The central nervous system sites mediating the orexigenic actions of ghrelin.

The peptide hormone ghrelin is important for both homeostatic and hedonic eating behaviors, and its orexigenic actions occur mainly via binding to the only known ghrelin receptor, the growth hormone secretagogue receptor (GHSR). GHSRs are located in several distinct regions of the central nervous system. This review discusses those central nervous system sites that have been found to play critical roles in the orexigenic actions of ghrelin, including hypothalamic nuclei, the hippocampus, the amygdala, the caudal brain stem, and midbrain dopaminergic neurons. Hopefully, this review can be used as a stepping stone for the reader wanting to gain a clearer understanding of the central nervous system sites of direct ghrelin action on feeding behavior, and as inspiration for future studies to provide an even-more-detailed map of the neurocircuitry controlling eating and body weight.

Diacylglycerol lipase ß inhibition reverses nociceptive behaviour in mouse models of inflammatory and neuropathic pain.

BACKGROUND AND PURPOSE: Inhibition of diacylglycerol lipase (DGL)ß prevents LPS-induced pro-inflammatory responses in mouse peritoneal macrophages. Thus, the present study tested whether DGLß inhibition reverses allodynic responses of mice in the LPS model of inflammatory pain, as well as in neuropathic pain models. EXPERIMENTAL APPROACH: Initial experiments examined the cellular expression of DGLß and inflammatory mediators within the LPS-injected paw pad. DAGL-ß (-/-) mice or wild-type mice treated with the DGLß inhibitor KT109 were assessed in the LPS model of inflammatory pain. Additional studies examined the locus of action for KT109-induced antinociception, its efficacy in chronic constrictive injury (CCI) of sciatic nerve and chemotherapy-induced neuropathic pain (CINP) models. KEY RESULTS: Intraplantar LPS evoked mechanical allodynia that was associated with increased expression of DGLß, which was co-localized with increased TNF-α and prostaglandins in paws. DAGL-ß (-/-) mice or KT109-treated wild-type mice displayed reductions in LPS-induced allodynia. Repeated KT109 administration prevented the expression of LPS-induced allodynia, without evidence of tolerance. Intraplantar injection of KT109 into the LPS-treated paw, but not the contralateral paw, reversed the allodynic responses. However, i.c.v. or i.t. administration of KT109 did not alter LPS-induced allodynia. Finally, KT109 also reversed allodynia in the CCI and CINP models and lacked discernible side effects (e.g. gross motor deficits, anxiogenic behaviour or gastric ulcers). CONCLUSIONS AND IMPLICATIONS: These findings suggest that local inhibition of DGLß at the site of inflammation represents a novel avenue to treat pathological pain, with no apparent untoward side effects.

Gastrointestinal permeability during exercise: effects of aspirin and energy-containing beverages.

The purpose of this study was to determine whether aspirin (A) ingestion combined with prolonged exercise increases gastrointestinal permeability and whether consumption of a carbohydrate-containing (CHO) or a CHO + glutamine-containing (CHO+G) beverage would reduce this effect. Seventeen subjects completed six experiments. They ingested A (1,300 mg) or placebo (P) pills the evening before and before running 60 min at 70% maximal oxygen uptake. Also, before running they ingested a solution containing 5 g lactulose (L), 5 g sucrose (S), and 2 g rhamnose (R). During each trial, either a 6% CHO beverage, a 6% CHO+G (0.6%; 41 mM) beverage, or a water placebo (WP) was consumed. For 4 h after a run, all urine was collected to measure urinary excretion of L, R, and S. S excretion (percentage of dose ingested; measure of gastroduodenal permeability) was significantly greater (P < 0.05) during the A trial while the subjects drank the WP compared with all other trials. Administration of A also significantly increased L/R (measure of intestinal permeability) for the CHO and WP trials compared with all P trials. Ingestion the CHO or CHO+G beverages significantly reduced S excretion and L excretion when A was administered, but it did not reduce L/R. These results indicate that gastroduodenal and intestinal permeability increase after A ingestion during prolonged running and that ingestion of a CHO beverage attenuates the gastroduodenal effect but not the intestinal effect. Furthermore, addition of G to the CHO beverage provided no additional benefit in reducing gastroduodenal or intestinal permeability.

Human plasma prekallikrein and high molecular weight kininogen decrease during parturition.

Prekallikrein and high molecular weight kininogen were measured in plasma taken from nine women during parturition. Prekallikrein decreased significantly (p less than 0.01) from 1.49 +/- 0.15 S-2302 U/ml (mean +/- SEM) in early labor to 1.26 +/- 0.13 S-2302 U/ml in the immediate postpartum period. Immunoreactive high molecular weight kininogen also significantly decreased from 76 +/- 5 micrograms/ml to 68 +/- 5 micrograms/ml one day postpartum (p less than 0.01). Both proteins rose to normal levels within two days. The data suggest that the kallikrein-kinin system is utilized during parturition.

Changes in the brain accumulation of glucocorticoids in abcb1a-deficient CF-1 mice.

The multidrug resistance transporter, P-glycoprotein (P-gp), contributes to highly lipophilic molecules penetrating the brain from the blood at a much lower rate than expected, and has numerous substrates, inhibitors and modulators. The drug-transporting isoform of P-gp is coded by a single human gene, ABCB1, and shares 80% homology with the murine drug-transporting isoforms, abcb1a and abcb1b, which share 92% homology with each other. Although these murine isoforms are highly similar, there are known affinity differences between the isoforms, and the localisation of the two isoforms in the brain is also disputed. Studies using mice genetically modified to be deficient in one or both isoforms of P-gp have also resulted in conflicting data. The contribution of the abcb1a isoform, which is considered to contribute most to the central nervous system (CNS)-protective role of P-gp, is investigated in the present study using CF-1-abcb1a(-/-) mice and the well-established brain/choroid plexus perfusion technique. Twenty-minute in situ brain/choroid plexus perfusions in CF-1-abcb1a(-/-) mice indicated the increased accumulation of [(3) H]cortisol, [(3) H]corticosterone and [(3) H]dexamethasone in most of the brain regions examined compared to CF-1-abcb1a(+/+) mice. Taken together with our earlier published studies in abcb1a/b(-/-) mice, these data strongly suggest that the in vivo CNS accumulation of glucocorticoids obtained using single knockout strains [e.g. abcb1a(-/-)] cannot be directly compared with those obtained in double knockout strains [e.g. abcb1a/b(-/-)].

Quantitative evaluation of phase-conjugate novelty filters.

A Michelson interferometer whose signal arm is terminated by a self-pumped BaTiO(3) phase conjugator records phase-change distributions in times short compared with the conjugator rewrite time that correspond quantitatively to a single (not a double) traversal of the phase disturbance. The proffered explanation, which is experimentally substantiated, also places limitations on the pictorial accuracy of intensity-change novelty filters.

Multimode fiber interferometry with and without phase conjugation.

It is generally assumed that fiber interferometers must be constructed from single-mode fibers. An alternative is to employ phase conjugation mirrors on the arms of a multimode fiber interferometer so that the spatial scrambling of modes is reversed, and good fringe contrast is maintained. When several interferometers all share the same self-conjugating BaTiO(3) crystal, however, the results show crosstalk between the separate fibers ranging from negligible to prohibitively large depending on the precise spatial configuration within the conjugator. A model accounting for these results is proposed. In addition it was observed in control experiments with conventional Mach-Zehnder configurations that good data could be obtained despite reduced contrast with both graded and step index multimode fibers.

Detection and characterization of hepatitis C virus RNA in immune globulins.

BACKGROUND: Hepatitis C virus (HCV) RNA was measured in immune globulins and its chemical and physical properties were characterized. STUDY DESIGN AND METHODS: The study examined 69 immune globulin lots from 7 manufacturers, including 44 intravenous and 25 intramuscular immune globulin preparations. In addition, 8 experimental intravenous immune globulin preparations were investigated. Detection and quantitation of HCV RNA were achieved by reverse transcription and nested polymerase chain reaction at limiting dilution. A multi-antigen anti-HCV enzyme immunoassay was also used to test these immune globulins. RESULTS: The highest level of HCV RNA was found in an experimental immune globulin lot derived from a plasma pool made up of 186 anti-c100-3-reactive units. HCV RNA was detected only in 1 of 7 manufacturers' experimental intravenous immune globulin preparations derived from a pool made up of 2887 anti-c100-3-negative units. It was also detected in commercial intravenous immune globulin lots prepared by the same manufacturer from source plasma, but not from recovered plasma. More than half of the commercial intramuscular immune globulin lots, including specific immune globulin products, were HCV RNA positive. All immune globulin products examined were reactive for anti-HCV. Certain similarities were found for HCV RNA present in an immune globulin product and plasma. Ethanol at 20 or 25 percent had no effect upon the buoyant density of HCV RNA. CONCLUSION: Many immune globulin preparations contained HCV RNA, with levels depending upon both the type of starting plasma and the manufacturing process. Exposure to ethanol did not appear to affect the physical characteristics of HCV RNA.

Plasma prekallikrein: quantitative determination by direct activation with Hageman factor fragment (beta-XIIa).

This report describes a plasma prekallikrein assay which, unlike methods that employ contact activation, is not affected by the factor XII or HMW kininogen content of the plasma analyzed. In this assay beta-XIIa, a potent fluid-phase activator of prekallikrein, is added to diluted plasma in the presence of 20% acetone (to inactivate kallikrein inhibitors) at 30 degrees C and the kallikrein generated is measured with the chromogenic substrate S-2302. Prekallikrein is fully activated under these conditions and the activity remains stable for at least 30 hr. The mean prekallikrein concentration in plasma samples from 24 healthy individuals was 1.50 +/- 0.35 (S.D.) S-2302 U/ml, corresponding to 20.3 +/- 4.7 micrograms/ml prekallikrein (the specific activity of highly purified human prekallikrein was determined to be 74 S-2302 U/mg). In contrast, the mean concentration in five plasma samples from patients deficient in HMW kininogen was 0.38 +/- 0.02 S-2302 U/ml. No activity was generated in prekallikrein-deficient plasma, and essentially normal levels (1.35 +/- 0.18 S-2302 U/ml) were measured in plasmas from three patients with factor XII deficiency. Plasma prekallikrein was also quantitated by radial immunodiffusion, which gave results similar to those obtained by functional assay with beta-XIIa. The determination of plasma prekallikrein by direct activation with beta-XIIa in the presence of acetone offers several advantages over the use of contact activators such as dextran sulfate. These advantages include complete inactivation of kallikrein inhibitors and total activation of prekallikrein (even in plasmas deficient in other contact factors) without simultaneous generation of plasmin.

Expression and characterization of the preS1 peptide of hepatitis B surface antigen in Escherichia coli.

The infectious particles of hepatitis B virus (HBV) contain 3 related surface antigens, i.e., small, medium, and large, all of which are encoded by one large open reading frame with multiple initiation codons. The large surface antigen (L-Ag) contains preS1, preS2, and S regions while both the middle and small surface antigens lack preS1. Several lines of evidence suggested that the preS1 region is involved in the binding of HBV to human hepatocytes as shown by its binding to HepG2 cells and isolated human hepatocyte membranes. To obtain large quantity of preS1 peptide, an expression vector was constructed containing a lac promoter, the 5' half of the beta-galactosidase gene, the Factor Xa tetrapeptide recognition sequence, and the coding region of preS1 plus preS2. This recombinant plasmid constitutively produced high concentration of a fusion protein in inclusion bodies in Escherichia coli. When the fusion protein was treated with Factor Xa, a peptide consisting of the N-terminal 91 amino acids of the preS1 region was released. This preS1 fragment purified by anion exchange chromatography was able to bind specifically to the isolated plasma membranes from human liver. Hence, this recombinant preS1 peptide can be used to identify and isolate hepatocyte receptors for HBV.

Contact-activated factors: contaminants of immunoglobulins preparations with coagulant and vasoactive properties.

The intramuscular or intravenous administration of ISG prepared from human plasma by ethanol fractionation can elicit such reactions as pain at the injection site, flushing, and even hypotension. Similar adverse reactions to plasma protein fraction, a volume expander also made by ethanol fractionation, have been associated with PKA (Hageman factor fragments) in the product. Twenty-five lots of commercial ISG were therefore analyzed for PKA and kallikrein, components of the contact activation system which could mediate such reactions through the generation of kinins in recipients. Kallikrein activity ranged from undetectable levels to > 60% of the total potential kallikrein activity in normal plasma. PKA, which was measured by its ability to catalyze the conversion of prekallikrein to kallikrein, ranged from 5% to 3950% of the activity in a reference plasma protein fraction that had caused hypotension. All but five lots increased vascular permeability in the guinea pig. The five lots which caused no increased were also the lowest in PKA and kallikrein activity. When ISG ws subjected to gel chromatography to separate the enzymic contaminants from immunoglobulin G, only the fractions containing PKA and/or kallikrein increased vascular permeability. Several lots of ISG shortened the nonactivated partial thromboplastin time of normal plasma fro 236 sec to 38 to 55 sec. During gel chromatography, coagulation activity was eluted in a position corresponding to a molecular weight of 150,000; it was inhibited by antibody to human factor XI. These data indicate that factor XIa is responsible for the coagulant activity observed and that PKA and/or kallikrein are potential mediators of vasoactive reactions to ISG.

Hypotension associated with prekallikrein activator (Hageman-factor fragments) in plasma protein fraction.

Thirteen lots of plasma protein fraction made by one manufacturer were implicated in 23 recent reports of hypotension in surgical patients. Four of these patients required resuscitation after rapid administration of the product in the postoperative period. All implicated lots had prekallikrein-activator activity but low levels of bradykinin and kallikrein. The prekallikrein activator was identified as Hageman-factor fragments by molecular weight (35,000 as estimated by gel chromatography), isoelectric point (4.2 to 4.4), and inhibition by antibody to Hageman factor. These data suggest that Hageman-factor fragments are potent hypotensive agents, presumably because they trigger the generation of bradykinin in recipients. Prekallikrein-activator activity, usually at levels lower than those in the initial 13 implicated lots, was frequently detected in plasma protein fraction made by other manufactures. Several of these lots were associated with additional reports of hypotension. Prekallikrein-activator activity rarely occurred in albumin.