Relationship between the ß-galactosidase activity in saliva and parameters associated with oral malodor.

Volatile sulfur compounds (VSCs) are produced by enzymes capable of transforming S-amino acids to corresponding sulfides. Protein degradation by periodontopathogens plays an important role in this process, and the proteolysis of glycoproteins depends on the initial removal of the carbohydrate side chains. In the present report, we tested the relationship between the ß-galactosidase activity in saliva and parameters that influence oral malodor, including daily habits and oral conditions. The prevalence of periodontopathic bacteria was also examined. Forty-nine saliva samples were collected from halitosis patients. Patients were examined for breath odor and other associated parameters. Their breath odor was assessed using an organoleptic test, a portable sulfide monitor and gas chromatography. The presence of periodontopathic bacteria in the saliva was also examined. ß-galactosidase activity was measured with the chromogenic substrates 5-bromo-4-chloro-3-indoyl-ß-d-galactopyranoside and isopropyl-ß-d-thiogalactopyranoside. ß-galactosidase activity was positively correlated with malodor strength (organoleptic score, portable sulfide monitor score and VSC concentrations). Enzyme activity was also correlated with the degree of observable tongue coating. However, it showed no relationship with periodontal condition, saliva flow, tooth decay, unfitted restorations or the color of any tongue coating. While there was no relationship with Porphyromonas gingivalis and Treponema denticola, there was a negative correlation with Prevotella intermedia. These results indicate that ß-galactosidase activity plays an important role in malodor production. Interestingly, the activity of this enzyme was not related to the presence of periodontopathic bacteria, which are the main malodor-producing organisms. The results obtained here may have been associated with physiologic halitosis, which is not necessarily associated with oral problems or with periodontopathic bacteria.

Reduced expression of Sytl 1 and Ccdc21 and impaired induction of Mt I by oxidative stress in SII-K1 knockout mice.

SII-K1 is a member of the transcription elongation factor S-II family. In the mouse, SII-K1 is expressed exclusively in the liver, kidney, heart, and skeletal muscle. Here, we report that deletion of the SII-K1 gene in mice resulted in the downregulation of the synaptotagmin-like 1 (Sytl 1) gene in liver and of the coiled-coil domain-containing 21 (Ccdc21) gene in liver and kidney. Moreover, the induction of the metallothionein I (Mt I) gene in SII-K1-deficient mice liver was impaired in diethyl maleate-induced oxidative stress conditions. Our results suggest that SII-K1 regulates these genes in vivo.

Hippocalcin protects hippocampal neurons against excitotoxin damage by enhancing calcium extrusion.

Hippocalcin, which is a member of the neuronal calcium-sensor protein family, is highly expressed in hippocampal pyramidal cells. Recently, it was demonstrated that hippocalcin deficit caused an increase in neuronal cell death in the field CA3 of Ammon's horn (CA3) region of the hippocampus following the systemic injection of kainic acid. Treatment with kainic acid results in seizure-induced cell death in CA3. In the present study, we injected quinolinic acid, which is an N-methyl-d-aspartate receptor agonist, into the hippocampal field CA1 of Ammon's horn (CA1) region in hippocalcin-knockout (-/-) mice, a procedure which mimics transient ischemia. Although significant pyknotic changes were observed at the injected site in wild-type (+/+) mice 24 h after injection, the area of pyknotic cells extended throughout the hippocampus in -/- mice. The quantification of cell numbers in Nissl-stained sections indicated that the cell damage in -/- mice was more severe than that in +/+ mice. The density of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling-positive cells roughly paralleled that of Nissl-stained pyknotic cells. Primary cultures of hippocampal neurons showed that the number of surviving neurons from -/- mice after 7 days in culture was smaller than the number from +/+ mice. The measurement of intracellular calcium concentrations in single cells revealed that the calcium extrusion from -/- neurons was slower than that from +/+ neurons. The involvement of hippocalcin in the upkeep of calcium extrusion was confirmed using hippocalcin-expressing COS7 cells. These results suggest that hippocalcin plays an important role in calcium extrusion from neurons and, in turn, helps to protect them against calcium-dependent excitotoxin damage in the hippocampus.

Real-Time PCR: Revolutionizing Detection and Expression Analysis of Genes.

Invention of polymerase chain reaction (PCR) technology by Kary Mullis in 1984 gave birth to real-time PCR. Real-time PCR - detection and expression analysis of gene(s) in real-time - has revolutionized the 21(st) century biological science due to its tremendous application in quantitative genotyping, genetic variation of inter and intra organisms, early diagnosis of disease, forensic, to name a few. We comprehensively review various aspects of real-time PCR, including technological refinement and application in all scientific fields ranging from medical to environmental issues, and to plant.

Hippocalcin-deficient mice display a defect in cAMP response element-binding protein activation associated with impaired spatial and associative memory.

Hippocalcin is a member of the neuronal calcium sensor (NCS) protein family that is highly expressed in hippocampal pyramidal cells and moderately expressed in the neurons of cerebral cortex, cerebellum and striatum. Here we examined the physiological roles of hippocalcin using targeted gene disruption. Hippocalcin-deficient (-/-) mice displayed no obvious structural abnormalities in the brain including hippocampal formation at the light microscopic level. Deletion of hippocalcin did not result in up-regulation of the hippocalcin-related proteins; neural visinin-like Ca(2+)-binding proteins (NVP) 1, 2, and 3. The synaptic excitability of hippocampal CA1 neurons appeared to be normal, as estimated by the shape of field excitatory postsynaptic potentials elicited by single- and paired-pulse stimuli, and by tetanic stimulation. However, N-methyl-d-aspartate stimulation- and depolarization-induced phosphorylation of cAMP-response element-binding protein (CREB) was significantly attenuated in -/- hippocampal neurons, suggesting an impairment in an activity-dependent gene expression cascade. In the Morris water maze test, the performance of -/- mice was comparable to that of wild-type littermates except in the probe test, where -/- mice crossed the previous location of the platform significantly less often than +/+ mice. Hippocalcin-deficient mice were also impaired on a discrimination learning task in which they needed to respond to a lamp illuminated on the left or right side to obtain food reinforcement. No abnormalities were observed in motor activity, anxiety behavior, or fear learning. These results suggest that hippocalcin plays a crucial role in the Ca(2+)-signaling pathway that underlies long-lasting neural plasticity and that leads to spatial and associative memory.

Validity of estimating limb muscle volume by bioelectrical impedance.

The present study aimed to investigate the validity of estimating muscle volume by bioelectrical impedance analysis. Bioelectrical impedance and series cross-sectional images of the forearm, upper arm, lower leg, and thigh on the right side were determined in 22 healthy young adult men using a specially designed bioelectrical impedance acquisition system and magnetic resonance imaging (MRI) method, respectively. The impedance index (L(2)/Z) for every segment, calculated as the ratio of segment length squared to the impedance, was significantly correlated to the muscle volume measured by MRI, with r = 0.902-0.976 (P < 0.05). In these relationships, the SE of estimation was 38.4 cm(3) for the forearm, 40.9 cm(3) for the upper arm, 107.2 cm(3) for the lower leg, and 362.3 cm(3) for the thigh. Moreover, isometric torque developed in elbow flexion or extension and knee flexion or extension was significantly correlated to the L(2)/Z values of the upper arm and thigh, respectively, with correlation coefficients of 0.770-0.937 (P < 0.05), which differed insignificantly from those (0.799-0.958; P < 0.05) in the corresponding relationships with the muscle volume measured by MRI of elbow flexors or extensors and knee flexors or extensors. Thus the present study indicates that bioelectrical impedance analysis may be useful to predict the muscle volume and to investigate possible relations between muscle size and strength capability in a limited segment of the upper and lower limbs.

Isolation and characterization of a human apoptosis-inducing gene with yeast two-hybrid system.

asy gene is a novel apoptosis-inducing gene, but its mechanism is unclear. To investigate the mechanism ofasy inducing apoptosis, a novel gene encoding ASY interacting protein (asyip) is isolated from human lung cell line (WI-38) cDNA library with yeast two-hybrid system. Theasyip gene is constitutively expressed as two mRNA transcripts with the size of 1.8 and 2.7 kb in various human tissues at different levels. Sequence analysis of full-length cDNA reveals that the two alternative transcripts ofasyip gene contain common 5' end and different 3' end, and share a common open reading frame encoding a polypeptide of 236 amino acids. Two protein kinase C phosphorylation sites and two casein kinase II phosphorylation sites are found in ASYIP amino acid sequence. Two highly hydrophobic regions encoding potentially two transmembrane domains are present. The ASYIP protein contains a C-terminal endoplasmic reticulum retrieval signal (Lys-Lys-Lys-Ala-Glu). Immunoprecipitation assay confirmed the interaction of ASY and ASYIP in mammalian cells. Compared withasy gene, overexpression ofasyip gene can inhibit growth of tumor cell Saos2 and induce cell apoptosis with a low efficiency.

A prolactin-releasing peptide in the brain.

Hypothalamic peptide hormones regulate the secretion of most of the anterior pituitary hormones, that is, growth hormone, follicle-stimulating hormone, luteinizing hormone, thyroid-stimulating hormone and adrenocorticotropin. These peptides do not regulate the secretion of prolactin, at least in a specific manner, however. The peptides act through specific receptors, which are referred to as seven-transmembrane-domain receptors or G-protein-coupled receptors. Although prolactin is important in pregnancy and lactation in mammals, and is involved in the development of the mammary glands and the promotion of milk synthesis, a specific prolactin-releasing hormone has remained unknown. Here we identify a potent candidate for such a hormone. We first proposed that there may still be unknown peptide hormone factors that control pituitary function through seven-transmembrane-domain receptors. We isolated the complementary DNA encoding an 'orphan' receptor (that is, one for which the ligand is unknown). This receptor, hGR3, is specifically expressed in the human pituitary. We then searched for the hGR3 ligand in the hypothalamus and identified a new peptide, which shares no sequence similarity with known peptides and proteins, as an endogenous ligand. We show that this ligand is a potent prolactin-releasing factor for rat anterior pituitary cells; we have therefore named this peptide prolactin-releasing peptide.

Pituitary adenylate cyclase-activating polypeptide rhythm in the rat pineal gland.

Pituitary adenylate cyclase-activating polypeptide (PACAP) was recently demonstrated to stimulate melatonin synthesis in the rat pineal gland. Circadian rhythms of melatonin concentration are well known. However, it has not been clarified whether PACAP contents in the pineal gland show circadian rhythm. In this study, we measured PACAP contents in the rat pineal gland throughout the day under 12:12 h light-dark cycle or constant dark conditions. A significant fluctuation was observed in the PACAP content under light-dark conditions but not under constant darkness. On the other hand, the pituitary gland showed no significant variation throughout the day under either conditions. These observations suggest that PACAP may participate in the modulation of melatonin synthesis depending on light conditions in the pineal gland.

A novel method for counting spontaneous motor activity in the rat.

Motor activity is a good index for studying the effects of pharmacological agents. Previous investigations have measured spontaneous motor activity by counting the number of times that an animal interrupts a magnetic field or photocell beam. Quite recently, a novel activity-monitoring system, Supermex, was developed. In this system, a sensor detects the radiated body heat of an animal. The Supermex method enables an investigator to perform multi-channel measurement at low cost. Any size home cage may be used, as long as its geometry cannot block the beam's contact with the animal. Operation is very simple and sensitivity adjustment is not required after the sensor-mount position and height from the cage floor are fixed. In the present study we first used the Supermex system to examine the effects of intracerebroventricular (i.c.v.) administration of a known stimulant, thyrotropin-releasing hormone (TRH). Our results confirmed the suitability of this system for testing spontaneous motor activity. We further studied the effects of pituitary adenylate cyclase-activating polypeptide (PACAP) and its related peptide, vasoactive intestinal polypeptide (VIP), on spontaneous motor activity.

Day-night variation of pituitary adenylate cyclase-activating polypeptide (PACAP) level in the rat suprachiasmatic nucleus.

Adenylate cyclase-activating polypeptide (PACAP) is synthesized in the retinal ganglion cells which terminate on vasoactive intestinal polypeptide neurons in the suprachiasmatic nucleus (SCN), the location of circadian clock. To examine whether PACAP exhibits daily variations in the rat SCN, we measured endogenous PACAP contents throughout the day under 12:12 h light-dark or constant dark conditions. PACAP level was low during the light periods, high during the dark periods, and was stable under constant dark conditions. In the periventricular nucleus of the hypothalamus and cerebral cortex, PACAP content did not show any significant variation throughout the day. Our findings suggest that PACAP content in the SCN may be changed by lighting conditions. Thus, PACAP-containing neurons may play certain roles in the entrainment of circadian rhythms.

Neurotensin and neuromedin N brain levels after fornix transection: evidence for an efficient neurotensin precursor processing in subicular neurons.

High levels of neurotensin/neuromedin N precursor mRNA, but few if any NT-positive perikarya have been detected in the dorsal subiculum of the adult rat or human hippocampus. This apparent discrepancy was tentatively ascribed to a lack of precursor mRNA translation or to a poor precursor posttranslational processing in neurons of the hippocampus. Another hypothesis is that in long neuronal pathways, maturation of neuropeptide precursors and derived peptides occurs during axonal transport to terminals, a process which accounts for the lack of peptide detection in cell bodies. In order to test this hypothesis, we performed surgical transection of the fornix to interrupt axonal transport of putative NT/NN products arising from the dorsal hippocampus and measured NT and NN levels in different brain regions. In the mamillary bodies, the main projection area of the dorsal subiculum, NN and NT levels were highly reduced 4 or 14 days after the septo-hippocampal transection which was correlated with a slight increase in NN and NT levels in the dorsal hippocampus and the retrosplenial cortex of 4 days lesioned animals. An increase in hypothalamic NN levels was also detected 14 days after the lesion. These data suggest that the peptide precursor processing can take place during the axonal transport, as shown here for neurotensin and neuromedin N from subicular neurons to their efferent brain areas such as the mamillary bodies.

Effects of intracerebroventricular administration of pituitary adenylate cyclase-activating polypeptide (PACAP) on the motor activity and reserpine-induced hypothermia in murines.

We investigated the effects of i.c.v. administration of pituitary adenylate cyclase-activating polypeptide (PACAP) on the spontaneous motor activity and reserpine-induced hypothermia in murines. The administration of PACAP (1 or 2 nmol) caused a dose-dependent increase in both spontaneous motor activity and rearing behavior in the rat. The peptide (0.1 or 0.2 nmol) counteracted reserpine-induced hypothermia in a dose-dependent manner in mice. On the other hand, i.c.v. injection of vasoactive intestinal polypeptide, which is structurally similar to PACAP, at a dose similar to that of PACAP (2 nmol in rats, 0.2 nmol n mice) did not show a significant effect on either behavior or body temperature. Therefore, the stimulating effect of PACAP observed here may be mediated by PACAP-specific (type I) receptors. PACAP was more potent and longer-lasting than a known potent stimulating peptide, thyrotropin-releasing hormone, in both stimulating motor activity and counteracting reserpine-induced hypothermia. Results of the present study, in combination with those of previous studies identifying endogenous PACAP in the brain, suggest that PACAP may play a important role in the CNS as a stimulant in regulating motor activity and body temperature.

Ontogeny of pituitary adenylate cyclase-activating polypeptide (PACAP) and its binding sites in the rat brain.

Endogenous levels of pituitary adenylate cyclase-activating polypeptide (PACAP) and its binding site densities were measured in eight brain regions in rats of different ages (2-240 days) by sandwich-enzyme immunoassay and autoradiography. PACAP levels were quite low at day 2, peaked in 30-60 days, and then remained constant in most regions. Such ontogenetic changes are similar to those of vasoactive intestinal polypeptide (VIP) and classical neurotransmitters. PACAP-binding sites were already dense at day 2 and varied only slightly up to day 240. These results suggest that PACAP may have modulatory effects on brain development.

Molecular cloning and functional expression of a cDNA encoding a human pituitary adenylate cyclase activating polypeptide receptor.

A functional cDNA clone for a human pituitary adenylate cyclase activating polypeptide (PACAP) receptor was isolated from a human pituitary cDNA library. The cDNA encoded a polypeptide consisting of 525 amino acids with putative seven hydrophobic domains. Chinese hamster ovary (CHO)-K1 cells transfected with the cDNA specifically bound PACAP and mediated PACAP-triggered intracellular accumulation of cAMP, indicating that this cDNA encoded a functional human PACAP Type I receptor. This receptor was structurally related to the vasoactive intestinal peptide (VIP), secretin, calcitonin and parathyroid hormone receptors and is much more homologous to a rat PACAP receptor. Northern blot analysis revealed that PACAP receptor mRNAs were expressed mainly in the brain and widely distributed in the central nervous system.

A survey of the cerebral regionalization and ontogeny of eight exo- and endopeptidases in murines.

We have established the cerebral regionalization and ontogeny of eight exo- and endopeptidases in murines. Aminopeptidases A, B, and M, post-proline dipeptidylaminopeptidase (DAP IV), and proline endopeptidase displayed a rather homogenous distribution within the brain regions with a three- to fourfold factor between the poorest and richest areas. Aminopeptidases M and B appeared maximal in the parietal cortex and nucleus accumbens, respectively, while proline endopeptidase was abundant in the piriform cortex. By contrast with the peptidases exhibiting a rather homogenous distribution, endopeptidase 24.11, angiotensin-converting enzyme, and, to a lesser extent, endopeptidase 24.15 appeared located in much more discrete cerebral zones. Angiotensin-converting enzyme activity was mainly restricted to the nigro-striatal axis. Such feature also stands for endopeptidase 24.11, which was also detected in additional zones corresponding to the globus pallidus and the nucleus accumbens. Endopeptidase 24.15 activity was maximal in the nucleus accumbens and particularly weak in the mamillary body. Neuropeptidases appeared differently regulated during development of mouse brain. Aminopeptidase M, DAP IV, and endopeptidase 24.15 were detected in utero, and their specific activities did not significantly vary until adulthood. Proline endopeptidase and endopeptidase 24.11 were detected in high quantity at day 9 before birth, then activity decreased until birth. Then, proline endopeptidase augmented and plateaued between day 3 and day 10, while endopeptidase 24.11 remained constant at a relatively low level. Finally, angiotensin-converting enzyme was virtually undetectable at early stages before parturition, then slightly increased after birth. The possibility that distinct cerebral regionalization and ontogeny of peptides could directly influence peptide physiology and/or reflect additional functions of the peptidases besides peptide degradation is discussed.

Effects of vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) on the spontaneous release of acetylcholine from the rat hippocampus by brain microdialysis.

Vasoactive intestinal polypeptide (VIP) has been suggested to have a presynaptic effect on cholinergic terminals in the rat hippocampus, which results in an activation of acetylcholine (ACh) synthesis. Recently, a VIP-related novel peptide, pituitary adenylate cyclase activating polypeptide (PACAP) was isolated from the ovine hypothalamus, and we previously demonstrated in the rat that PACAP binding site densities were high in the hippocampus. In the present study, we investigated the effects of VIP and PACAP on the release of ACh from the rat hippocampus. We succeeded in detecting the spontaneous release of ACh from the dorsal hippocampus in the conscious rat using microdialysis and HPLC-ECD. VIP, PACAP38 and PACAP27 were applied through a microinjection cannula placed in a region adjacent to the tip of a microdialysis tube. Injections of VIP, PACAP38 and PACAP27 (12, 120 pmol) resulted in dose-related increases in ACh release. The ability to enhance ACh release was VIP > PACAP38 > PACAP27. The increased release of ACh caused by these peptides was highly calcium-dependent. Tetrodotoxin (10(-6) M) added to the perfusion medium significantly reduced both the release of ACh enhanced by these peptides and the basal release. The present results suggest that VIP, PACAP38 and PACAP27 presynaptically stimulate cholinergic activity in the hippocampus, which may be reflected by an increase in ACh synthesis to maintain releasable terminal stores of ACh.

Regional distribution of pituitary adenylate cyclase activating polypeptide (PACAP) in the rat central nervous system as determined by sandwich-enzyme immunoassay.

We investigated endogenous levels of a novel peptide, pituitary adenylate cyclase activating polypeptide (PACAP), in the rat central nervous system. The amount of PACAP was measured by means of highly specific and sensitive sandwich-enzyme immunoassay. This assay system following HPLC analysis revealed that PACAP38 was a major portion of the total PACAP immunoreactivity and PACAP27 levels were negligibly low in the brain. Therefore, we measured the amount of PACAP38 in 62 regions punched out from frozen tissue sections. High amounts of PACAP38 were found in the lateral septal nucleus (intermediate part), diagonal band, central amygdaloid nucleus, several parts of the hypothalamus (suprachiasmatic, supraoptic, periventricular and arcuate nuclei), central gray, interpeduncular nucleus and dorsal raphe. The suprachiasmatic, paraventricular and periventricular hypothalamic nuclei showed the highest levels. A moderate amount of the peptide was observed in the lateral septal nucleus (dorsal part), medial septal nucleus, medial amygdaloid nucleus, thalamus (paraventricular, paratenial, central medial, ventromedial, reuniens and rhomboid nuclei), hypothalamus (lateral hypothalamic area and mammillary body), ventral tegmental area, interfascicular nucleus and in the locus coeruleus. Such a distribution of endogenous PACAP38 did not parallel the localization of PACAP binding sites which we had demonstrated recently. Moreover, the topographical distribution of PACAP38 observed in the present study differed from that of VIP which had been previously reported. The present results suggest that PACAP38 may have a neurotransmitter/neuromodulator role which is different from that of VIP in the central nervous system.

Design and synthesis of ETA receptor antagonists and study of ETA receptor distribution.

Many oligopeptides were designed to find ETA receptor antagonists on the hypothesis that an ETA receptor can recognize two hydrophobic parts of ET-1, i.e., Val-Tyr-Phe and Ile-Ile-Trp, over a short distance. They were synthesized from the benzyl ester of the C-terminal amino acid by stepwise chain elongation using the solution method. The binding affinity of the synthetic peptides to the endothelin receptors was examined in porcine cardiac ventricular muscle membrane for ETA receptor and in bovine whole brain membrane for ETB receptor. Hexamethyleneiminocarbonyl-Leu-trp-ala-beta ala-tyr-phe (TTA-386) was selected as an ETA receptor-selective competitive antagonist to ET-1. It competed against ET-1 at ETA receptor sites and showed one-third the binding affinity of ET-1 for ETA receptor and < 1/10,000 the affinity for ETB receptor. It inhibited the ET-1-induced increase of cytosolic free calcium concentration in A-10 cells. The 125I-labeled hexapeptide (125I-TTA-386) was prepared to distinguish the distribution of ETA receptor from ETB receptor. Scatchard plot analysis of saturation binding of 125I-TTA-386 to porcine cardiac membranes showed the same Bmax value as that of 125I-ET-1. Autoradiographic studies showed that ETA receptors are most abundant in the cardiac muscle, intestine, large bowel, spleen, and testis.