Dietary methionine requirement of Jing Brown layer hens from 9 to 17 weeks of age.

This study was conducted to evaluate the effect of dietary methionine (Met) supplementation in growth performance and reproductive performance of Jing Brown layer hens. A total of 375 9-week-old Jing Brown layer hens were allocated equally to five treatments consisting of 5 replicates with 15 hens. Hens were fed with a diet of corn and soya bean meal supplemented with 0.23%, 0.27%, 0.31%, 0.35% and 0.39% Met respectively. Different Met levels did not significantly affect average daily feed intake (ADFI), average daily gain (ADG) and feed/gain ratio (F/G) (p > 0.05), whereas flock uniformity (FU) and jejunum index were significantly different (p < 0.05), and the largest FU was observed in 0.31% Met. Dietary supplementation of Met significantly affected reproductive system development (p < 0.05), and 0.27-0.31% Met obtained optimal reproductive system development. Different Met levels significantly affected serum uric acid and alkaline phosphatase. Moreover, the relatively higher reproductive hormones in serum were observed in 0.27% Met. Analysis of quadratic curve estimation of flock uniformity, the total number of follicles, the primary follicles and the secondary follicles showed that the optimal Met levels were 0.293%, 0.286%, 0.286% and 0.288%, which could be averaged to 0.288%. These results suggested that the optimal Met requirement for Jing Brown layer hens from 9 to 17 weeks old is 0.29%.

Effects of dietary threonine levels on growth performance, serum biochemical indexes, antioxidant capacities, and gut morphology in broiler chickens.

A dose-response experiment was conducted to investigate the impacts of dietary threonine (Thr) levels on growth performance, serum biochemical indices, antioxidant capacities, and gut morphology of broiler chickens. Four hundred and thirty-two 1-d-old commercial broilers were allocated to 4 treatments consisting of 6 replicates of 18 birds. The experimental treatments received the same Thr-deficient basal diet and were labeled as follows: 85%, 100%, 125%, and 150% of NRC (1994) recommendations. The results demonstrated that on 21 d and 42 d, average daily weight gain (ADG, 22 to 42 d, 0 to 42 d) increased quadratically or cubically as the inclusion of Thr increased, while feed conversion ratio (FCR, 0 to 21 d, 0 to 42 d) decreased quadratically or cubically as dietary Thr increase from 85% to 150%. Excess dietary Thr levels triggered plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities. The concentrations of total protein (TP) and globulin (GLO) increased quadratically with increasing Thr level, and the highest concentrations of TP and GLO were obtained at the 125% Thr level. Moreover, the plasma uric acid (UA) concentration decreased linearly or quadratically with the increase in dietary Thr level. Likewise, the serum glutathione peroxidase (GSH-Px) and total superoxide dismutases (T-SOD) activities increased quadratically as dietary Thr increased, and the highest activity of GSH-Px was obtained at the 125% Thr level, while the highest T-SOD level occurred in the 100% Thr group. Gut morphology of birds showed significant response to different graded concentrations of Thr level. Villus height (VH), crypt depth (CD), and VH:CD ratio (VH/CD) were increased linearly or quadratically by Thr supplementation. Therefore, the present study suggests that the NRC (1994) recommendations Thr level that was optimum for growth performance, and 125% of the NRC (1994) recommendations Thr level had better effects on biochemical indices, antioxidant function, and gut morphology of broilers.

Etretinate augments interferon beta-1b effects on suppressor cells in multiple sclerosis.

BACKGROUND: Interferon beta treatment is only partially effective in multiple sclerosis (MS) suggesting a potential role for adjunctive therapies. Retinoids can augment the clinical efficacy of type 1 interferons in patients with cancer. We reasoned that the same might hold in MS. Interferon beta-1b added to peripheral blood mononuclear cells in vitro partially reverses the CD8 suppressor cell defect of patients with MS. All-trans retinoic acid added to peripheral blood mononuclear cells from untreated patients with MS or from controls potentiates this ability of interferon beta-1b to augment CD8 suppressor cell function in vitro. OBJECTIVE: To determine whether retinoid administration to patients with MS who are being treated with interferon beta-1b augments their CD8 suppressor cell function. SETTING: A university hospital MS clinic. PARTICIPANTS: Patients with MS who were being treated with interferon beta-1b, 14 patients with secondary progressive MS and 3 patients with relapsing remitting MS. RESULTS: Seventeen patients with MS received etretinate treatment for up to 6 months. Planned dosing was 10 mg 3 times daily for the first month, 25 mg twice daily for the second and third months, and 10 mg twice daily thereafter. The 25-mg twice daily dose was not well tolerated and of the 14 patients who remained in the phase 1 clinical trial through month 3 dose reduction to 10 mg thrice daily was required in 1 patient and to 10 mg twice daily in 4 patients. Eleven patients completed the trial. Etretinate treatment significantly augmented suppressor function over baseline values at 1, 3, and 6 months. No meaningful change was noted in disability or quality of life over the course of the phase 1 clinical trial. Neuropsychological testing of completers suggested improvement on selected aspects of verbal memory at 6 months compared with baseline values. CONCLUSIONS: Etretinate treatment at a dose of 10 mg twice or three times daily augments suppressor cell function in patients with MS receiving interferon beta-1b. Higher dose etretinate treatment (25 mg twice daily) is poorly tolerated by patients with MS. Even at 10 mg twice daily adverse experiences involving the mucous membranes and the skin become troublesome for some, but not all, patients. Whether pulse therapy or administration of retinoid restricted to the day of interferon beta dosing will also augment suppressor function, while being better tolerated, remains to be determined.

All-trans retinoic acid potentiates the ability of interferon beta-1b to augment suppressor cell function in multiple sclerosis.

OBJECTIVE: To determine the effects of combination all-trans retinoic acid (RA) and interferon beta-1b therapy on immune system functions potentially relevant to multiple sclerosis (MS). DESIGN: Interferon gamma-secreting cells, T suppressor cell function, and lymphocyte proliferative responses were assayed using peripheral blood mononuclear cells from patients with MS and control subjects under control conditions and in the presence of interferon beta-1b, RA, and the 2 combined. SETTING: A university hospital MS clinic. PARTICIPANTS: Seventeen patients with secondarily progressive MS and 25 control subjects. RESULTS: Interferon beta-1b use increased interferon gamma-secreting cell counts, augmented T suppressor cell function, and inhibited T-cell proliferation. Therapy with RA decreased interferon gamma-secreting cell counts, had a minimal positive effect on T suppressor cell function, and had no effect on T-cell proliferation. When RA and interferon beta-1b were combined, the inhibitory effect of RA on interferon gamma-secreting cells predominated, T suppressor cell function increased synergistically over the increment observed with interferon beta-1b use alone, and the inhibitory effect of interferon beta-1b alone on T-cell proliferation remained unchanged. CONCLUSIONS: Treatment with interferon beta-1b partially restores defective T suppressor cell function in patients with MS. This potentially beneficial action is synergistically potentiated by RA. Interferon beta-1b increases the number of interferon gamma-secreting cells in the circulation when treatment is initiated. A similar increment in interferon gamma-secreting cells is observed when interferon beta-1b is added to cultural peripheral blood mononuclear cells in vitro. This potentially deleterious action of interferon beta-1b is reversed by RA. Interferon beta-1b inhibits lymphocyte proliferation modestly but reproducibly. This action of interferon beta-1b is unaltered by RA. These data provide a rationale for a trial of combination treatment with interferon beta-1b and RA in patients with MS.

GM1 ganglioside and darkly staining neurons in brains of rats subjected to neonatal hypoxia-ischemia.

Rat pups, seven days old, with right carotid artery ligations were exposed to an atmosphere of oxygen 8% remainder nitrogen for 2 hr. The animals that survived for three weeks after the hypoxic-ischemic episode had clusters of darkly stained (hematoxylin-eosin) neurons in the cortex and reduced uptake of dopamine (frontal cortex) and choline (frontal cortex, hippocampus and striatum) in preparations of synaptosomes. Treatment with GM1 ganglioside partially corrected the loss of uptake activity and increased the number of darkly stained neurons.

Effect of U-50488h, a selective opioid kappa receptor agonist, on vascular injury after spinal cord trauma.

U-50488H, a selective opioid kappa receptor agonist has been shown to be a neuroprotective agent in animal models of spinal cord injury. The mechanism of action of U-50488H is not known. Methylprednisolone, the only neuroprotective drug proven in patients with acute spinal cord injury may prevent the secondary injury after an initial trauma. Secondary vascular injury develops after experimental spinal cord trauma. In this study we examined the effects of U-50488H on post-traumatic vascular injury based on the measurement of vascular permeability, edema and neutrophil infiltration in a rat spinal cord injury model. Vascular permeability was assessed by vascular extravasation of fluorescein isothiocyanate conjugated dextran (FITC-D), a macromolecular tracer. Tissue edema was determined by percentage water content and neutrophil infiltration by myeloperoxidase (MPO) activity, a marker enzyme for neutrophils. U-50488H at doses of 5, 10, 20 and 40 mg/kg i.p. administered twice (0.5 h before and 0.5 h after trauma) reduced vascular permeability in a dose-dependent manner. More frequent dosing (10 mg/kg, 0.5 h before and 0.5, 2, 8 and 22 h after injury) reduced vascular permeability 24 h after injury. U-50488H also reduced edema formation but did not affect neutrophil infiltration. Results from this study raise the possibility that the neuroprotective effect of U-50488H involves a secondary vascular event.

Dynorphin A (1-13) potentiates dynorphin A (1-17) on loss of the tail-flick reflex after intrathecal injection in the rat.

Dynorphin A (1-13) or dynorphin A (1-17) administered intrathecally to rats induces a dose-dependent loss of the tail-flick reflex and a reversible hindlimb paralysis. Although their potency is comparable, dynorphin A (1-17) is less efficacious in producing loss of the tail-flick reflex. These data suggest that dynorphin A (1-17) acts as a partial agonist or exerts its neurotoxic effect in the presence of an endogenous non-competitive inhibitor. To determine whether these substances act at similar sites we performed isobolographic analysis. This analysis revealed that dose-additivity exists for paralysis whereas deviation from dose-additivity exists for loss of the tail-flick reflex.

Receptor-linked hydrolysis of phosphoinositides and production of prostacyclin in cerebral endothelial cells.

The receptor agonist-mediated hydrolysis of phosphoinositides and production of prostacyclin were studied in murine cerebral endothelial cells (MCEC). Of 11 neurotransmitters and neuromodulators examined, carbachol, noradrenaline (NE), bradykinin, and thrombin significantly increased 3H-inositol phosphate accumulation in the presence of LiCl (20 mM). The maximal stimulation of [3H]inositol monophosphate ([3H]IP1) reached approximately 11, 11, seven, and four times the basal levels for carbachol, NE, bradykinin, and thrombin, respectively. The EC50 values of IP1 accumulation for carbachol and NE were 34 and 0.16 microM, respectively. The muscarinic antagonists, atropine and pirenzepine, blocked the carbachol-induced IP1 accumulation with Ki values of 0.3 and 30 nM, respectively. The adrenergic antagonist, prazosin, blocked NE-induced IP1 accumulation with a Ki of 0.1 nM. The calcium ionophore A23187, histamine, glutamate, vasopressin, serotonin, platelet activating factor, and substance P did not stimulate IP1 accumulation. A23187, bradykinin, and thrombin stimulated prostacyclin release to approximately four, four, and two times the basal levels, respectively, whereas carbachol and NE had little effect upon prostacyclin release. These results suggest that the activation of phospholipase C and of phospholipase A2 in MCEC are regulated separately.

Protective effect of methylprednisolone on vascular injury in rat spinal cord injury.

High-dose methylprednisolone (MP) given to patients within 8 h of traumatic spinal cord improved neural function at 6 and 12 months, suggesting a probable secondary injury process that may be amenable to therapeutic intervention. Vascular injury plays an important role in the secondary injury process of CNS trauma. We have examined the effect of MP on vascular changes, including tissue edema, vascular permeability, and polymorphonuclear (PMN) cell infiltration in a rat model of spinal cord impact injury. MP significantly reduced extravasation of fluorescein isothiocyanate dextran (FITC-D), a macromolecular tracer, by 64.3% and 50.7% with trauma forces of 20 and 40 g-cm, respectively, when MP was administered IV immediately after trauma at a bolus of 165 mg/kg, with a subsequent continuous MP infusion at 31.5 mg/kg/h for 23 h. MP reduced the water content in the 40 g-cm traumatic cord lesion to 73.0% compared to the traumatic control (74.3%, p < 0.001) at the same schedule of large dose 24-h infusion. The same doses of MP showed a trend to decrease the extent of neutrophil infiltration as determined by myeloperoxidase (MPO) activity, but the change was not significant. MP had little effect in decreasing FITC-D extravasation and cord edema when given at a lower dose (bolus of 30 mg/kg with continued infusion of 1.3 mg/kg/h for 23 h). MP did not reduce extravasation of FITC-D and edema when administered IV as one bolus injection at high (165 mg/kg) or low (30 mg/kg) doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential changes of retina dopamine binding sites and adenylyl cyclase responses following 6-hydroxydopamine treatment.

Both dopamine D1 and D2 receptors are present in rat retina. D1 receptors are positively coupled to adenylyl cyclase, while D2 receptors are negatively coupled. After intraocular administration of the neurotoxin 6-hydroxydopamine (6-OHDA) there is depletion of retinal dopamine by about 90%, as well as a decrease of the number of D1 and D2 receptor binding sites. Following the 6-OHDA lesion, there is an enhancement of the D1 receptor-stimulated accumulation of cyclic-AMP and a loss of D2 receptor-inhibited accumulation of cyclic-AMP. Our results suggest that some of the retinal D2 receptors are coupled to adenylyl cyclase and some are not.

A sensitive fluorometric method for measurement of vascular permeability in spinal cord injury.

A sensitive fluorometric method was modified for the evaluation of drug action upon vascular permeability in rat spinal cord injury. Fluorescein isothiocyanate-conjugated dextran (FITC-D MW 71,200), used as a macromolecular tracer, was injected iv 2 hours before sacrifice. The optimal pH for FITC-D fluorescence was 8.2. The recovery in spinal cord was 101.4 +/- 4.0% (mean +/- SD). The extent of FITC-D extravasation, expressed as the vascular injury index (VII), was increased in proportion to the trauma force. The peak of VII after trauma was at 2 hours. This fluorometric method is sensitive, simple, and reliable for evaluation of drug effects upon vascular permeability in CNS trauma.

Pharmacological characterization of rat retinal dopamine receptors.

The dopamine (DA) D-1 and D-2 receptors coupled to adenylate cyclase in the rat retina were characterized pharmacologically. In confirmation of reports using other neural tissues, activation of D-1 receptors with DA, apomorphine or SKF 38393 resulted in activation of adenylate cyclase and enhanced accumulation of cyclic AMP (cAMP). The response to DA was blocked by SCH 23390, a D-1 receptor antagonist. D-2 receptors negatively coupled to adenylate cyclase were demonstrated by preincubating retina with SCH 23390 and then with DA or apomorphine. D-2 receptor responses were also elicited with quinpirole or bromocriptine, D-2 receptor agonists, in the absence of SCH 23390. (+)-Butaclamol, but not (-)-butaclamol, blocked the D-2 receptor-induced decrease of cAMP. Moreover, I-sulpiride was more active than d-sulpiride in reversing the DA-induced inhibition of cAMP accumulation. D-1 and D-2 receptor responses were also evident in forskolin-activated retina. The intraocular injection of pertussis toxin prevented the fall of cAMP and enhanced the rise of cAMP by DA, indirectly implicating the need for a guanine nucleotide regulatory protein in the process. Our results demonstrate that retinal tissue contains DA receptors that are similar to those found in brain and they imply that therapeutic agents that interact with the receptors in brain might interact with the receptors in retina.

Pharmacological characterization of muscarinic receptors mediating inhibition of adenylate cyclase activity in the rat retina.

Incubation of the rat retina with acetylcholine resulted in about a 20 to 30% decrease of basal cyclic AMP accumulation. Oxotremorine, arecoline, [4-hydroxy-2-butynyl]trimethylammonium chloride, m-chlorocarbanilate and carbachol also inhibited cyclic AMP accumulation. Nicotine had no effect. The response was blocked by atropine and pirenzepine but not gallamine. Intraocular injection of pertussis toxin 72 hr before testing also blocked the response to acetylcholine. The presence of forskolin exaggerated the response to acetylcholine. Intraocular injection of the cholinotoxin AF64A resulted in apparent supersensitivity of the response to acetylcholine. Our results suggest that rat retina contains muscarinic M1 receptors that are coupled negatively to adenylate cyclase.

MPP+ depletes retinal dopamine and induces D-1 receptor supersensitivity.

Intraocular administration of 1-methyl-4-phenylpyridinium ion (MPP+) to mice resulted in a dose-dependent depletion of retinal dopamine (DA) and 3,4-dihydroxyphenylacetic acid. Pretreatment with benztropine partially prevented the MPP+-induced depletion. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was less active than MPP+ for depleting DA when administered by the same route and pretreatment with deprenyl partially prevented the depletion. Supersensitivity of retinal D-1 receptors resulted following MPP+-induced DA depletion.

Expression and characterization of a truncated murine Fc gamma receptor.

We have isolated a recombinant secreted Fc gamma R beta molecule by deletion of the transmembrane and cytoplasmic domains encoding sequence from a Fc gamma R beta 1 cDNA clone, and insertion of the truncated cDNA into a eukaryotic expression vector, pcEXV-3. To express and amplify the production of the truncated Fc gamma R beta molecule, we transfected the truncated cDNA plasmid into a dihydrofolate reductase-minus CHO cell line along with a dhfr minigene, and amplified the gene products with methotrexate. The resulting cell line secretes 2-3 micrograms/ml/24 h of truncated Fc gamma R beta, which can be readily purified by affinity chromatography on IgG-Sepharose. The truncated Fc gamma R beta has a Mr of 31-33,000 on SDS-PAGE and is glycosylated. N-glycosidase F cleavage reduces the Mr to 19,000, consistent with the size of the truncated product, 176 amino acid residues. There are two disulfide bonds in the protein. Binding of immune complexes formed between DNP20BSA and anti-DNP mAbs reveals better binding of IgG1 aggregates than that of IgG2b and IgG2a aggregates. The binding of the immune complexes was somewhat better at more acidic pH, in contrast to previous experiments with binding of purified Fc gamma R to immune complex-coated beads. We were surprised to observe that the truncated Fc gamma R beta did not react with the anti-Fc gamma R mAb 6B7C. Previous work had shown that 6B7C reacts with Fc gamma R on immunoblots, fails to bind to the surface of resting B cells and peritoneal macrophages, but does bind to macrophage cell lines and LPS-stimulated B cells. We show, by binding of mAb 6B7C to a peptide conjugate, that the 6B7C epitope lies within residues 169-183 of the intact Fc gamma R beta, which is just outside the plasma membrane. The availability of the truncated Fc gamma R beta in microgram quantities should facilitate further analysis of structure and function of these receptors.