters were rather distinct. Peak cluster at 9-11 min was identified as adenosine, guanosine and uridine, and shared a similarity between natural and cultured products. In contrast, the peak cluster at 5-7 min was characteristic of natural Cordyceps, regardless of hosts and sources. By using the peak characteristics of CE profiles of different Cordyceps samples, hierarchical clustering analysis was performed. The result shows that those samples of natural Cordyceps were grouped together distinct from the cultured and commercial products. Thus, the CE profiles could serve as fingerprints for the quality control of Cordyceps.

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.

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.

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.

The morphological remodeling of neuronal cells influences neurogenesis and brain functions. We hypothesize that psychoactive and neurotoxic effects of cannabinoids may be mediated, at least in part, by their morphoregulatory activities. In the present study, mouse neuroblastoma N1E-115 cells were used as an in vitro model to investigate cannabinoid-induced neurite remodeling effects and to identify the involvement of cannabinoid receptors in this neurite remodeling process. Using reverse transcription-polymerase chain reaction and immunofluorescence microscopy, the endogenously expressed CB1, but not CB2, cannabinoid receptors were detected in morphologically differentiated N1E-115 cells. Activation of these natively expressed CB1 cannabinoid receptors by cannabinoid agonist HU-210 led to a concentration-dependent inhibition of adenylate cyclase activity. Importantly, HU-210 treatment induced neurite retraction in a concentration-dependent manner. Pretreatment of N1E-115 cells with a CB1 antisense oligodeoxynucleotide (ODN) suppressed HU-210-induced inhibition of forskolin-stimulated cAMP accumulation, indicating that the knocking down of functional CB1 cannabinoid receptor expression was achieved. Antisense ODN pretreatment also abolished HU-210-induced neurite retraction, demonstrating the involvement of CB1 cannabinoid receptors in mediating the neurite remodeling effects of HU-210. In addition, reversing HU-210-induced intracellular cAMP declination by 8-Br-cAMP partially prevented HU-210-induced neurite retraction, indicating the involvement of cAMP-dependent signaling pathways in mediating the neurite remodeling function of CB1 cannabinoid receptors in N1E-115 cells. These data demonstrate that neurite remodeling is a newly discovered function of CB1 cannabinoid receptors. This morphoregulatory function of CB1 cannabinoid receptors might be a new mechanism that mediates the psychoactive and neurotoxic effects of cannabinoids in developing and adult brain., (Copyright 2001 Wiley-Liss, Inc.)

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.

Aim: To study the metabolic process of phenylethanoid glycosides (PhGs) in the gastrointestine of beagle dogs that were administered intragastrially process, and develop some new methods of biopharmacology on the effective position of traditional Chinese medicine., Methods: High-performance liquid chromatography was used to purify constituents from faeces and analyze relative contents of the three main compounds in the gastrointestinal tract at different times and in the faeces of dogs. Every sample was collected, extracted with methanol and analyzed with integration., Results: Four compounds, based on reference substances, were identified as echinacoside, acteoside, isoacteoside, and 2'-acetylacteoside from extraction of faces of dogs. Quantitative "with HPLC" analysis reveals that variation of ratios of the three main compounds is not distinct when moving in the gastrointestinal tract 7 h, that is quite different from those in faeces, in which the content of echinacoside fell from 48.0% to 16.0%, and acteoside increased from 11.0% to 34.7%., Conclusion: PhGs are mainly metabolized in large intestine. Among them, a portion of echinacoside is transformed into aceteoside.

Aim: To determine vitamin B1 in pharmaceutical preparations by chemiluminescence (CL) flow sensor., Methods: When 200 microL of Na3PO4 was passed through an anion exchange column, K3Fe(CN)6 was eluted from the resin and then mixed with the vitamin B1 stream containing NaOH to product CL, by the fast oxidation reaction between vitamin B1 and K3Fe(CN)6 in alkaline solution., Results: The CL emission intensity was correlated with the vitamin B1 concentration in the range 1.0 x 10(-5) to 1.0 x 10(-3) mol.L-1 and the detection limit was 8.0 x 10(-6) mol.L-1 (3 sigma). A complete analysis, including sampling and washing, can be performed within 2 min with a relative standard deviation of less than 4.0%., Conclusion: The vitamin B1 flow sensor was stable for over 200 analyses and has been applied successfully to determination of vitamin B1 in pharmaceutical preparations.

Echinacoside, one kind of phenylethanoid glycosides (PhGs), was isolated from the stems of Cistanche tubulosa (Schenk) R. Wight with a series of steps, including solvent extraction, D101 polymer adsorption column separation, Sephadex LH-20 separation, C18 column reversed-phase preparative high performance liquid chromatographic (RP-prep-HPLC) preparation and polyamide thin-layer chromatographic detection. The purity of the product was over 98%. The chemical structure of echinacoside was identified by 1H NMR and 13C NMR. To find out the optimum condition of mobile phase of RP-prep-HPLC, several systems were used in this work. Finally acetonitrile-1% HCOOH water (18:82, V/V) system was found to be the best. On the other hand D101 polymer adsorption column and Sephadex LH-20 were also effective for PhGs separation.

Objective: To investigate the chemical constituents of Cistanche tubulosa., Method: The chemical constituents were isolated by solvent extraction together with various chromatographic techniques including preparative HPLC. The structures were elucidated on the basis of chemical evidence and spectral data., Results: Four iridoid glycosides, one lignan glycoside and one monoterpenoid were isolated from the 95% ethanol extract from the stem of C. tubulosa and identified as adoxosidic acid(I), 8-epiloganic acid(II), geniposidic acid (III), mussaenosidic acid(IV), (+)-syringaresinol-O-beta-D-gluco pyranoside(V) and 8-hydroxygeraniol(VI)., Conclusion: Compounds I and VI were isolated from the genus of Cistanche for the first time. Compounds III, IV and V were isolated from this plant for the first time.

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)*.

Recent studies have suggested that cell migratory responses are often mediated by G(i) protein-coupled receptors. Because it is known that CB1 cannabinoid receptors are coupled to pertussis toxin-sensitive G proteins, we proposed that CB1 may mediate cell migration. To test this hypothesis, modified Boyden chamber assays were used to investigate cell migration mediated by CB1 cannabinoid receptors. HU-210, WIN55212-2, and anandamide, three cannabinoid agonists with distinct chemical structures, induced migration of human embryonic kidney 293 cells stably transfected with human CB1 gene, but not 293 cells transfected with an empty expression vector. These migratory responses were concentration-dependent. The EC(50) values for HU-210, WIN55212-2, and anandamide were 0.19 +/- 0.04, 12. 2 +/- 1.4, and 39.9 +/- 3.7 nM, respectively. The maximal migration index for HU-210, WIN55212-2, and anandamide were 8.9 +/- 1.6, 9.5 +/- 1.6, and 8.8 +/- 1.3, respectively. Pretreating cells with 100 ng/ml pertussis toxin eliminated the cannabinoid agonist-induced cell migration. SR141716A, a selective antagonist for CB1, inhibited the cannabinoid agonist-induced migratory responses in a concentration-dependent manner. Checkerboard analysis demonstrated that anandamide-induced cell migrations are due to chemotaxis as well as chemokinesis. Furthermore, anandamide-induced migratory responses were inhibited, in a concentration-dependent manner, by PD098059, an inhibitor of mitogen-activated protein kinase activation, but not by 8-bromoadenosine-3',5'-cyclic monophosphate, a cell-permeable cAMP analog. These data demonstrate that cannabinoid agonists are able to induce chemotaxis and chemokinesis, and that these migratory responses are mediated by G protein-coupled, CB1 cannabinoid receptors. In addition, these data suggest that activation of mitogen-activated protein kinase plays an important role, whereas inhibition of adenylate cyclase is probably not involved in the cell migration mediated by CB1.

It is known that marijuana smoking and administration of natural cannabinoids reduce intraocular pressure. However, it has not been established whether the intraocular pressure-lowering effects of cannabinoids are mediated by cannabinoid receptors. Aminoalkylindoles are a new class of cannabimimetics with structures entirely different from those of natural cannabinoids. WIN55212-2, a prototypic aminoalkylindole, has been shown to bind cannabinoid receptors and to exhibit cannabinoid-like activities. The objective of this study was to determine whether aminoalkylindoles lower intraocular pressure and whether the effects of aminoalkylindoles are mediated by ocular cannabinoid receptors. The intraocular pressure of New Zealand White rabbits was measured with the use of applanation pneumatonography. After the measurement of baseline intraocular pressure, drugs were applied topically and the intraocular pressure was monitored. The topical application of WIN55212-2 significantly reduced intraocular pressure in the treated eyes. The intraocular pressure-lowering effects of WIN55212-2 were time and dose dependent, and the maximal reduction was 4.7 +/- 0.5 mm Hg at a dose of 100 microg. In contrast to treated eyes, the intraocular pressure on the contralateral eyes was not significantly affected. The topical application of WIN55212-3, the enantiomer of WIN55212-2, had no effect on intraocular pressure. Furthermore, the intraocular pressure-lowering effects of WIN55212-2 were significantly reduced by topically administered SR141716A, a selective antagonist for the CB1 cannabinoid receptor. The dose-response curve of WIN55212-2 is shifted parallel to the right by SR141716A. These data demonstrate that like natural cannabinoids, WIN55212-2 also reduces intraocular pressure, and the effects of WIN55212-2 are mediated at least in part by the CB1 cannabinoid receptors in the eye.

It has been reported that WIN55212-2, a prototypic aminoalkylindole, has higher affinity for CB(2) than for CB(1). To explain the selectivity of WIN55212-2 for CB(2), molecular modeling studies were performed to probe the interacting sites between WIN55212-2 and cannabinoid receptors. In TMH5 the position 5.46 is a Phe in CB(2) versus a Val in CB(1). Docking of WIN55212-2 into the models of CB(1) and CB(2) predicts that F5.46 will result in a greater aromatic stacking of CB(2) with WIN55212-2. Using site-directed mutagenesis, this hypothesis was tested by exchanging the amino acids at position 5.46 between CB(1) and CB(2). Two mutations, including a Phe to Val mutation at the position 5.46 in CB(2) (CB2F5. 46V), and a corresponding Val to Phe mutation at the position 5.46 in CB(1) (CB(1)V5.46F), were made. The mutant receptors were transfected into 293 cells, and stable cell lines expressing similar numbers of receptors as wild-type receptors were chosen for additional ligand binding and cAMP accumulation studies. In ligand- binding assays, the CB(2)F5.46V mutation decreased the affinity of WIN55212-2 for CB(2) by 14-fold. In contrast, the CB(1)V5.46F mutation increased the affinity of WIN55212-2 for CB(1) by 12-fold. However, these mutations did not change the affinity of HU-210, CP-55940, and anandamide for CB(1) and CB(2). In cAMP accumulation assays, the changes in EC(50) values of WIN55212-2 were consistent with the changes in its binding affinity caused by the mutations. These results strongly support the hypothesis that the selectivity of WIN55212-2 for CB(2) over CB(1) is attributable to the change from Val in CB(1) at position 5.46 to Phe in CB(2).

The thermogenesis curves of the germination of different rice and tree seeds were determined and studied by using a newly constructed microcalorimeter. The thermogenesis curves of the germination of the seeds demonstrate the existence of physiological triphasic patterns, which include imbibition, activation and growth stages in the germination process. The thermodynamics and thermokinetics of the main growth phase of the growth stage in the germination process have been studied. The growth heat effect (deltaH), the growth rate constant (k), the growth inhibitory factor (s) and deceleration rate constant (beta) have been determined and calculated, In addition, the experimental thermokinetic equations of the growth stage in the seed germination process have been established.

Lys192 in the third transmembrane domain of the human CB1 cannabinoid receptor was converted to an alanine to study its role in receptor recognition and activation by agonists. HU-210, CP-55940, WIN55212-2, and anandamide, four cannabinoid agonists with distinct chemical structures, were used to characterize the wild-type and the mutant receptors. In human embryonal kidney 293 cells stably expressing the wild-type receptor, specific binding to [3H]WIN55212-2 and inhibition of cAMP accumulation by cannabinoid agonists were demonstrated, with different ligands exhibiting the expected rank orders of potency and stereoselectivity in competition binding and functional assays. In cells expressing the mutant receptor, the binding affinity of the receptor for [3H]WIN55212-2 was only slightly affected (the Kd for the mutant receptor was twice that of the wild-type), and the ability of WIN55212-2 to inhibit cAMP accumulation was unchanged. However, HU-210, CP-55940, and anandamide were unable to compete for [3H]WIN55212-2 binding to the mutant receptor. In addition, the potencies of HU-210, CP-55940, and anandamide in inhibiting cAMP accumulation were reduced by > 100-fold. These results demonstrate that Lys192 is critical for receptor binding by HU-210, CP-55940, and anandamide. Because Lys192 is not important for receptor binding and activation by WIN55212-2, WIN55212-2 must interact with the cannabinoid receptor through at least one point of interaction that is distinct from those of the three other agonists.

A novel multiplexing scheme for dense holographic storage in photorefractive crystals, spatioangular multiplexing, is described in detail. Compared with spatial multiplexing, spatioangular multiplexing increases the storage capacity by fully utilizing the volume of the storage medium. On the other hand, spatioangular multiplexing reduces the number of holograms overlapping any one hologram in a given volume and so increases the diffraction efficiency achievable as compared with angular multiplexing. Using this scheme, we succeeded in storing 756 high-resolution patterns in an Fe:LiNbO(3) crystal of volume 1 cm(3), with an average diffraction efficiency of 0.5%. This large database is designed for practical use in a novel associative-memory system, called a high-order feedback neural network.

Cosmids containing human genes for three orphan G protein-coupled receptors, GPR12, GPR6, and GPR3, were isolated using their rat homologs as probes. Previous studies of the mouse and rat cDNAs have shown the receptors to be expressed primarily in brain but have failed to identify their ligands. The three receptor proteins of 334, 363, and 330 amino acids, respectively, are encoded by a single exon in each gene. Excluding the divergent sequences preceding the first transmembrane domain, they have approximately 60% amino acid identity with each other. Fluorescence in situ hybridization of GPR12, GPR6, and GPR3 localized these three genes to human chromosomal regions 13q12, 6q21, and 1p34.3-p36.1, respectively.

80 cases with acquired infantile mental retardation caused by perinatal brain injury was treated with large doses of hyoscyamus, self made Retarded Recovery Pill and acupuncture. At the same time, 50 cases were taken as control group treated with Nao An Tai, Nao Fu Kang, etc. After 3 month treatment, the Intelligence Quotient (IQ) increased by 15 points in 29 cases of treatment group, while 3 cases in control group, chi 2 = 15.2, P < 0.01. After treatment for 6 month the IQ increased in 41 and 5 cases respectively, t = 5.53, P < 0.01. In 58.75% of the faculty of speech and motion greatly improved. It revealed that to treat the acquired infantile mental retardation, the combined therapy of traditional Chinese and Western medicine is effective.

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.

Corticotropin-releasing factor (CRF) has been shown to release endogenous opioid peptides from several rat brain regions. Since we have demonstrated previously that the actions produced by intrathecally administered CRF in mice involve spinal kappa opioid receptors, experiments were conducted in this study to test the possibility that CRF may release dynorphin A, a putative endogenous kappa opioid agonist, from the mouse spinal cord. Using a superfusion system in vitro, mouse spinal cords were superfused with aerated (95% O2, 5% CO2) Krebs-Ringer buffer. Fractions of superfusion were collected and dynorphin A levels in each fraction were monitored by radioimmunoassay. The presence of CRF in the perfusion buffer stimulated significantly the release of immunoreactive dynorphin A. The releasing rate of immunoreactive dynorphin A returned to the basal level after withdrawing CRF from the superfusion buffer. The stimulatory effect of CRF on the release of immunoreactive dynorphin A was abolished by alpha-helical CRF-(9-41), a CRF receptor antagonist, indicating that the dynorphin-releasing effect of CRF was mediated by CRF receptors in the spinal cord. Also the dynorphin-releasing effect of CRF was a concentration-related phenomenon, with an estimated EC50 value of 5.3 nM. The results from this study support the hypothesis that intrathecally administered CRF may produce its effects by releasing endogenous dynorphin from the terminals of dynorphin-containing neurons in the spinal cord. This study also provides evidence to support the notion that there is a close communication between CRF- and opioid peptide-containing neuronal pathways in the central nervous system.

Intrathecally (i.t.) administered corticotropin-releasing factor (CRF) has been shown to produce antinociception in the mouse abdominal stretching (writhing) assay. It also has been demonstrated that spinal kappa opioid receptors as well as CRF receptors are involved in the antinociception induced by CRF. In the present study, the role of CRF i.t. in modulating nociception was assessed further using the mouse tail-flick test. In addition, the modulatory effect of i.t. administered CRF on the antinociceptive activity of morphine was studied. Despite its potent and long-lasting antinociceptive effect in the writhing assay, CRF injected i.t. produced no consistent antinociception in the tail-flick test at doses up to 20 times the antinociceptive ED50 of CRF in the writhing test. In contrast, i.t. injection of CRF significantly attenuated the antinociceptive action of s.c. administered morphine. CRF at doses of 0.1 and 0.2 nmol/mouse i.t. increased the antinociceptive ED50 of s.c. morphine by 2- and 4-fold, respectively. The antagonistic action of CRF peaked between 15 min and 1 hr after i.t. injection and was still observable 4 hr after injection, demonstrating a time course similar to that of the antinociceptive effect of CRF in the writhing test. Intrathecal injection of alpha-helical CRF(9-41), a competitive CRF receptor antagonist, was able to inhibit, in a dose-dependent manner, the antagonistic activity of CRF. The antagonistic action of CRF also was attenuated dose-dependently by i.t. injection of nor-binaltorphimine (nor-BNI), a highly selective kappa opioid receptor antagonist. However, intrathecal injection of (+)-1-nor-BNI, an inactive enantiomer of nor-BNI, did not affect the antagonistic action of CRF. These data suggest that spinal kappa opioid receptors as well as CRF receptors are involved in the antagonistic effect of CRF against morphine antinociception.

The modulatory effects of intrathecally (i.t.) administered dynorphin A(1-17) and dynorphin A(1-13) on morphine antinociception have been studied previously in rats by other investigators. However, both potentiating and attenuating effects have been reported. In this study, the modulatory effects of i.t. administered dynorphin A(1-17) as well as the smaller fragment, dynorphin A(1-8), were studied in mice. In addition, nor-binaltorphimine (nor-BNI), a highly selective kappa opioid receptor antagonist, and naltrindole (NTI), a highly selective delta opioid receptor antagonist, were used to characterize the possible involvement of spinal kappa and delta opioid receptors in the modulatory effects of the dynorphins. Dynorphin A(1-17) and dynorphin A(1-8) administered i.t. at doses that did not alter tail-flick latencies, were both able to antagonize in a dose-dependent manner, the antinociceptive action of s.c. administered morphine sulfate. The antinociceptive ED50 of morphine sulfate was increased 3.9- and 5.3-fold by 0.4 nmol/mouse of dynorphin A(1-17) and dynorphin A(1-8), respectively. Injections of 0.4 and 0.8 nmol/mouse of nor-BNI i.t., but not its inactive enantiomer (+)-1-nor-BNI, inhibited dose-dependently the antagonistic effects of the dynorphins. These doses of nor-BNI alone did not affect the antinociceptive action of morphine sulfate. Intrathecal administration of 5 nmol/mouse of NTI also did not affect the modulatory effects of dynorphins. These observations that dynorphins exert their antagonistic effects on morphine-induced antinociception stereoselectively through spinal kappa opioid receptors may suggest a coupling between spinal kappa and mu opioid receptors.

Corticotropin-releasing factor (CRF), a putative stress transmitter, in previous studies failed to show any antinociceptive effect after central intracerebroventricular administration. In this study, a possible role of intrathecally (i.t.) administered CRF in modulating nociception and the involvement of opioid receptors were examined. CRF (i.t.) produced a dose-dependent antinociceptive effect in the mouse abdominal stretching (writhing) assay, with an ED50 of 22.1 (18.9-25.8) pmol/mouse. This antinociceptive effect of CRF peaked from 10 min to 1 hr after i.t. administration, although a significant antinociceptive effect was still observable 4 hr after injection. The dose-response curve of CRF antinociception was shifted to the right by i.t. administered alpha-helical CRF (9-41), a CRF antagonist, and the ED50 was raised by 3.7-fold. Naloxone, an opioid antagonist, also dose-dependently shifted the antinociceptive dose-response curve of CRF to the right. Naloxone at 1 and 10 mg/kg s.c. increased the ED50 value of CRF by 2- and 4-fold, respectively. In contrast, s.c. injection of 10 mg/kg of (+)-naltrexone, an inactive enantiomer of naltrexone, did not affect significantly the antinociception of i.t. administered CRF. Intrathecal injection of nor-binaltorphimine, a highly selective kappa opioid antagonist, increased the antinociceptive ED50 of CRF by 5.6-fold. However, the antinociceptive activity of CRF (i.t.) was not significantly affected by either beta-funaltrexamine, an irreversible mu selective opioid antagonist, or naltrindole, a highly selective delta opioid antagonist. Furthermore, the antinociceptive ED50 of CRF was not altered by (+)-1-nor-binaltorphimine, an inactive enantiomer of nor-binaltorphimine.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of the guinea pig ileum (GPI) and mouse vas deferens (MVD) to quantify opiate tolerance and physical dependence in the same preparation was examined. In the GPI, tolerance to and physical dependence on various opiates were induced by direct incubation with an agonist and tolerance and abstinence were assessed after its rapid removal by extensive washing. Dependence, as evidenced by an increase in twitch height and after removal of the agonist as compared to that before incubation, provided an index of the degree of physical dependence. The extent of the supersensitive response after agonist removal was noted to be related directly to the agonist concentration and the degree of tolerance development. Furthermore, the supersensitive response was found to be stereospecific, naloxone reversible and preferentially mu receptor-mediated. Likewise, tolerance to opiates could be demonstrated in the MVD by direct incubation with an agonist. Unlike the GPI, rapid removal of the agonist by washing did not produce a supersensitive response to electrical stimulation. However, a supersensitive response was demonstrable in the presence of naloxone. Thus, after a tolerant/dependent state was induced by incubation of the vas with a predetermined dose of [D-Ala2, D-Leu5]enkephalin that completely abolished the twitch response, the subsequent addition of naloxone resulted in a supersensitive response to electrical stimulation that was concentration-dependent. The maximum twitch response in the presence of naloxone, compared to that obtained before incubation with the agonist, provided an index of the extent of dependence.(ABSTRACT TRUNCATED AT 250 WORDS)

The fundamental growth thermograms of bacteria have been determined by using the microcalorimetric method. These perfect thermogram curves reflect the changes of bacterial growth patterns (including the lag phase of growth, log growth, stationary phase and the decline phase of growth). In our experiments, highly characteristic and reproducible growth patterns are observed under the same condition, therefore one can use these thermograms as "finger print" to discriminate bacteria. On the other hand, there thermogram curves contain ample information, which are very significant for the studies on microorganism metabolism, bio-thermokinetic and clinical fields.