Enzymatic effects of Pleurotus ostreatus spent substrate on whole-plant corn silage and performance of lactating goats.

Pleurotus ostreatus (oyster mushroom) synthesizes enzymes that degrade lignin, cellulose, and hemicellulose. The objectives of this study were to evaluate the effect of Pleurotus ostreatus spent substrate (POSS) on whole-plant corn silage (WPCS) chemical composition, antioxidant capacity, lignin monomers, and in vitro digestibility, as well as the performance of lactating goats fed corn silage treated with different levels of POSS. In experiment 1, 4 levels of lignocellulolytic enzymes were tested in a complete randomized design: 0, 10, 20, and 30 mg of lignocellulosic enzymes per kilogram of fresh matter, 4 replicates per treatment (vacuum-sealed bags). The bags were opened 60 d after ensiling. In experiment 2, corn silage treated with 3 enzyme levels (0, 10, or 30 mg/kg of fresh matter) was fed to lactating goats as part of the total mixed ration. Nine lactating Saanen goats (62.68 ± 7.62 kg BW; 44 ± 8 d in milk; 2.91 ± 0.81 kg of milk/day, mean ± SD) were assigned to three 3 × 3 Latin squares. Data were analyzed using the GLIMMIX procedure of SAS (version 9.4, SAS Institute Inc.), and means were compared by linear and quadratic orthogonal contrast. In experiment 1, neutral detergent fiber (NDF), acid detergent fiber (ADF), lignin, and cellulose quadratically decreased in the WPCS treated with POSS. At the nadir point, POSS decreased NDF by 14.1%, ADF by 19.5%, lignin by 9.07%, and cellulose by 22.1% compared with the untreated silage. Therefore, POSS led to a quadratic increase in in vitro dry matter digestibility of WPCS (+8.88% at the vertex) compared with the untreated silage. In experiment 2, POSS quadratically increased the in vivo total-tract ADF digestibility. Also, the concentration of polyphenols in the milk of goats linearly increased with the addition of POSS, and no differences were observed among treatments for milk yield and composition. In summary, adding 10 mg of lignocellulolytic enzymes from POSS per kilogram of fresh matter of whole-plant corn at ensiling had a more evident reduction in lignin and cellulose concentration, leading to greater in vitro digestibility, as well as greater in vivo ADF digestibility; however, milk yield was not different among treatments.

Associations of neonatal adiponectin and leptin with growth and body composition in African American infants.

BACKGROUND: Cord blood adiponectin and leptin concentrations are associated with birth weight and adiposity. Birth size and rate of infant weight gain are associated with future obesity risk. However, it is unclear whether biomarkers reflecting the intrauterine environment are predictive of infant prospective body composition change. OBJECTIVES: To examine whether cord blood adiponectin and leptin are predictive of neonatal adiposity and fat mass (FM) accrual to 3 months of age. METHODS: Participants (n = 36) were healthy African American infants. Leptin and adiponectin concentrations were measured in umbilical cord blood. At 2 weeks and 3 months, infant body composition was assessed via air displacement plethysmography. Weight-for-length z-scores (WLZ) were calculated using World Health Organization standards. Multiple linear regression was used to examine associations of cord blood adiponectin and leptin with birth WLZ; WLZ, FM and fat-free mass at 2 weeks, and the conditional change in these variables from 2 weeks to 3 months (body composition at 3 months adjusted for body composition at 2 weeks). RESULTS: Adiponectin was positively associated with FM at 2 weeks (r = 0.45, P < 0.01), but inversely associated with conditional FM change from 2 weeks to 3 months of age (r = -0.38, P < 0.05). Leptin was not significantly associated with infant body composition. CONCLUSIONS: Adiponectin may be a marker for FM accrual in African American infants, a relatively understudied population with a high long-term obesity risk. Mechanistic studies are needed to determine whether adiponectin directly influences infant growth or is simply a maker reflective of other ongoing biological changes after birth.

Association of early pregnancy body mass index with post-partum weight change among African-American women.

Post-partum weight retention is relatively common and increases the risk for future obesity. Women who are overweight or obese prior to pregnancy, or who gain excessively during pregnancy, are more likely to retain weight post-partum. Much of the existing research is limited by a single post-partum body-weight measure and therefore cannot distinguish post-partum weight retention from post-partum weight accrual. This study tested the hypothesis that early pregnancy body mass index (BMI) is positively associated with post-partum weight change, independent of gestational weight gain (GWG) and breastfeeding (BF) among African-American women, a demographic group with greater risk for obesity. Healthy African-American women (n = 32) were weighed at 2 weeks and 3 months post-partum to derive post-partum weight change. Data from prenatal care records were retrieved to calculate BMI at the first prenatal care visit and GWG. BF status at 2 weeks post-partum was self-reported. Early pregnancy BMI was positively associated with post-partum weight change (partial r = 0.53, P < 0.005), independent of GWG and BF status at 2 weeks post-partum. These results extend the literature by suggesting that the association between early pregnancy BMI and post-partum weight retention may be at least partially attributable to the accrual of new weight during the post-partum period. Future research in a larger and more diverse cohort is warranted and should explore potential mechanisms contributing to post-partum weight change.

Quantitative 3D micro-CT imaging of human lung tissue.

PURPOSE: To assess the feasibility of micro-CT for obtaining quantitative volumetric and morphologic information of changes in soft tissue, respiratory tracts and vascularization in fibrotic, emphysematous and non-diseased human lung specimens. MATERIALS AND METHODS: Specimens from autopsy or lung explantation with lung fibrosis of UIP pattern (n = 22) or centrilobular emphysema (n = 10) were scanned by micro-CT and compared to controls (n = 22). Imaging was performed subsequent to intravascular contrast enhancement for the assessment of the vascular volume fraction. The soft tissue and air fraction were quantified after the fixation of ventilated lungs followed by tissue contrast enhancement using osmium. Aiming an artifact-free 3 D reconstruction of lung acini, synchrotron-based micro-CT scans of specimens with emphysema (n = 5) and non-diseased tissue (n = 6) was performed. Micro-CT imaging was complemented by histology for the demonstration of comparable findings. RESULTS: Quantitative analysis showed a significant increase of the soft tissue fraction, equivalent to a decrease of the air fraction in fibrotic lungs compared to controls (p < 0.001) and a significant reduction of the vascular volume fraction compared to controls (p < 0.02). Specimens with emphysema demonstrated a significant increase of the air fraction with a decrease in soft tissue compared to controls (p < 0.001). 3 D reconstructions of lung acini worked successfully in non-diseased tissue but failed in fibrotic and emphysematous lungs. CONCLUSION: Our findings indicate micro-CT's technical feasibility to assess quantitative and morphological data from diseased and non-diseased human lung specimens.

Modulation of gastrointestinal function by MuDelta, a mixed µ opioid receptor agonist/ µ opioid receptor antagonist.

BACKGROUND & PURPOSE: Loperamide is a selective µ opioid receptor agonist acting locally in the gastrointestinal (GI) tract as an effective anti-diarrhoeal but can cause constipation. We tested whether modulating µ opioid receptor agonism with δ opioid receptor antagonism, by combining reference compounds or using a novel compound ('MuDelta'), could normalize GI motility without constipation. EXPERIMENTAL APPROACH: MuDelta was characterized in vitro as a potent µ opioid receptor agonist and high-affinity δ opioid receptor antagonist. Reference compounds, MuDelta and loperamide were assessed in the following ex vivo and in vivo experiments: guinea pig intestinal smooth muscle contractility, mouse intestinal epithelial ion transport and upper GI tract transit, entire GI transit or faecal output in novel environment stressed mice, or four weeks after intracolonic mustard oil (post-inflammatory). Colonic δ opioid receptor immunoreactivity was quantified. KEY RESULTS: δ Opioid receptor antagonism opposed µ opioid receptor agonist inhibition of intestinal contractility and motility. MuDelta reduced intestinal contractility and inhibited neurogenically-mediated secretion. Very low plasma levels of MuDelta were detected after oral administration. Stress up-regulated δ opioid receptor expression in colonic epithelial cells. In stressed mice, MuDelta normalized GI transit and faecal output to control levels over a wide dose range, whereas loperamide had a narrow dose range. MuDelta and loperamide reduced upper GI transit in the post-inflammatory model. CONCLUSIONS AND IMPLICATIONS: MuDelta normalizes, but does not prevent, perturbed GI transit over a wide dose-range in mice. These data support the subsequent assessment of MuDelta in a clinical phase II trial in patients with diarrhoea-predominant irritable bowel syndrome.

Stimulation of neuronal receptors, neuropeptides and cytokines during experimental oil of mustard colitis.

Oil of mustard (OM), administered intracolonically, produces severe colitis in mice that is maximized within 3 days. The purpose of this study was to characterize the cytokine response, and to establish expression patterns of enteric neuronal mediators and neuronal receptors affected during active colitis. We measured the changes in the mRNA levels for neuronal receptors and mediators by real-time PCR, and cytokine and chemokine protein levels in the affected tissue. Significant increases in neuronal receptors, such as transient receptor potential A1 (TRPA1), cannabinoid type 1 receptor, neurokinin 1 receptor (NK1R) and delta-opioid receptor; prokineticin-1 receptor; and soluble mediators, such as prodynorphin, proenkephalin1, NK1, prokineticin-1 and secretory leukocyte protease inhibitor, occurred. Significant increases in cytokines, such as interleukin (IL)-1beta, IL-6 and granulocyte macrophage colony stimulating factor (GM-CSF), and in chemokines, such as macrophage chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1 (MIP-1alpha) and Kupffer cell derived chemokine (KC), were detected, with no changes in T-cell-derived cytokines. Furthermore, immunodeficient C57Bl/6 RAG2(-/-) mice exhibited OM colitis of equal severity as seen in wt C57Bl/6 and CD-1 mice. The results demonstrate rapidly increased levels of mRNA for neuronal receptors and soluble mediators associated with pain and inflammation, and increases in cytokines associated with macrophage and neutrophil activation and recruitment. Collectively, the data support a neurogenic component in OM colitis coupled with a myeloid cell-related, T- and B-cell-independent inflammatory component.

Methicillin-resistant Staphylococcus aureus infection in liver transplantation: a matched controlled study.

UNLABELLED: The purpose of this study was to evaluate the clinical impact of methicillin-resistant Staphylococcus aureus (MRSA) infections on transplant recipients. METHODS: Liver and kidney recipients with MRSA infections were retrospectively identified and compared to an age, gender, UNOS status, organ transplanted, and transplant date matched (2:1) non-MRSA-infected recipient control group. All MRSA infections were initially treated with vancomycin, and four (33%) liver recipients were converted to linezolid therapy after failing to improve with vancomycin. RESULTS: The overall MRSA infection incidence was 1.4% (24/1770) with MRSA more common in liver (3.75%; 12/320) than kidney transplants (0.8%; 12/1450) (P < .001). The most common sites of MRSA infection were blood (42%), lung (38%), and abdomen (29%). The MRSA group had a greater percentage of prior antibiotic usage (79% vs 40%; P < .0015). The MRSA group experienced more posttransplant complications (52% vs 19%; P < .011)), and exhibited a trend toward greater length of stay in the intensive care unit (7.8 vs 4.6 days; P = .09), but not overall length of stay. Survival was similar in MRSA and non-MRSA groups (75% vs 88%; P = .17). No significant differences in mortality were noted between liver and kidney recipients infected with MRSA (P = .6). CONCLUSION: MRSA infection is associated with a higher incidence of posttransplant complications and antibiotic usage in both liver and kidney recipients compared to patients with MRSA infection.

Vanilloid receptor 1 antagonists attenuate disease severity in dextran sulphate sodium-induced colitis in mice.

Neurogenic mechanisms have been implicated in the induction of inflammatory bowel disease (IBD). Vanilloid receptor type 1 (TRPV1) has been visualized on nerve terminals of intrinsic and extrinsic afferent neurones innervating the gastrointestinal tract and local administration of a TRPV1 antagonist, capsazepine, reduces the severity of dextran sulphate sodium (DSS)-induced colitis in rats (Gut 2003; 52: 713-9(1)). Our aim was to test whether systemically or orally administered TRPV1 antagonists attenuate experimental colitis induced by 5% DSS in Balb/c mice. Intraperitoneal capsazepine (2.5 mg kg(-1), bid), significantly reduced the overall macroscopic damage severity compared with vehicle-treated animals (80% inhibition, P < 0.05); however, there was no effect on myeloperoxidase (MPO) levels. An experimental TRPV1 antagonist given orally was tested against DSS-induced colitis, and shown to reverse the macroscopic damage score at doses of 0.5 and 5.0 mg kg(-1). Epithelial damage assessed microscopically was significantly reduced. MPO levels were attenuated by approximately 50%, and diarrhoea scores were reduced by as much as 70%. These results suggest that pharmacological modulation of TRPV1 attenuates indices of experimental colitis in mice, and that development of orally active TRPV1 antagonists might have therapeutic potential for the treatment of IBD.

Regulation of M-type potassium current by intracellular nucleotide phosphates.

The effects of intracellular application of various concentrations of adenine nucleoside phosphates and nucleotide analogs on the M-type K current (IM) of single neurons isolated from sympathetic ganglia were studied. With 1 mM MgATP intracellularly IM decreased to 25% of its initial level 39 min after the start of whole-cell recording. In the absence of ATP the current decreased more rapidly. Addition of glucose and pyruvate extracellularly was equivalent to adding 1 mM MgATP intracellularly. AMP-PNP, a nonhydrolyzable ATP analog, at a concentration of 1 or 3 mM was unable to maintain IM in the absence of ATP. When ATP and AMP-PNP were combined in the pipette, however, the maintenance of IM was prolonged. A series of nucleotides and analogs have been combined with ATP to test for their ability to maintain IM and to alter calcineurin phosphatase activity. There was a positive correlation between the ability of a nucleotide to prevent the rundown of IM and its ability to inhibit calcineurin phosphatase activity. These findings show that the amplitude of IM is dually regulated by cellular levels of adenine nucleotide diphosphates and triphosphates. A hydrolyzable form of ATP is necessary to maintain the M current. The maintenance of IM is further enhanced by the simultaneous presence of ADP or other adenine nucleotides that alter calcineurin activity, but not by higher concentrations of ATP alone. These results are consistent with regulation of IM by phosphorylation events that maintain IM and dephosphorylation events that lead to current rundown.

Molecular characterization and functional expression of a substance P receptor from the sympathetic ganglion of Rana catesbeiana.

Substance P is an important neuropeptide neurotransmitter in the central, autonomic and enteric nervous systems. In sympathetic ganglia, substance P is thought to play a role in modulating synaptic transmission. Release of substance P by neuronal stimulation or direct application of substance P to ganglionic neurons increases neuronal excitability. An amphibian substance P receptor complementary DNA has been cloned and characterized from bullfrog, Rana catesbeiana, sympathetic ganglion complementary DNA libraries. The deduced primary structure contains features indicative of a seven transmembrane domain G-protein-coupled receptor. The deduced protein sequence shows 69% identity to previously cloned mammalian substance P receptors. In situ hybridization analysis performed on bullfrog sympathetic ganglia using digoxigenin-labelled complementary RNA probe demonstrated that approximately 75% of the principal neurons displayed reaction product above background levels. Radioligand binding studies were performed on stably transfected cells with [(125)I]Tyr-1-substance P as the ligand. Substance P had an IC50 of 16 nM and the agonist potency profile was substance P>neurokinin A >> neurokinin B. The order of potency for three tachykinins to increase intracellular calcium when applied to a stably transfected clonal cell line was substance P>neurokinin A >> neurokinin B. This order of agonist potency also held for inhibition of the M-type potassium current in intact bullfrog sympathetic neurons. The non-peptide substance P antagonists CP-96345 and RP-67580 at concentrations that block mammalian substance P receptors had little or no effect on the responses to substance P at the bullfrog receptor. Overall, these results demonstrate that the cloned sequence has the features consistent with and characteristic of a substance P receptor. The results are discussed with reference to the established pharmacology of the bullfrog substance P receptor and known structure activity relationships of mammalian tachykinin receptors.

Hyperpolarizing shift of the M-current activation curve after washout of muscarine in bullfrog sympathetic neurons.

The mechanism underlying the over-recovery of an M-type potassium current following the washout of muscarine (20 microM) has been examined. Whole-cell recordings were made from single neurons dissociated from bullfrog sympathetic ganglia. During over-recovery, the maximum M-conductance decreased by about 2.8 nS while the steady-state M-current activation curve was displaced in the hyperpolarizing direction by about 13 mV. These data suggest that a hyperpolarizing shift in the kinetics of M-current causes over-recovery in amphibian autonomic neurons.

Activation of cytokine production and adhesion molecule expression on THP-1 myelomonocytic cells by macrophage colony-stimulating factor in combination with interferon-gamma.

THP-1 myelomonocytic leukemia cells cultured with either macrophage colony-stimulating factor (M-CSF) or interferon-gamma (IFN-gamma) alone produce, at best, only low levels of interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha). However, combinations of the two factors resulted in at least 3- to 20-fold greater amounts of IL-1 beta and TNF-alpha than would have been predicted by additive mechanisms. This enhanced cytokine production was observed when M-CSF and IFN-gamma were added simultaneously or when M-CSF was added 24 h after addition of IFN-gamma to the cells. Similar results were obtained with fresh human peripheral blood cells treated with IFN-gamma + M-CSF. Cycloheximide treatment of the cultures containing M-CSF and IFN-gamma inhibited the production of IL-1 beta and TNF-alpha. Northern blotting studies revealed no effect of IFN-gamma alone on IL-1 beta or TNF-alpha mRNA production. IL-1 beta and TNF-alpha mRNA expression was observed at 2 and 6 h after treatment with M-CSF or IFN-gamma + M-CSF. Higher TNF-alpha mRNA expression was observed at 2 and 6 h after treatment with IFN-gamma + M-CSF, and higher IL-1 beta mRNA expression was observed at 2 h after treatment with IFN-gamma + M-CSF compared with mRNA levels observed for cells cultured only with M-CSF. These results suggest that the augmented cytokine production resulting from treatments with combinations of M-CSF and IFN-gamma occurs due to increased cytokine mRNA and increased cytokine protein synthesis. In addition to up-regulating cytokines, combinations of IFN-gamma and M-CSF resulted in augmented cell surface expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. This was accompanied by morphological and functional changes that included plastic adherence, extensive homotypic aggregation, and a macrophage-like appearance. These phenotypic changes and enhancements in cytokine expression and cell surface molecule expression may be related to activation of monocytic cells to become cytotoxic effectors by M-CSF and IFN-gamma combinations. In vitro cytotoxicity against A-375 melanoma cells was greatest for cultures that contained M-CSF and IFN-gamma in combination.

Inhibition by wortmannin of M-current in bullfrog sympathetic neurones.

1. The actions of wortmannin, an inhibitor of myosin light chain kinase (MLCK), on M-type potassium current of dissociated bullfrog sympathetic neurones have been examined. 2. The amplitude of M-current was measured by whole cell recordings from cells pretreated with wortmannin (0.01-10 microM) or the wortmannin vehicle, dimethylsulphoxide (0.0001-0.1 vol%), for 30 min. Internal (recording pipette) solutions having three different pCa values (6, 7 and 8) were used for the measurements. 3. Irrespective of the pCa, M-current was not detectable when the cells were pretreated with 10 microM wortmannin. Wortmannin, 3 microM, produced 85-95% inhibition of the M-current. Pretreatment with 10-30 nM wortmannin was without effect on M-current. 4. The M-current inhibition by wortmannin at concentrations of 0.1-1 microM depended on the pCa of the internal solution. Inhibition occurred only when the calcium-rich (pCa = 6) internal solution was used. 5. Pre-treatment of the cells with wortmannin (10 microM) did not affect rapidly-inactivating A-type or delayed rectifier-type potassium currents not did it alter inwardly rectifying sodium-potassium current (IH). 6. These observations show that M-current inhibition by wortmannin has two pharmacological profiles. One is calcium-dependent and occurs at lower concentrations (0.1-1 microM), and is attributed to inhibition of MLCK by wortmannin. At higher concentrations (3-10 microM), wortmannin has an additional, calcium-independent action, inhibiting the M-current by an unknown mechanism.

Regulation of the responses to gonadotropin-releasing hormone, muscarine and substance P in sympathetic neurons by changes in cellular constituents and intracellular application of peptide fragments of the substance P receptor.

The effects of alterations of intracellular constituents on the actions of three agonists [gonadotropin-releasing hormone, muscarine and substance P (SP)] on the M-type potassium current in bullfrog sympathetic neurons were examined. Application of maximal concentrations of each agonist resulted in inhibition of M-current followed by desensitization. Desensitization was greatest during SP application, less with gonadotropin-releasing hormone and least after muscarine. Recovery after agonist washout was greatest for SP, less for muscarine and least for gonadotropin-releasing hormone. The effects of varying intrapipette pH, [ATP], Ca buffer and free Ca on inhibition by each of the agonists, desensitization and recovery were tested. Comparison of the effects of different intracellular constituents showed that desensitization and recovery are distinct phenomena. Desensitization was greatest with 3 mM ATP in the pipette and was enhanced when pyruvate and glucose were added extracellularly. Two synthetic peptides, comprising amino acids 325-360 and 361-375, respectively, of the carboxyl tail of the rat SP receptor inhibited desensitization to SP, but not to the other agonists. A third peptide homologous to residues 376-407 and a peptide from the extracellular portion of the receptor (residues 168-179) did not affect desensitization. This suggests that the portion of the carboxyl tail of the SP receptor from amino acids 325-375 is involved in desensitization.

Myosin light chain kinase occurs in bullfrog sympathetic neurons and may modulate voltage-dependent potassium currents.

A polyclonal antibody against myosin light chain kinase (MLCK) of chicken gizzard recognized a 130 kd peptide of bullfrog sympathetic ganglia as MLCK. MLCK immunoreactivity was confined to the neuronal cell body. A synthetic peptide corresponding to an inhibitory domain of MLCK (Ala783-Gly804) was applied intracellularly to isolated sympathetic neurons during whole-cell recordings of ionic currents. The peptide inhibitor reversibly decreased M-type potassium current (IM) while not affecting A-type of delayed rectifier-type potassium currents. Intracellular application of an active fragment of MLCK enhanced IM, whereas application of an inactive MLCK fragment did not. The results suggest that IM can be modulated by MLCK-catalyzed phosphorylation.

Amyloid beta peptides act directly on single neurons.

A peptide consisting of residues 25-35 of the amyloid beta protein was applied to single neurons while monitoring membrane current by whole cell voltage clamp recording. Within minutes of direct exposure of a neuron to the amyloid beta peptide, a paroxysmal increase in neuronal membrane conductance was observed. This conductance does not resemble previously described ionic conductances in terms of its time-dependence, voltage-dependence or sensitivity to changes in extracellular or intracellular ionic constituents. The effect of the amyloid beta peptide was not mimicked or blocked by substance P nor was it prevented by low intracellular or extracellular Ca. The increased membrane permeability elicited by the peptides may lead to the neuropathology observed in Alzheimer's disease.

Levamisole causes differential cytokine expression by elicited mouse peritoneal macrophages.

Thioglycolate-elicited peritoneal macrophages from normal C57B1/6J mice were examined in vitro for bacterial lipopolysaccharide (LPS)-stimulated interleukin-1 (IL-1), IL-6, and tumor necrosis factor (TNF) production. Macrophages from mice administered a single oral dose of levamisole (3 mg/kg) 1 to 4 days prior to macrophage harvest demonstrated a twofold enhancement of IL-1 production compared to vehicle-treated mice. In contrast, IL-6 production and TNF production by the same macrophages were inhibited up to 36 and 62%, respectively, compared to production by macrophages harvested from vehicle-treated mice. Similar results were observed when IL-1 production and TNF production were followed in peritoneal exidate cells directly stimulated with levamisole in vitro. The ex vivo LPS-stimulated IL-1 production was enhanced 4 days after macrophage elicitation, whereas TNF and IL-6 production returned to baseline by 72 h after macrophage recruitment and augmentation. No evidence could be found for the presence of inhibitors of TNF or IL-6. The specificity of the IL-1, IL-6, and TNF bioactivities was demonstrated by neutralization with specific antisera. Immunoprecipitation studies of supernatants from biosynthetically labeled macrophages also revealed augmented IL-1 production and decreased IL-6 and TNF, indicating that levamisole may have affected cytokine production at the translational level. Kinetics studies revealed that ex vivo release of IL-6 and TNF by macrophages from levamisole-dosed mice was delayed compared to production of these cytokines by macrophages harvested from mice given vehicle only. The results may explain, in part, the reported ability of levamisole to ameliorate cases of rheumatoid arthritis or other autoimmune and inflammatory diseases by affecting the relative levels of cytokines produced by macrophages recruited to sites of injury, which are associated with inflammation and acute-phase protein synthesis.

Enhancement of in vitro lipopolysaccharide-stimulated interleukin-1 production by levamisole.

The production of interleukin-1 (IL-1) by the P388D1 mouse macrophage cell line and by adherent peritoneal exudate cells (PMs) was examined. In vitro IL-1 production by P388D1 cells treated with lipopolysaccharide (LPS) was enhanced by coculture with levamisole (0.1 to 10 microM). Oral administration of levamisole (3 mg/kg) to mice also resulted in potentiation of in vitro IL-1 production by thioglycollate-elicited peritoneal macrophages in response to in vitro LPS stimulation. Potentiation was approximately twofold. IL-1 production in the absence of LPS by either the P388D1 cells or the PMs was nil, and levamisole did not directly stimulate IL-1 production in these cases. IL-1 activity in the culture supernatants was measured by thymocyte comitogenic assays. The immunochemical identify of the thymocyte comitogenic activity as IL-1 alpha was confirmed by neutralization with a specific goat anti-mouse IL-1 alpha antiserum. These results suggest that one mechanism by which levamisole acts to normalize and restore immune responses may be enhancing the signals which enable activated macrophages to secrete IL-1.

Ion dependence of neurotransmitter uptake: inhibitory effects of ion substitutes.

Several ions commonly used as substitutes for Na+ or Cl- were found to inhibit directly the high-affinity uptake of norepinephrine, dopamine, serotonin, and gamma-aminobutyric acid, but not glutamate or glutamine. When Na+ was partially replaced by any of several different cations or sucrose the uptake of all neurotransmitters studied except that of serotonin was reduced more than could be accounted for by just the inhibitory effect of the cation substitute. In contrast, when Cl- was partially replaced by any of several anions only the uptake of dopamine was reduced more than could be accounted for by the inhibitory effect of the anion substitute. These results suggest that for most neurotransmitters the electrochemical potential for Na+, but not for Cl-, contributes to the uptake driving force. When either Na+ or Cl- was totally replaced by an ion substitute or by sucrose the high-affinity uptake was virtually abolished, an exception being that glutamate uptake was not affected when isethionate was substituted for Cl-. The lack of uptake in the absence of either Na+ or Cl- may reflect a specific role for these ions in either increasing the affinity between the substrate and the carrier, or facilitating the translocation process. Alternatively, the transport carriers may undergo a nonspecific conformational change to an inactive form in the absence of Na+ or Cl-. A partial substitution of Na+ with Li+ or sucrose differentially affected the kinetics of uptake in that replacement with Li+, but not sucrose, usually resulted in a marked increase in the Km values.(ABSTRACT TRUNCATED AT 250 WORDS)

Pyrroloisoquinoline antidepressants. 2. In-depth exploration of structure-activity relationships.

A series of pyrrolo[2,1-a]isoquinolines, and related compounds, were examined for antidepressant-like activity, by virtue of their antagonism of tetrabenazine-induced ptosis and sedation, and inhibition of biogenic amine uptake. Thus, we have identified some of the most potent antagonists of TBZ-induced ptosis and some of the most potent inhibitors of the uptake of dopamine, norepinephrine, and serotonin (in rat brain synaptosomes) ever reported. Compounds of particular note, in this regard, are 52b, 29b, 22b, and 48b, respectively. Biological activity was chiefly manifested by the trans isomeric class. Also, through resolution of four compounds, 7b, 24b, 37b, and 48b, biological activity was found to be associated with the (+) enantiomer subgroup (salts measured at 589 nm in MeOH), corresponding to the 6S, 10bR absolute configuration for 7b, 37b, and 48b, and the 6R,10bR configuration for 24b. An X-ray determination on (+)-24b X HBr established its absolute configuration; configurations for the other compounds were verified by enantiospecific synthesis starting with (+)-(R)-2-phenylpyrrolidine. Regarding the pendant phenyl ring, diverse substitution patterns were investigated. Those substitutions that were particularly unfavorable were 3',4',5'-trimethoxy (20b), 2',3',4',5',6'-pentafluoro (34b), 2'-trifluoromethyl (38b), 3',5'-bis(trifluoromethyl) (42b), 4'-n-butyl (44b), 2'-cyano (47b), 4'-methylsulfonyl (50b), and 2'-carboxy (58b). Exceedingly potent compounds, in one way or another, were 10b-12b, 22b, 23b, 25b, 28b, 29b, 33b, 45b, 48b, 51b-53b. The pattern of aromatic substitution had a strong impact on selectivity in the uptake tests (NE vs. DA vs. 5-HT). Activity was significantly diminished by methyl substitution of 7b at the 5 (65, 66), 6 (61b), or 10b (60b) position, by changing the phenyl group of 7b to cyclohexyl (67b), benzyl (68b), or H (72), by moving the phenyl group of 7b to the 5 (69) or 10b (70) position, by expansion of ring B to an azepine (78b), and by modification of ring C to an azetidine (77b), piperidine (75b), or azepine (74b). The interaction of selected analogues with various CNS receptors is reported. Little affinity was shown for the muscarinic cholinergic receptor, suggesting a lack of anticholinergic side effects. Interestingly, 24b and 33b displayed a high affinity for the serotonin-2 receptor, analogous to mianserin and clomipramine. After the body of data was reviewed, derivatives 24b and 48b were chosen for advanced development.