Identification and characterization of novel mammalian neuropeptide FF-like peptides that attenuate morphine-induced antinociception.

The two mammalian neuropeptides NPFF and NPAF have been shown to have important roles in nociception, anxiety, learning and memory, and cardiovascular reflex. Two receptors (FF1 and FF2) have been molecularly identified for NPFF and NPAF. We have now characterized a novel gene designated NPVF that encodes two neuropeptides highly similar to NPFF. NPVF mRNA was detected specifically in a region between the dorsomedial and ventromedial hypothalamic nuclei. NPVF-derived peptides displayed higher affinity for FF1 than NPFF-derived peptides, but showed poor agonist activity for FF2. Following intracerebral ventricular administration, a NPVF-derived peptide blocked morphine-induced analgesia more potently than NPFF in both acute and inflammatory models of pain. In situ hybridization analysis revealed distinct expression patterns of FF1 and FF2 in the rat central nervous system. FF1 was broadly distributed, with the highest levels found in specific regions of the limbic system and the brainstem where NPVF-producing neurons were shown to project. FF2, in contrast, was mostly expressed in the spinal cord and some regions of the thalamus. These results indicate that the endogenous ligands for FF1 and FF2 are NPVF- and NPFF-derived peptides, respectively, and suggest that the NPVF/FF1 system may be an important part of endogenous anti-opioid mechanism.

Identification and characterization of a second melanin-concentrating hormone receptor, MCH-2R.

Melanin-concentrating hormone (MCH) is a 19-aa cyclic neuropeptide originally isolated from chum salmon pituitaries. Besides its effects on the aggregation of melanophores in fish several lines of evidence suggest that in mammals MCH functions as a regulator of energy homeostasis. Recently, several groups reported the identification of an orphan G protein-coupled receptor as a receptor for MCH (MCH-1R). We hereby report the identification of a second human MCH receptor termed MCH-2R, which shares about 38% amino acid identity with MCH-1R. MCH-2R displayed high-affinity MCH binding, resulting in inositol phosphate turnover and release of intracellular calcium in mammalian cells. In contrast to MCH-1R, MCH-2R signaling is not sensitive to pertussis toxin and MCH-2R cannot reduce forskolin-stimulated cAMP production, suggesting an exclusive G(alpha)q coupling of the MCH-2R in cell-based systems. Northern blot and in situ hybridization analysis of human and monkey tissue shows that expression of MCH-2R mRNA is restricted to several regions of the brain, including the arcuate nucleus and the ventral medial hypothalamus, areas implicated in regulation of body weight. In addition, the human MCH-2R gene was mapped to the long arm of chromosome 6 at band 6q16.2-16.3, a region reported to be associated with cytogenetic abnormalities of obese patients. The characterization of a second mammalian G protein-coupled receptor for MCH potentially indicates that the control of energy homeostasis in mammals by the MCH neuropeptide system may be more complex than initially anticipated.

FMRFamide-related neuropeptides are agonists of the orphan G-protein-coupled receptor GPR54.

We have isolated and determined the coding sequences of human and mouse orthologs of the rat orphan G-protein-coupled receptor GPR54. Mouse and rat GPR54 are nearly 95% identical to each other, and both are approximately 85% identical to human GPR54 at the amino acid level. Screening of agonists for GPR54 identified several invertebrate neuropeptides of the RFamide and RWamide family that were able to activate GPR54 at microM range through the G(alpha)q pathway. Substitution analysis showed that the C-terminal optimal sequence of GPR54-activating peptides is Gly-Leu-Arg-Trp-NH2. Northern analysis of human GPR54 detected expression in several peripheral tissues and many regions of the central nervous system.

The path to adoption for children of color.

OBJECTIVE: This article focuses on the path to adoption for children involved in the public child welfare system. METHOD: Descriptive and event history analyses were conducted of 1,550 children who had been removed from their homes and placed in out of home care in the child welfare system in Kansas and have adoption as a goal. RESULTS: African American children consistently took longer to reach significant milestones, including adoption placement and finalization. CONCLUSION: African American children are over-represented throughout the progression from substantiated abuse to adoption. Future research in other states should focus on whether this trend is unique to Kansas or applicable in other states. Additionally, efforts should be devoted to investigating the social, psychological, cultural, and systemic factors contributing to this differential treatment. Finally, there is a dire need to develop and evaluate interventions targeted at meeting the specialized needs of African American children in this system.

The postadoption experience: child, parent, and family predictors of family adjustment to adoption.

Eighteen to 24 months after they adopted children in state custody, 159 parents were surveyed regarding their postadoption experiences. Most children remained with their new families, with parents reporting the adoption experience to be "about as hard as expected," and rating the past year of caring for the child as being more "smooth" than "stressful." Parents saw how the child contributed positively to their lives, were less positive about available social supports, and expressed dissatisfaction with the speed of the adoption, the availability of community supports, and the ability to link with supports. Children's special needs (e.g., older age at adoption) were predictive of postplacement adjustment.

Cysteine residues in the D-galactose-H+ symport protein of Escherichia coli: effects of mutagenesis on transport, reaction with N-ethylmaleimide and antibiotic binding.

The galactose-H(+) membrane-transport protein, GalP, of Escherichia coli is similar in substrate specificity and susceptibility to cytochalasin B and forskolin, to the human GLUT1 sugar-transport protein; furthermore, they are about 30% identical in amino acid sequence. Transport activities of both GalP and GLUT1 are inhibited by the thiol-group-specific reagent, N-ethylmaleimide. GalP contains only three cysteine residues at positions 19, 374 and 389, each of which we have mutated, singly and in combination, to serine. Each single change of Cys-->Ser has only a minor effect on transport activity, whereas alteration of all three simultaneously profoundly diminishes V(max) for transport. The high level of expression of the GalP protein facilitates measurements of the reactivity of each mutant with N-ethylmaleimide or eosin 5-maleimide, which conclusively demonstrate that Cys(374) is the site of covalent modification by the reagents. By comparing the reactivity of Cys(374) in right-side-out and inside-out vesicles it appears that Cys(374) is located on the cytoplasmic face of the GalP protein. Although impaired in transport activity, the 'Cys-free' mutant, with all three cysteine residues mutated into serine, binds cytochalasin B and forskolin with wild-type affinities. All these results are interpreted in terms of a 12-helix model of the folding of the protein, in which the relative orientations of helix 10, containing the reactive Cys(374) residue, and helix 11, containing the unreactive Cys(389) residue, can now be defined.

Identification of receptors for neuromedin U and its role in feeding.

Neuromedin U (NMU) is a neuropeptide with potent activity on smooth muscle which was isolated first from porcine spinal cord and later from other species. It is widely distributed in the gut and central nervous system. Peripheral activities of NMU include stimulation of smooth muscle, increase of blood pressure, alteration of ion transport in the gut, control of local blood flow and regulation of adrenocortical function. An NMU receptor has not been molecularly identified. Here we show that the previously described orphan G-protein-coupled receptor FM-3 (ref. 15) and a newly discovered one (FM-4) are cognate receptors for NMU. FM-3, designated NMU1R, is abundantly expressed in peripheral tissues whereas FM-4, designated NMU2R, is expressed in specific regions of the brain. NMU is expressed in the ventromedial hypothalamus in the rat brain, and its level is significantly reduced following fasting. Intracerebroventricular administration of NMU markedly suppresses food intake in rats. These findings provide a molecular basis for the biochemical activities of NMU and may indicate that NMU is involved in the central control of feeding.

Identification of urotensin II as the endogenous ligand for the orphan G-protein-coupled receptor GPR14.

Urotensin II (UII) is a neuropeptide with potent cardiovascular effects. Its sequence is strongly conserved among different species and has structural similarity to somatostatin. No receptor for UII has been molecularly identified from any species so far. GPR14 was cloned as an orphan G protein-coupled receptor with similarity to members of the somatostatin/opioid receptor family. We have now demonstrated that GPR14 is a high affinity receptor for UII and designate it UII-R1a. HEK293 cells and COS-7 cells transfected with rat GPR14 showed strong, dose-dependent calcium mobilization in response to fish, frog, and human UII. Radioligand binding analysis showed high affinity binding of UII to membrane preparations isolated from HEK293 cells stably expressing rat GPR14. In situ hybridization analysis showed that GPR14 was expressed in motor neurons of the spinal cord, smooth muscle cells of the bladder, and muscle cells of the heart. The identification of the first receptor for UII will allow better understanding of the physiological and pharmacological roles of UII.

Predicting caregiver stress: an ecological perspective.

The family caregiving process for children with severe emotional disorders is conceptualized from an ecological perspective and examined in light of outcomes for caregivers. This study of 259 families assessed the relative importance, individually and in combination, of various clusters of caregiver, child, family, and environmental characteristics and responses as predictors of caregiver stress. Findings raise questions about current practice and policy, and suggest directions for future research.

Video-assisted thoracoscopic resection of an epiphrenic diverticulum with esophagomyotomy and partial fundoplication.

Videothoracoscopic stapled diverticulectomy with distal esophageal myotomy and partial fundoplasty was successfully done for a 65-year-old woman who had a large, symptomatic epiphrenic diverticulum associated with achalasia. This minimally invasive approach resulted in good symptomatic relief of dysphagia, minimal postoperative pain, a 1-day hospital stay, and early return to normal activity.

Desensitization of the human vasoactive intestinal peptide receptor (hVIP2/PACAP R): evidence for agonist-induced receptor phosphorylation and internalization.

To investigate the role of phosphorylation and internalization in the desensitization of the hVIP2/PACAP receptor, we expressed a C-terminal epitope-tagged (hemagglutinin; YPYDVPDYASL) receptor in COS7 and HEK293 cell lines. Radiolabeling experiments demonstrated that exposure to agonist induced receptor phosphorylation significantly above basal levels. This receptor phosphorylation was greater than that induced by receptor-independent activation of PKA with forskolin and that induced by co-application of forskolin and agonist. This suggests that receptor occupancy promotes phosphorylation and also that receptor phosphorylation may involve a specific G protein-coupled receptor kinase in addition to PKA. Immunocytochemical analysis showed that the receptor was internalized in response to agonist to a single site of accumulation within the cell and this was dependent on temperature, agonist concentration, and time. Further studies will focus on identifying phosphorylation sites and endocytic signals within the hVIP2/PACAP R.

C3H mice have larger spleens, lower platelet counts, and shorter platelet lifespans than C57BL mice: an animal model for the study of hypersplenism.

Several studies have shown that C3H mice have a modal megakaryocyte ploidy class of 32N as opposed to the 16N found in all other mouse strains tested. In addition to higher megakaryocyte DNA content, C3H mice are characterized by greater 35S-sulfate incorporation into platelets but somewhat lower platelet counts than C57BL mice. The aim of this study was to determine the etiology of mild thrombocytopenia in C3H mice. Several studies have indicated that an increase in platelet 35S incorporation parallels increased megakaryocyte ploidy. Thus the higher 35S incorporation by C3H mouse platelets is likely related to their higher megakaryocyte ploidy, but these differences do not explain the lower platelet counts observed in C3H mice. To elucidate the paradox of relatively low platelet counts in C3H mice despite elevated platelet 35S incorporation, we performed a hematological evaluation (including assessment of platelet counts and lifespans) and measured (relative) spleen size in C3H and C57BL mice. Our data confirm earlier reports of significantly lower platelet counts in C3H mice (p < 0.0005) compared with C57BL mice. We also found that C3H mice have larger spleens (p < 0.0005) than C57BL mice. Other groups of C3H and C57BL mice were splenectomized to determine the role of the spleen in decreasing platelet counts in C3H mice compared with C57BL mice. C3H mice had an average platelet lifespan of 4.8 days, which was significantly shorter (p < 0.01) than the 5.7-day platelet lifespan observed in normal C57BL mice. Platelet lifespans for splenectomized C3H mice were significantly (p < 0.005) longer than those found in intact C3H control mice, but splenectomy of C57BL mice had no effect on their platelet lifespans (p < 0.40). The lengthened platelet lifespans of splenectomized C3H mice indicate that the spleens of C3H mice were sequestering or destroying platelets at a faster rate than the spleens of C57BL mice. These data lead to the hypothesis that the relatively large spleens of C3H mice produce mild thrombocytopenia with increased platelet production (shorter platelet lifespan) compared with spleens of C57BL mice.

Asparagine 394 in putative helix 11 of the galactose-H+ symport protein (GalP) from Escherichia coli is associated with the internal binding site for cytochalasin B and sugar.

The galactose-H+ symport protein (GalP) of Escherichia coli is very similar to the human glucose transport protein, GLUT1, and both contain a highly conserved Asn residue in predicted helix 11 that is different in a cytochalasin B-resistant member of this sugar transport family (XylE). The role of the Asn394 residue (which is predicted to be in putative trans-membrane alpha-helix 11) in the structure/activity relationship of the D-galactose-H+ symporter (GalP) was therefore assessed by measuring the interaction of sugar substrates and the inhibitory antibiotics, cytochalasin B, and forskolin with the wild-type and Asn394 --> Gln mutant proteins. Steady-state fluorescence quenching experiments show that the mutant protein binds cytochalasin B with a Kd 37-53-fold higher than the wild type. This low affinity binding was not detected with equilibrium binding or photolabeling experiments. In contrast, the mutant protein binds forskolin with a Kd similar to that of the wild type and is photolabeled by 3-125I-4-azido-phenethylamido-7-O-succinyl-desacetyl-forskolin. The mutant protein displays an increased amount of steady-state fluorescence quenching with the binding of forskolin, suggesting that the substitution of the Asn residue has altered the environment of a tryptophan, probably Trp395, in a conformationally active region of the protein. Time-resolved fluorescence measurements on the mutant protein provided association and dissociation rate constants (k2 and k-2), describing the initial interaction of cytochalasin B to the inward-facing binding site (Ti), that are decreased (9-fold) and increased (4.9-fold) compared with the wild type. This yielded a dissociation constant (K2) for cytochalasin B to the inward-facing binding site 44-fold higher than that of the wild type. The binding of forskolin gave values for k2 and k-2 3.9- and 3.6-fold lower, respectively, yielding a K2 value for Ti similar to that of the wild type. The low overall affinity (high Kd) of the mutant protein for cytochalasin B is due mainly to a disruption in binding to the Ti conformation. It is proposed that Asn394 forms either a direct binding interaction with cytochalasin B or is part of the immediate environment of the binding site and that Asn394 is in the immediate environment, but not part, of the forskolin binding site. The ability of the mutant protein to catalyze energized transport is only mildly impaired with 4.8- and 2.1-fold reduction in Vmax/Km values for D-galactose and D-glucose, respectively. In stark contrast, the overall Kd describing binding of D-galactose and D-glucose to the inward-facing conformation of the mutant and their subsequent translocation across the membrane is substantially increased (64-fold for D-galactose and 163.3-fold for D-glucose). These data indicate that Asn394 is associated with both the cytochalasin B and internal sugar binding sites. This conclusion is also supported by data showing that the sugar specificity of the mutant protein has been altered for D-xylose. This work powerfully illustrates how comparisons of the aligned amino acid sequences of homologous membrane proteins of unknown structure and characterization of their phenotypes can be used to map substrate and ligand binding sites.

Successful determination of platelet lifespan in C3H mice by in vivo biotinylation.

Mice are frequently used as test animals in blood platelet research studies, but safe and convenient methods are not available to easily assess lifespan. Therefore we have improved an existing method for measuring platelet lifespan by determining the disappearance of biotin-labeled platelets from the circulation of mice with immunofluorescence and flow cytometry. In vivo labeling of platelets with Sulfo-NHS-Biotin, a water-soluble moiety, was used as a direct method for labeling the entire platelet population by direct infusion into mice. Platelet samples were collected daily, washed, labeled with a fluorescent detection agent, and analyzed by flow cytometry to measure the percentage of biotinylated cells. This method allowed a rapid determination of murine platelet lifespan without requiring radioactive labels. Using this method, we have estimated that the platelet lifespan in C3H male mice is approximately 4.8 days.

Determination of the lifespan of erythrocytes from greyhounds, using an in vitro biotinylation technique.

OBJECTIVE: To determine the RBC lifespan of Greyhounds, using an in vitro labeling technique. DESIGN: RBC from dogs were labeled with NHS-biotin and their disappearance measured over time to determine RBC lifespan. SAMPLE POPULATION: 5 Greyhounds that had been vaccinated against distemper, adenovirus 1 and 2 infections, parainfluenza, leptospirosis, parvovirus, and coronavirus infections, Bordetella bronchiseptica infection, and rabies the previous year; 3 sexually intact 14-month-old Beagles served as controls. PROCEDURE: After venipuncture for CBC, catheters were inserted in the cephalic vein of each dog. Butorphanol was then administered to achieve mild sedation and analgesia, and glycopyrrolate was administered to ensure maintenance of adequate heart rate during phlebotomy. Dogs were positioned in lateral recumbency; blood was removed via jugular venipuncture, using a standard laboratory donor blood bag containing citrate-phosphate-dextrose solution. Blood was transferred aseptically into sterile polystyrene containers and NHS-biotin was added. After incubation, the labeled RBC were reinfused into the dogs and the blood was allowed to recirculate for 1 hour before the first postinfusion sample was taken. At frequent intervals, blood to be analyzed was taken by jugular venipuncture, and the percentage of labeled cells was determined by flow cytometry. RESULTS: The mean RBC lifespan of non-Greyhounds was significantly longer than that of Greyhounds (104.3 +/- 2.2 days vs 53.6 +/- 6.5 days; P = 0.001). A negative linear correlation was also found between age of the Greyhounds and their RBC lifespan (P = 0.01, R2 = 0.91). CONCLUSIONS: The shorter RBC lifespan of the Greyhounds may explain the finding of macrocytosis reported in earlier work. The reason for the shorter RBC lifespan in Greyhounds may be caused by differences in Greyhound RBC membrane structure or accelerated RBC removal from the circulation.

The role of tryptophans 371 and 395 in the binding of antibiotics and the transport of sugars by the D-galactose-H+ symport protein (GalP) from Escherichia coli.

The interactions between the D-galactose-H+ symporter (GalP) from Escherichia coli and the inhibitory antibiotics, cytochalasin B and forskolin, and the substrates, D-galactose and H+, have been investigated for the wild-type protein and the mutants Trp-371-->Phe and Trp-395-->Phe, so that the roles of these residues in the structure-activity relationship could be assessed. Neither mutation prevented photolabeling by either [4-3H]cytochalasin B or by 3-[125I]iodo-4-azidophenethyl-amido-7-O-succinyldesacetylforskolin ([125I]APS-forskolin). However, measurements of protein fluorescence show that both residues are in structural domains, the conformations of which are perturbed by the binding of cytochalasin B or forskolin. Moreover, both mutations cause a substantial decrease in the affinity of the inward-facing site of the GalP protein for cytochalasin B, 10- and 43-fold, respectively, but have little effect upon the affinity of this site for forskolin, 0.8- and 2.6-fold reductions, respectively. Both these mutations change the equilibrium between the putative outward- (T1) and inward-facing (T2) conformations, so that the inward-facing form is more favored. They also stabilize a different conformational state, "T3-antibiotic," in which the initial interactions between the protein and antibiotics are tightened. Overall, this has the effect of compensating for the reduction in affinity for cytochalasin B, so that the respective overall Kd values are 0.74- and 3.5-fold that of the wild type, while causing a slight increase, 1.5- and 3.2-fold, respectively, in affinity of the mutants for forskolin. The Trp-371-->Phe mutation causes a 15-fold reduction in the affinity of the inward-facing site for D-galactose, suggesting that this residue forms part of the sugar binding site. In contrast, the Trp-395-->Phe mutation has no effect upon the affinity of the inward-facing site for D-galactose. These effects may be related to the reduction in galactose-H+ symport activity only in the Trp-371-->Phe mutant, although it still effects active transport to the same extent as the Trp395-->Phe mutant. However, there is a 10-20-fold increase in the Km values for energized transport of D-galactose for both mutants.

Cloning, sequencing, and expression of the araE gene of Klebsiella oxytoca 8017, which encodes arabinose-H+ symport activity.

Southern analysis of the genomic DNA from species of the family Enterobacteriaceae, using a probe derived from the Escherichia coli araE gene, which encodes an arabinose-H+ symporter, detected araE in Salmonella, Citrobacter, Klebsiella, and Enterobacter spp. The Klebsiella oxytoca araE gene was cloned, sequenced, and expressed to compare its properties with those of araE from E. coli.

Evaluation of murine leukemia virus infection as a model for thrombocytopenia of HIV/AIDS: mechanism of thrombocytopenia and modulation of thrombocytopenia by thrombopoietin.

Infection of mice with the murine leukemia virus (LP-BM5) was evaluated as a model for the thrombocytopenia of HIV/AIDS. Percent 35S incorporation into platelets, platelet size, platelet count, platelet-associated immunoglobulins (PAIgG), and megakaryocyte size and number were evaluated over a period of 3-9 weeks postinfection (PI). Thrombopoietin from human embryonic kidney cells was administered to mice 9 weeks PI, and similar indices of platelet production were measured 2, 3, and 4 days after treatment with a biological preparation of thrombopoietin (thrombocytopoiesis-stimulating factor, or TSF). Platelet counts decreased in a time-dependent fashion (p = 0.0006) following infection, reaching a nadir at 8 weeks PI (82% of control values). Percent 35S incorporation into platelets also decreased over the 9-week period (p = 0.0001), falling to 63% of control values by week 9. Additionally, platelet volume increased in a linear fashion (p = 0.01), rising to 105% of control values by week 9. No changes in PAIgG were noted over the 9-week period. Megakaryocyte numbers in the femoral marrow were decreased at 8 weeks PI (p = 0.02, 78% of control values), while increased mean megakaryocyte size (p = 0.007, 116% of controls) was noted in the same animals. Increased numbers of naked megakaryocyte nuclei were observed at 3 weeks PI (p < 0.05, 208% of control values). Administration of 2 U/mouse of a highly purified preparation of TSF to virus-infected, thrombocytopenic mice resulted in increased thrombocytopoiesis, as compared to human serum albumin-treated, virus-infected controls.(ABSTRACT TRUNCATED AT 250 WORDS)