Scoring tools to identify TB patients facing catastrophic costs in the Philippines.

BACKGROUND: This study was to meet a practical need to design a simple tool to identify TB patients who may potentially be facing catastrophic costs while seeking TB care in the public sector. Such a tool may help prevent and address catastrophic costs among individual patients. METHODS: We used data from the national TB patient cost survey in the Philippines. We randomly allocated TB patients to either the derivation or validation sample. Using adjusted odds ratios (ORs) and ß coefficients of logistic regression, we developed four scoring systems to identify TB patients who may be facing catastrophic costs from the derivation sample. We validated each scoring system in the validation sample. RESULTS: We identified a total of 12 factors as predictive indicators associated with catastrophic costs. Using all 12 factors, the ß coefficients-based scoring system (area under the curve [AUC] 0.783, 95% CI 0.754-0.812) had a high validity. Even with seven selected factors with OR > 2.0, the validity remained in the acceptable range (ß coefficients-based: AUC 0.767, 95% CI 0.737-0.798). CONCLUSION: The ß coefficients-based scoring systems in this analysis can be used to identify those at high risk of facing catastrophic costs due to TB in the Philippines. Operational feasibility needs to be investigated further to implement this in routine TB surveillance.

CONTEXTE: Cette étude visait à répondre à un besoin pratique de concevoir un outil simple pour identifier les patients atteints de TB qui pourraient potentiellement être confrontés à des coûts catastrophiques lorsqu'ils recherchent des soins de TB dans le secteur public. Un tel outil pourrait aider à prévenir et à traiter les coûts catastrophiques chez les patients individuels. MÉTHODES: Nous avons utilisé des données de l'enquête nationale sur les coûts des patients atteints de TB aux Philippines. Nous avons réparti aléatoirement les patients atteints de TB dans l'échantillon de dérivation ou de validation. À l'aide des odds ratio (OR) ajustés et des coefficients ß de la régression logistique, nous avons développé quatre systèmes de notation pour identifier les patients atteints de TB qui pourraient être confrontés à des coûts catastrophiques à partir de l'échantillon de dérivation. Nous avons validé chaque système de notation dans l'échantillon de validation. RÉSULTATS: Nous avons identifié un total de 12 facteurs en tant qu'indicateurs prédictifs associés à des coûts catastrophiques. En utilisant les 12 facteurs, le système de notation basé sur les coefficients ß (aire sous la courbe [AUC] 0,783 ; IC 95% 0,754­0,812) avait une validité élevée. Même avec sept facteurs sélectionnés avec OR > 2,0, la validité est restée dans la plage acceptable (basée sur les coefficients ß : AUC 0,767 ; IC 95% 0,737­0,798). CONCLUSION: Les systèmes de notation basés sur les coefficients ß dans cette analyse peuvent être utilisés pour identifier les personnes à haut risque de faire face à des coûts catastrophiques liés à la TB aux Philippines. La faisabilité opérationnelle doit être étudiée plus avant pour mettre en œuvre cela dans la surveillance de routine de la TB.

Novel excitatory neuropeptides isolated from a prosobranch gastropod, Thais clavigera: The molluscan counterpart of the annelidan GGNG peptides.

The GGNG peptides are excitatory neuropeptides identified from earthworms, leeches and polychaeta. Two structurally related peptides were purified and characterized from a mollusk, Thais clavigera (prosobranch gastropod). The peptides designated as Thais excitatory peptide-1 (TEP-1) (KCSGKWAIHACWGGN-NH2) and TEP-2 (KCYGKWAMHACWGGN-NH2) are pentadecapeptides having one disulfide bond and C-terminal GGN-NH2 structures, which are shared by most GGNG peptides. TEP augmented the motilities of Thais esophagus and penial complex. TEP-like immunoreactivity is distributed in both the neurons of the central nervous system and nerve endings in the penial complex. Thus, the involvement of TEP in the contraction of the digestive and reproductive systems is suggested. Substitution of amino acids in TEP revealed that two tryptophan residues in TEP are important for maintaining bioactivity.

PRQFVamide, a novel pentapeptide identified from the CNS and gut of Aplysia.

We have purified a novel pentapeptide from the Aplysia nervous system using bioassay on gut contractions. The structure of the peptide is Pro-Arg-Gln-Phe-Val-amide (PRQFVa). The precursor for PRQFVa was found to code for 33 copies of PRQFVamide and four related pentapeptides. Peaks corresponding to the predicted masses of all five pentapeptides were detected in Aplysia neurons by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Northern analysis revealed that expression of the precursor is abundant in the abdominal ganglion, much less in the pedal and cerebral ganglia, and rarely seen in the buccal and pleural ganglia. PRQFVa-positive neurons, mapped by immunohistochemistry and in situ hybridization, were present in all the central ganglia. PRQFVa immunopositive processes were observed in the gut, particularly in association with the vasculature. Some arteries and other highly vascularized tissues, such as the gill and the kidney, also contain numerous PRQFVa immunopositive processes. Application of synthetic PRQFVa suppresses not only contractions of the gut but also contractions of vasculature. PRQFVa is expressed in some of the neurons within the feeding circuitry and application of synthetic PRQFVa was found to decrease the excitability of some (B4/5 and B31/32) but not all (B8) neurons of the buccal feeding circuit. Our findings suggest that PRQFVa may act as a modulator within the feeding system as well as in other systems of Aplysia.

Identification of a vasopressin-like immunoreactive substance in hydra.

Vasopressin (VP)-like immunoreactivity has long been known in the hydra nervous system, but has not yet been structurally identified. In this study, using HPLC fractionation and an immunological assay, we have purified two peptides, FPQSFLPRGamide and SFLPRGamide, from Hydra magnipapillata. Both the peptides shared the same C-terminal structure, -PRGamide, with Arg-VP. The nonapeptide proved to be Hym-355, a peptide that stimulates neuronal differentiation in hydra. Detailed evaluation by competitive enzyme-linked immunosorbent assay (ELISA) and double immunostaining using anti-VP and anti-Hym-355 antibodies enabled us to conclude that the two peptides account for a major part of the VP-like immunoreactivity in hydra nerve cells.

Deamidase inactivates a D-amino acid-containing Aplysia neuropeptide.

Membrane-catalyzed degradation of the cardioexcitatory peptide, Asn-D-Trp-Phe-NH(2) (N(d)WF-NH(2)), which was previously isolated from Aplysia, was investigated in relation to its inactivation mechanism. The principal degradation was deamidation of the C-terminal amide, producing biologically inert Asn-D-Trp-Phe-OH (N(d)WF-OH). Among membrane fractions prepared from different organs, the fraction from the ganglia showed the highest specific activity of the deamidation reaction. The deamidase activity was inhibited by Ebelactone B and the serine protease inhibitor, phenylmethanesulfonyl fluoride (PMSF), while the degradation of the synthetic stereoisomer, Asn-Trp-Phe-NH(2) (N(l)WF-NH(2)), was sensitive to the divalent cation-chelating agent, o-phenanthroline, and aminopeptidase inhibitors, amastatin and bestatin. The presence of D-Trp residue in the second position of N(d)WF-NH(2) endows this peptide not only with stereospecific bioactivity but also peptidase stability. The deamidation reaction seems to be the major inactivation mechanism for this peptide.

Distribution and function of an Aplysia cardioexcitatory peptide, NdWFamide, in pulmonate snails.

The distribution and function of an Aplysia cardioexcitatory peptide, NdWFamide, were examined in the nervous system of pulmonate snails. We chemically identified the authentic NdWFamide from a land snail (Euhadra congenita) and a freshwater snail (Lymnaea stagnalis). NdWFamide potentiated the heartbeat of those snails. Immunohistochemistry using anti-NdWFamide antibody demonstrated the distribution of NdWFamide-containing neurons and fibers in the central nervous system, as well as peripheral tissues, such as the cardiovascular region and accessory sex organs. These results suggest that NdWFamide is a neuropeptide mediating the neural regulation of the activity of the cardiovascular and reproductive systems of snails.

A novel GGNG-related neuropeptide from the polychaete Perinereis vancaurica.

The GGNG peptides are myoactive peptides so far identified from earthworms and leeches, which are the earthworm excitatory peptides (EEP) and the leech excitatory peptide (LEP), respectively. A novel GGNG peptide was isolated and structurally determined from a marine polychaete, Perinereis vancaurica, using a combination of immunological assay and high performance liquid chromatography (HPLC). The peptide was a pentadecapeptide whose amino acid sequence was similar to that of EEP and LEP, and showed myoactivity on isolated esophagus of P. vancaurica with a threshold concentration of 10(-10)M. The peptide was designated as polychaete excitatory peptide (PEP). Amidation of the alpha-carboxyl group of C-terminal residue occurred in PEP. This is the case for LEP, but not for EEP. The cDNA cloning revealed that the structure of the PEP precursor is more similar to the EEP precursor than to the LEP precursor. Immunohistochemical staining showed the presence of PEP in several neurons of central nervous system (CNS) as somata and neuropile structure, epithelial cells of the pharynx and epidermal cells throughout the body wall. Altogether these results support the physiological significance of PEP in regulation of the CNS neural activity and the peripheral myoactivity.

The enterins: a novel family of neuropeptides isolated from the enteric nervous system and CNS of Aplysia.

To identify neuropeptides that have a broad spectrum of actions on the feeding system of Aplysia, we searched for bioactive peptides that are present in both the gut and the CNS. We identified a family of structurally related nonapeptides and decapeptides (enterins) that are present in the gut and CNS of Aplysia, and most of which share the HSFVamide sequence at the C terminus. The structure of the enterin precursor deduced from cDNA cloning predicts 35 copies of 20 different enterins. Northern analysis, in situ hybridization, and immunocytochemistry show that the enterins are abundantly present in the CNS and the gut of Aplysia. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry we characterized the enterin-precursor processing, demonstrated that all of the precursor-predicted enterins are present, and determined post-translational modifications of various enterins. Enterin-positive neuronal somata and processes were found in the gut, and enterins inhibited contractions of the gut. In the CNS, the cerebral and buccal ganglia, which control feeding, contained the enterins. Enterin was also present in the nerve that connects these two ganglia. Enterins reduced the firing of interneurons B4/5 during feeding motor programs. Such enterin-induced reduction of firing also occurred when excitability of B4/5 was tested directly. Because reduction of B4/5 activity corresponds to a switch from egestive to ingestive behaviors, enterin may contribute to such program switching. Furthermore, because enterins are present throughout the nervous system, they may also play a regulatory role in nonfeeding behaviors of Aplysia.

NdWFamide: a novel excitatory peptide involved in cardiovascular regulation of Aplysia.

Although diverse peptides are known to affect invertebrate cardiac activity, the peptidergic regulation of the cardiovascular system of Aplysia is still poorly understood. Asn-D-Trp-Phe-NH(2) (NdWFamide) is a recently purified cardioactive peptide in Aplysia. Pharmacological experiments showed that NdWFamide was one of the most potent cardioexcitatory peptides among the known endogenous cardioactive peptides in Aplysia. NdWFamide-immunopositive neuronal processes were abundant in the cardiovascular region of Aplysia, and many of them originated from neurosecretory cells in the abdominal ganglion (R3-R13 cells). The data suggest that NdWFamide is a cardioexcitatory peptide utilized by R3-R13 cells of Aplysia.

Purification and some properties of alanine racemase from a bivalve mollusc Corbicula japonica.

A brackish-water mollusc, Corbicula japonica, uses large quantities of D- and L-alanine as intracellular osmotically active solutes, osmolytes, for regulation of intracellular osmolarity. We purified alanine racemase from the mantle of C. japonica to characterize its enzymological properties. The molecular masses of the enzyme were estimated to be 41 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 140 kDa by gel filtration on high-performance liquid chromatography, suggesting the trimeric or tetrameric nature of the enzyme. Neither dialysis nor chromatographic procedures in the absence of pyridoxal 5'-phosphate led to loss of enzyme activity, although carbonyl reagents, hydroxylamine and phenylhydrazine, inhibited the activity. These results suggest that alanine racemase of the animal may bind pyridoxal 5'-phosphate tightly as a cofactor. Kinetic experiments using the partially purified enzyme revealed that alanine was the sole substrate among 17 kinds of L-amino acids tested. The Lineweaver-Burk plot for L-alanine as substrate resulted in Km value of 22.6 mM, and the value for D-alanine was 9.2 mM. Together with the previous evidence that D- and L-alanine levels of this animal change with the external salinity maintaining the D-/L-alanine ratio at unity, the present results seem to indicate that the physiological role of alanine racemase in this animal is to supply D-alanine as a main intracellular osmolyte. J. Exp. Zool. 289:1-9, 2001.

Enhancement of foot formation in Hydra by a novel epitheliopeptide, Hym-323.

During the course of a systematic screening of peptide signaling molecules in Hydra magnipapillata, a novel peptide, Hym-323, which enhances foot regeneration was identified. The peptide is 16 amino acids long, and is encoded in the precursor protein as a single copy. Northern blot analysis, in situ hybridization analysis and immunohistochemistry showed that it was expressed in both ectodermal and endodermal epithelial cells throughout the body, except for the basal disk and the head region. The peptide enhanced foot regeneration by acting on epithelial cells. Lateral transplantation experiments indicated that the foot activation potential was increased in the peptide-treated tissue. These results suggest that Hym-323 is a peptide involved in a foot-patterning process in Hydra.

Comparative aspects of invertebrate neuropeptides.

1. We searched for bioactive peptides, most of which were considered to be neuropeptides, in various animals of several phyla. These peptides were compared with each other and with peptides identified by many other investigators. Consequently, we found that structures of neuropeptides are generally conserved in each phylum. 2. We also found some exceptional interesting aspects. First, there are a number of peptide groups whose members are distributed among several phyla. Second, there are many structural similarities between molluscan and annelidan peptides as if molluscs and annelids were the animals in a phylum. Third, certain toxic peptides of invertebrates are closely related to vertebrate neuropeptides. 3. In addition to the above phylogenetic aspects, we found some other interesting aspects. A wide structural variety of members of a peptide group is generally found in invertebrate species. Invertebrate muscles seem to be generally regulated not only by some or several classical non-peptidic neuromediators but also by various peptidic neuromediators. Peptides containing a D-amino acid residue are not rare.

Carassius RFamide, a novel FMRFa-related peptide, is produced within the retina and involved in retinal information processing in cyprinid fish.

Carassius RFamide (C-RFa) is a novel peptide, isolated originally from the brain of the Japanese crucian carp and sharing homologies with mammalian prolactin-releasing peptide (PrRP). It has been demonstrated previously that C-RFa mRNA is abundant in the proximal half (fundus) of the Japanese crucian carp eye. In the present work, we localized C-RFa by immunohistochemistry mainly to perikarya, in the proximal half of the inner nuclear layer (amacrine cell layer). This distribution is different from that of FMRFamide, which is confined to axon terminals of terminal nerve efferent fibers in the inner plexiform layer. Electrophysiological recording revealed that C-RFa depolarized some amacrine cells and hyperpolarized L-type horizontal cells in the carp. These results suggest that C-RFa is produced within the cyprinid retina and functions as a transmitter or neuromodulator in retinal image processing.

Separation of drugs by packed-column supercritical fluid chromatography.

Packed-column supercritical fluid chromatography (pSFC) has been expected to analyze various kinds of compounds. Many researchers have expected a new chromatographic technique that overcomes the limitations of other techniques, HPLC and GC. In pharmaceutical development, chromatography plays an important role in the evaluation of safety and efficacy of a new compound. This article provides an overview of the separation of drugs by pSFC. The effects of the chromatographic parameters were studied for the separation of steroids. In chiral separation, the successful results were shown and compared with HPLC.

Immunohistochemical Localization of C-RFamide, a FMRF-related Peptide, in the Brain of the Goldfish, Carassius auratus.

Carassius RFamide (C-RFa) is a novel peptide found in the brain of the Japanese crucian carp. It has been demonstrated that mRNA of C-RFa is present in the telencephalon, optic tectum, medulla oblongata, and proximal half of the eyeball in abundance. Immunohistochemical methods were employed to elucidate the distribution of the peptide in the brain of the goldfish (Carassius auratus) in detail. C-RFaimmunoreactive perikarya were observed in the olfactory bulb, the area ventralis telencephali pars dorsalis and lateralis, nucleus preopticus, nucleus preopticus periventricularis, nucleus lateralis tuberis pars posterioris, nucleus posterioris periventricularis, nucleus ventromedialis thalami, nucleus posterioris thalami, nucleus anterior tuberis, the oculomotor nucleus, nucleus reticularis superior and inferior, facial lobe, and vagal lobe. C-RFa immunoreactive fibers and nerve endings were present in the olfactory bulb, olfactory tract, area dorsalis telencephali pars centralis and medialis, area ventralis telencephali, midbrain tegmentum, diencephalon, medulla oblongata and pituitary. However, in the optic tectum the immunopositive perikarya and fibers were less abundant. Based on these results, some possible functions of C-RFa in the nervous system were discussed.

The Aplysia mytilus inhibitory peptide-related peptides: identification, cloning, processing, distribution, and action.

Neuropeptides are a ubiquitous class of signaling molecules. In our attempt to understand the generation of feeding behavior in Aplysia, we have sought to identify and fully characterize the neuropeptides operating in this system. Preliminary evidence indicated that Mytilus inhibitory peptide (MIP)-like peptides are present and operating in the circuitry that generates feeding in Aplysia. MIPs were originally isolated from the bivalve mollusc Mytilus edulis, and related peptides have been identified in other invertebrate species, but no precursor has been identified. In this study, we describe the isolation and characterization of novel Aplysia MIP-related peptides (AMRPs) and their precursor. Several AMRPs appear to have some structural and functional features similar to vertebrate opioid peptides. We use matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to confirm that all 14 AMRPs predicted by the precursor are processed in isolated neurons. Northern analysis, whole-mount in situ hybridization, and immunohistochemistry are used to map the abundant expression of these peptides in the CNS and peripheral tissues such as the digestive tract, vasculature, and the reproductive organs. Physiological studies demonstrate that the rank order of the inhibitory actions of these peptides is different for three target muscles. These results underscore the importance of using a multidisciplinary approach to identifying and characterizing the actions of neuropeptides in an effort to gain understanding of their role in systems of interest. The widespread distribution of the AMRPs indicates that they may be operating in many different systems of Aplysia.

Possible functions of oxytocin/vasopressin-superfamily peptides in annelids with special reference to reproduction and osmoregulation.

Annetocin is an earthworm oxytocin-related peptide that we previously isolated from the whole body of a lumbricid earthworm Eisenia foetida. We have reported that annetocin induces egg-laying-like behaviors in E. foetida and a gnathobdellid leech, Whitmania pigra, when it is injected into the respective animals. The present study was undertaken to probe physiological functions of invertebrate oxytocin-vasopressin-superfamily peptides with special reference to reproductive and osmoregulatory events in which vertebrate peptides of this superfamily are involved. Annetocin, Lys-conopressin (a leech vasopressin-related peptide) and two analog peptides, [Tyr(3)]-annetocin ((3)Y-annetocin) and [Phe(3)]-annetocin ((3)F-annetocin), were compared for their activities to induce egg-laying-like behavior and to change body weight as a measure of water balance in the leech W. pigra. Injection of annetocin, Lys-conopressin, and (3)F-annetocin caused both egg-laying-like behavior and reduction of body weight in the animals, but (3)Y-annetocin induced neither. Furthermore, leeches in the non-breeding season responded to peptides less conspicuously than those in the breeding season. Such a concomitant induction of egg-laying-like behavior and body-weight reduction suggests that these two phenomena are unitary and might be accounted for by the fact that egg-laying in leeches and earthworms is accompanied by secretion of a large quantity of mucus, which should significantly contribute to body-weight loss. J. Exp. Zool. 284:401-406, 1999.

beta-Adrenergic receptor subtypes in melanophores of the marine gobies Tridentiger trigonocephalus and Chasmichthys gulosus.

The subtype of beta-adrenergic receptors in melanophores of the marine gobies Tridentiger trigonocephalus and Chasmichthys gulosus was studied. Pigment of denervated melanophores in isolated, split caudal fins was preliminarily aggregated by incubating the specimens in a physiological saline containing 10 microM phentolamine and 30-100 microM verapamil or 2-10 nM melatonin, and the responses of the melanophores to a beta-adrenergic agonist added to the incubating medium were recorded photoelectrically. The beta-adrenergic agonists noradrenaline, adrenaline, isoproterenol, salbutamol and, dobutamine were all effective in evoking a dispersion of melanophore pigment in the presence of phentolamine and verapamil or melatonin. The pigment-dispersing effect of noradrenaline (beta 1-selective agonist) was inhibited by metoprolol (beta 1-selective antagonist), propranolol,- and butoxamine. Whereas, the effect of salbutamol (beta 2-selective agonist) was hardly inhibited by metoprolol, though it was considerably inhibited by propranolol and ICI-118551. It was estimated that beta 1- and beta 2-adrenergic receptors coexist at ratios of 8.6:91.4, in the melanophore of Tridentiger trigonocephalus, and 25:75, in the melanophore of Chasmichthys gulosus, through the analyses of Hofstee plots of the effects of the beta-adrenergic drugs. It was suggested that the relation between the pigment-dispersing effect of a beta-adrenergic agonist on the melanophores and the concentration of the drug follows mass action kinetics, when the effect is mainly caused by the activation of beta 2-adrenergic receptors of the melanophores. However, when it is mainly caused by the activation of beta 1-adrenergic receptors of the melanophores, the relation does not follow mass action kinetics.

Localization of leech excitatory peptide, a member of the GGNG peptides, in the central nervous system of a leech (Whitmania pigra) by immunohistochemistry and in situ hybridization.

We have recently isolated a myoactive peptide, called leech excitatory peptide, belonging to the GGNG peptide family from two species of leeches, Hirudo nipponia and Whitmania pigra. Immunohistochemistry and in situ hybridization were employed to localize leech excitatory peptide-like peptide(s) and its gene expression in the central nervous system of W. pigra. A pair of neuronal somata were stained by both immunohistochemistry and in situ hybridization in the supraesophageal, subesophageal, and segmental ganglia. In addition, several other neurons showed positive signals by either immunohistochemistry or in situ hybridization in these ganglia. An immunoreactive fiber was observed to run in the anterior root of segmental ganglion 6, which is known to send axons to the sexual organs, though we failed to detect immunoreactivity in possible target tissues. Antiserum specificity was established by enzyme-linked immunosorbent assay using different leech excitatory peptide-related peptides. Leech excitatory peptide elicited muscular contraction of isolated preparations of penis and intestine at concentrations of 10(-8 )M. These results suggest that leech excitatory peptide is a neuropeptide modulating neuromuscular transmission in multiple systems, including regulation of reproductive behavior.

Release of proteins and polysaccharides from the albumen gland of the freshwater snail Helisoma duryi: effect of cAMP and brain extracts.

The albumen gland is a compound tubular exocrine gland found in the female reproductive tract of freshwater pulmonate snails such as Helisoma duryi. It secretes a perivitelline fluid, composed of protein and polysaccharide complexes, and coats each fertilized egg. A 288-kDa native glycoprotein, composed of several 66-kDa subunits, was identified in soluble extracts of albumen gland. Forskolin stimulates the release of secretory granules, containing both proteins and polysaccharides, from the cytoplasm of the glandular cells. An acid extract of the central nervous system or the adenosine-3', 5'-cyclic monophosphate (cAMP) analogue 8-bromo cAMP, stimulates protein secretion from the gland. Pretreatment of the albumen gland with cAMP antagonist (Rp isomer of cAMP) inhibits the stimulatory effect of a brain extract. Digestion of brain extract with proteolytic enzymes abolishes its activity, suggesting the factor from the brain is peptidergic. The neuroactive agents serotonin, Phe-Met-Arg-Phe-amide, Tyr-Gly-Gly-Phe-Met-Arg-Phe-amide, small cardioactive peptide B, and caudodorsal cell hormone were also tested for potential secretion-promoting ability. Brain extracts were partially purified with a Sep-Pak C18 reverse-phase cartridge and indicate the peptide is relatively hydrophobic. These results suggest that a brain peptide promotes the secretion of perivitelline fluid, and this is mediated by the adenylate cyclase/cAMP signal transduction pathway.