Why adductor magnus muscle is large: the function based on muscle morphology in cadavers.

The aim of this study was to examine anatomical properties of the adductor magnus through a detailed classification, and to hypothesize its function and size to gather enough information about morphology. Ten cadaveric specimens of the adductor magnus were used. The muscle was separated into four portios (AM1-AM4) based on the courses of the corresponding perforating arteries, and its volume, muscle length, muscle fiber length and physiological cross-sectional area were assessed. The architectural characteristics of these four portions of the adductor magnus were then classified with the aid of principal component analysis. The results led us into demarcating the most proximal part of the adductor magnus (AM1) from the remaining parts (AM2, AM3, and AM4). Classification of the adductor magnus in terms of architectural characteristics differed from the more traditional anatomical distinction. The AM2, AM3, and AM4, having longer muscle fiber lengths than the AM1, appear to be designed as displacers for moving the thigh through a large range of motion. The AM1 appears instead to be oriented principally toward stabilizing the hip joint. The large mass of the adductor magnus should thus be regarded as a complex of functionally differentiable muscle portions.

Interface between intramembranous and endochondral ossification in human foetuses.

In the head and neck of human mid-term foetuses, the interface between areas of endochondral ossification and adjacent membranous (intramembranous) ossification is extensive. Using 8 foetal heads at 15-16 weeks, we have demonstrated differences in the matrices and composite cells between these 2 ossification processes, especially in the occipital squama and pterygoid process. Aggrecan-positive cartilage was shown to be invaded by a primitive bony matrix that was negative for aggrecan. At the interface, the periosteum was continuous with the perichondrium without any clear demarcation, but tenascin-c expression was restricted to the periosteum. In contrast, the interface between the epiphysis and shaft of the femur showed no clear localisation of tenascin-c. Versican expression tended to be restricted to the perichondrium. In the pterygoid process, the density of CD34-positive vessels was much higher in endochondral than in membranous ossification. The membranous part of the occipital was considered most likely to contribute to growth of the skull to accommodate the increased volume of the brain, while the membranous part of the pterygoid process seemed to be suitable for extreme flattening under pressure from the pterygoid muscles.

Immunosuppressive effect of prolactin-induced protein: a new insight into its local and systemic role in chronic allergic contact dermatitis.

BACKGROUND: Prolactin-induced protein (PIP) has been shown to bind to CD4 and is speculated to block CD4-HLA-DR interaction. However, the immunomodulatory effect of PIP on chronic allergic contact dermatitis (ACD) remains to be elucidated. OBJECTIVES: To define the role of PIP during the immunoresponse. METHODS: Using a low-dose oxazolone-induced mouse chronic ACD model, expression of PIP was examined immunohistologically. Furthermore, effects of continued exposure to a peptide mimicking the major binding site of PIP (amino acids 106-132) for CD4 was examined in a mouse chronic ACD model. RESULTS: We clarified that keratinocytes, dermal infiltrating cells and spleen infiltrating mononuclear cells are positively stained with anti-PIP antibody. The PIP peptide significantly downregulated oxazolone-induced mouse ACD compared with controls. We also found that inflammation of the control ear, to which the PIP peptide had not been applied, was also suppressed in a synchronized manner in the late phase of ACD. CONCLUSIONS: These findings suggest that PIP might have a local and systemic immunosuppressive effect in mouse chronic ACD.

Centrally administered neuromedin S inhibits feeding behavior and gastroduodenal motility in mice.

Neuromedin S (NMS) was recently identified as an endogenous ligand for the FM-4/TGR-1 receptor in the rat hypothalamus. No previous studies have examined the effect of NMS on gut motility. We examined the effects of intracerebroventricular administration of NMS on food intake in food-deprived and free-feeding mice, and on gastroduodenal motility by using a manometric method, and gastric emptying in mice. We found that NMS decreased food intake and the gastric emptying rate. It also disrupted the motor activity in the antrum and duodenum of conscious food-deprived mice. These results suggest that NMS influences gut motility as well as feeding behavior.

Modest overexpression of neuropeptide Y in the brain leads to obesity after high-sucrose feeding.

Neuropeptide Y (NPY), one of the most abundant peptide transmitters in the mammalian brain, is assumed to play an important role in feeding and body weight regulation. However, there is little genetic evidence that overexpression or knockout of the NPY gene leads to altered body weight regulation. Previously, we developed NPY-overexpressing mice by using the Thy-1 promoter, which restricts NPY expression strictly within neurons in the central nervous system, but we failed to observe the obese phenotype in the heterozygote. Here we report that in the homozygous mice, overexpression of NPY leads to an obese phenotype, but only after appropriate dietary exposure. NPY-overexpressing mice exhibited significantly increased body weight gain with transiently increased food intake after 50% sucrose--loaded diet, and later they developed hyperglycemia and hyperinsulinemia without altered glucose excursion during 1 year of our observation period.

Insulin production in a neuroectodermal tumor that expresses islet factor-1, but not pancreatic-duodenal homeobox 1.

We studied a 60-yr-old female with a brain tumor who showed severe symptoms of hypoglycemia (plasma glucose, 2.2 mmol/L) and hyperinsulinemia (1.28 nmol/L) after radiotherapy. The cystic brain tumor contained proinsulin and insulin at concentrations of 13.6 and 1.22 nmol/L, respectively. Immunohistochemical studies showed the tumor cells were ectodermal in origin but not endodermal, based on three diagnostic features of neuroectodermal tumors 1) pseudorosette formation noted under light microscopy, 2) finding of a small number of dense core neurosecretory granules on electron microscopy, and 3) positive immunostaining for both neuronal specific enolase and protein gene product 9.5. These cells also expressed the transcription factor, neurogenin-3, NeuroD/beta 2, and islet factor I, which are believed to be transcription factors in neuroectoderm as well as in pancreatic islet cells, but not pancreatic-duodenal homeobox 1, Pax4, or Nkx2.2. In addition, they did not express glucagon, somatostatin, or glucagon-like peptide-1. Our results show the presence of proinsulin in an ectoderm cell brain tumor that does not express the homeobox gene, pancreatic-duodenal homeobox 1, but expresses other transcription factors, i.e. neurogenin3, NeuroD/beta 2, and islet factor-1, which are related to insulin gene expression in the brain tumor.

Thioperamide, a histamine H3 receptor antagonist, powerfully suppresses peptide YY-induced food intake in rats.

BACKGROUND: Whether or not peptide YY (PYY)-induced hyperphagia is modified by the histaminergic system in the brain is not yet known. METHODS: We investigated the effect on feeding of intracerebroventricular (ICV) administration of a specific histamine H3 receptor antagonist prior to ICV administration of PYY in rats. RESULTS: PYY (1, 3, and 10 micrograms/10 microL) strongly induced feeding behavior in a dose-dependent manner in sated rats. The 4-hour food intake induced by 3 micrograms/10 microL of PYY was equal to that induced by a 16-hour fast. The ICV administration of thioperamide (40.8, 122.4, and 408.5 micrograms/10 microL) did not suppress the 4-hour food intake induced by 16-hour fasting; however, thioperamide produced dose-dependent and strong inhibition of hyperphagia induced by a 3-microgram dose of PYY. CONCLUSIONS: These results suggest that the effect of PYY on appetite is different than that induced by fasting and may involve a histaminergic mechanism.

Postnatal development of serotonin nerve fibers in the somatosensory cortex of mice studied by immunohistochemistry.

Postnatal serotonin (5HT) innervation in the cerebral cortex of mice has been studied by 5HT immunohistochemistry. 5HT-like immunoreactive (5HT-LI) nerve fibers and terminals appeared to increase transiently, particularly in the somatosensory (Sm) cortex during early postnatal days. As pups grow, 5HT afferent inputs decreased rapidly to reach a similar pattern of distribution to that in adult animals. Since the transient increase was seen at a critical period (seventh postnatal day) for the differentiation of layer IV, it is suggested that increased 5HT concentrations might have an effect on thalamocortical inputs and/or cortical lamination of the developing brains.

Immunohistochemical localization of tryptophan hydroxylase in the human and rat gastrointestinal tracts.

Because few previous studies have shown the immunohistochemical localization of tryptophan 5-hydroxylase (TPH) in the gastrointestinal tract, we developed a specific antibody against TPH purified from mouse mastocytoma P-815 and stained human and rat gastrointestinal tracts. The specificity of the antibody was examined by Western blotting and by immunohistochemistry in brain sections. Human ileum and colon specimens, rat stomach, duodenum, jejunum, ileum and colon specimens, with and without colchicine treatment were prepared for immunohistochemistry. Immunoelectron microscopic double staining of TPH and serotonin/chromogranin A and immunofluorescence double staining of TPH and serotonin were performed to identify the cell types. Epithelial enterochromaffin (EC) cells, mast cells in the lamina propria and submucosa, and varicose fibers in the submucosa and muscle layer showed positive immunoreactivity in all segments examined from human and normal rat specimens. In colchicine-treated rat specimens, nerve cell bodies in the myenteric plexus were stained. Because the antibody does not cross react with tyrosine hydroxylase as defined in Western blotting or brain sections, these positive structures may contain TPH. The present results show evidence that EC cells, mast cells, and nerve cell bodies and fibers in the gastrointestinal tracts of both the human and the rat contain TPH and therefore may have the ability to synthesize serotonin from tryptophan.

Transient expression of [D-Ala2] deltorphin I-like immunoreactivity in prenatal rat small intestine.

We studied the distribution of immunoreactive elements for [D-Ala2] deltorphin I (DADTI), a delta-opioid receptor ligand, in fetal and postnatal rat small intestine. DADTI-like immunoreactive cells were detected transiently on embryonic Days 20 and 21. Electron microscopic examination revealed that positive staining occurred in mucous epithelial cells, either mature goblet cells or undifferentiated cells containing only a few mucous granules. Positive immunoreaction products in mature goblet cells were confined in their apical cytoplasm to the luminal parts of mucous granule aggregates. The result suggests that a DADTI-like molecule(s) is synthesized in rat intestinal goblet cells and is secreted in a diacrine fashion into the intestinal lumen at a late fetal period. The molecule(s) thus secreted may be important for the intestine of rats just before birth, because DADTI-like immunopositive goblet cells are no longer seen at any postnatal period.

Effects of endothelial impairment by saponin on the responses to vasodilators and nitrergic nerve stimulation in isolated canine corpus cavernosum.

1. Responsiveness to EDRF-releasing substances and inhibitory nerve stimulation of canine isolated penile corpus cavernosum with and without saponin treatment were investigated. 2. Histological studies demonstrated that saponin did not detach endothelial cells from underlying tissues, but induced degenerative changes in the endothelial cells selectively. 3. In the cavernous strips contracted with phenylephrine, addition of acetylcholine, sodium nitroprusside, ATP and Ca2+ ionophore A23187 induced relaxations, but substance P and bradykinin did not change the muscle tone. 4. Acetylcholine-induced relaxation was significantly attenuated but not abolished by NG-nitro-L-arginine (L-NOARG). L-arginine restored the response inhibited by L-NOARG. The L-NOARG resistant relaxation was not influenced by 1H[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) but was suppressed in the strips contracted with K+. Treatment with saponin abolished the relaxation elicited by acetylcholine and A23187 but did not influence the response to nitroprusside and ATP. The ATP-induced relaxation was attenuated by aminophylline. 5. Transmural electrical stimulation at 2-20 Hz produced endothelium-independent relaxations which were abolished by tetrodotoxin and L-NOARG but unaffected by treatment with saponin. In saponin-treated cavernous strips, the neurogenic relaxation was not affected by acetylcholine, physostigmine, atropine and vasoactive intestinal peptide (VIP) but was abolished by ODQ. 6. It is concluded that acetylcholine-induced relaxations are endothelium-dependent and mediated partly by NO and also by other substances from the endothelium. The endothelium-independent relaxation to ATP is likely to be mediated by P1 purinoceptors. The function of nitrergic nerve does not seem to be prejunctionally modulated by acetylcholine and VIP.

Nitric oxide formation in the dog sphincter of Oddi from nitric oxide donors as measured with in vivo micro-dialysis.

BACKGROUND: Nitric oxide (NO) is known to play an important role in neurally mediated relaxation of the sphincter of Oddi. AIM: We investigated whether NO donors, such as nitroglycerin or zwitterionic polyamine/NO, applied into the common bile duct or intravenously, may induce the relaxation of the sphincter of Oddi by producing NO in the anaesthetized dog. METHODS: NO production in the sphincter of Oddi was measured by detecting NO oxidation products (NO2- and NO3-) using micro-dialysis methods. RESULTS: Zwitterionic polyamine/NO and nitroglycerin applied into the common bile duct induced a marked increase in NO2- but not NO3-, in the sphincter of Oddi. Intravenous infusion of zwitterionic polyamine/NO and nitroglycerin induced little or no increase in NO2- formation. Nitroglycerin infused into either the common bile duct or intravenously administered produced relaxation of the sphincter of Oddi, but zwitterionic polyamine/NO had no effect on the sphincter of Oddi in spite of the increase in NO2- levels. CONCLUSIONS: Locally or systemically applied NO donors induce relaxation of the sphincter of Oddi by producing NO, although their mode of action differs in different analogues.

Immunocytochemical localization of serotonin and serotonin transporter (SET) in taste buds of rat.

We used an immunocytochemical approach to study the localization of serotonin and its termination system, serotonin transporter (SET), in the taste buds of rats using specific antibodies against serotonin and SET. Under confocal laser scanning microscopy, both serotonin and SET immunoreactivity were detected in the taste buds of rat vallate papillae. Serotonin immunoreactivity was seen in the spindle-shaped cells with apical processes that seemed to be light (Type II) taste cells. SET-immunoreactivity was mainly localized in the periphery or interfaces between the taste cells. Double staining studies revealed that all serotonin-containing taste cells were immunoreactive for SET, while a subclass of SET-positive cells showed serotonin immunoreactivity. These data support the hypothesis that serotonin plays a transmitter role in taste receptor cells and suggest that the serotonin-induced sensation of taste is terminated by serotonin uptake through serotonin transporter.

Immunocytochemical localization of a neuron-specific thrombospondin-1-like protein, NELL2: light and electron microscopic studies in the rat brain.

We have studied the cellular and intracellular localization of NELL2, a neural thrombospondin-1-like protein. NELL2 protein was detected as doublet bands of 140 and 90 kDa with the use of the specific antibodies raised against the C-terminal region of NELL2 and was recognized only in the brain but not in the peripheral tissues. Within the brain, NELL2 was abundantly present in the hippocampus and cerebral cortex, found moderately in the olfactory bulb and hypothalamus, and at a low level in the thalamus, cerebellum, and medulla. Immunocytochemically, NELL2 was seen only in neurons but not in glial cells or in the white matter. NELL2-immunoreactive cells were distributed throughout the brain with the highest density in the hippocampus and cerebral cortex. NELL2 was mainly found in the cell bodies of neurons and the immunoreactivity was often seen as dots in the perikarya. The distribution of NELL2 immunoreactivity did not completely correspond to that of any subtypes of protein kinase C (PKC). Under electron microscopy, NELL2 protein was associated with the endoplasmic reticulum (ER), especially with rough ER. NELL2 immunoreactivity was found in the restricted parts of the ER and found commonly inside the ER. These results suggest that NELL2 protein is synthesized by neurons and may be secreted from the neurons involved in certain neuronal functions.

Localization of human placental glucose transporter 1 during pregnancy. An immunohistochemical study.

To elucidate the potential roles of glucose transporter 1 (GLUT1) in human placenta during pregnancy, we examined the localization of GLUT1 in human placenta at various stages by immunohistochemistry with an anti-GLUT1 antibody by use of both light and electron microscopy. Specific staining for GLUT1 was localized on the apical brush border and along the basal plasma membrane of the syncytiotrophoblasts. The staining at the apical side was more intense than that at the basal side during the early stages of gestation. In later gestational stages, however, the staining pattern at the apical side became blurred and the staining intensity at the basal side increased. The cytotrophoblasts, seen embedded in the basal part of the syncytiotrophoblasts, seemed to show immunoreactivity for GLUT1 along the plasma membranes at the light-microscopic level. However, immuno-electron microscopic analysis with either pre- or post-embedding methods revealed that specific staining for GLUT1 was hardly observed on the cytotrophoblasts, but the cytotrophoblasts were often surrounded by immunoreactive processes of syncytiotrophoblasts. The blood capillaries and erythrocytes in the stroma of placental villi were always immunoreactive for GLUT1 throughout pregnancy. These findings suggest that GLUT1 may play a vital role in human pregnancy.

Effect of VIP and PACAP on vascular and luminal release of serotonin from isolated perfused rat duodenum.

Effects of CCK, VIP, PACAP38, and PACAP27 on the release of 5HT into the intestinal lumen and into the portal circulation were examined in in vivo experiments of isolated rat duodenum perfused vascularly and luminally. VIP, PACAP 38 and 27 reduced the release of 5HT into the lumen but did not affect the vascular release of 5HT. These effects were not affected by the presence of atropine, hexamethonium, or TTX, suggesting that VIP, PACAP 38 and 27 exert a direct inhibitory effect on the luminal release of 5HT from the EC cells. Nitric oxide synthase inhibitor, NG-nitro-L-arginine, antagonized the inhibitory effects of VIP, PACAP 38 and 27, suggesting that nitric oxide seems to be essential to exert the inhibitory action of VIP and PACAPs on the release of 5HT into the intestinal lumen from the EC cells.

Peptidergic regulation of gastrointestinal motility in rodents.

Peptides involved in the endocrine and enteric nervous systems as well as in the central nervous system exert concerted action on gastrointestinal motility. Mechanical and chemical stimuli which induce peptide release from the epithelial endocrine cells are the earliest step in the initiation of peristaltic activities. Gut peptides exert hormonal effects, but peptide-containing stimulatory (Ach/substance P/tachykinin) and inhibitory (VIP/PACAP/NO) neurons are also involved in the induction of ascending contraction and descending relaxation, respectively. The dorsal vagal complex (DVC), located in the medulla of the brainstem, constitutes the basic neural circuitry of vago-vagal reflex control of gastrointestinal motility. Several gut peptides act on the DVC to modify vagal cholinergic reflexes directly (PYY and PP) or indirectly via afferent fibers in the periphery (CCK and GLP-1). The DVC is also a primary site of action of many neuropeptides (such as TRH and NPY) in mediating gastrointestinal motor activities. The identification over the last few years of a number of neuropeptide systems has greatly changed the field of feeding and body weight regulation. By exploring the brain and gut systems that employ recently identified peptidergic molecules, it will be possible to elaborate on the central and peripheral pathways involved in the regulation of gastrointestinal motility.

Emerging functions of neuropeptide Y Y(2) receptors in the brain.

The Y(2) receptor is the predominant neuropeptide Y (NPY) receptor subtype in the brain. Y(2) receptor mRNA is discretely distributed in the brain, including specific subregions of the hippocampus and the hypothalamus, and is largely consistent with the distribution of Y(2) receptor protein demonstrated by radioligand-binding methods. Y(2) receptor-mediated effects have been reported principally based on the observations using the C-terminal fragments of NPY. Recent studies indicate an involvement of the receptor in food intake, gastrointestinal motility, cardiovascular regulation, and neuronal excitability. Very recently, Y(2) receptor selective antagonist has been developed and Y(2) receptor-deficient animals have been created. These new pharmacological tools will help to clarify the roles of this receptor in brain functions.

Change in subcellular localization of gastrin-like immunoreactivity in epithelial cells of rat duodenum induced by carbachol.

The present study provides morphological evidence to support the contention of exocrine secretion from duodenal gastrin-containing cells. The isolated vascularly perfused duodenal preparation with or without carbachol stimulation was used. At the end of perfusion, tissue was fixed and prepared for electron microscopic examination. For immunoelectron microscopic study for gastrin, postembedding immunogold reaction combined with preembedding DAB staining was used. In saline-treated controls, DAB reaction was restricted to the basal cytoplasm and immunogold labeling was concentrated over electron-dense cores of secretory granules packed at the basal cytoplasm. However, in carbachol-stimulated animals, immunogold labeling as well as DAB reactions were accumulated at the apical portion of the cytoplasm, suggesting that a high concentration of gastrin was involved in the apical cytoplasm. In carbachol-stimulated cells, aggregation of small vesicles was observed beneath the microvilli, and most of these vesicles had no cores but were similar in size to the basal secretory granules. Immunogold particles were diffusely scattered at the cytoplasm outside these vesicles. These findings suggest that the gastrin-like immunoreactivity was pooled at the matrix of apical cytoplasm in carbachol-stimulated cells, which might be derived from the secretory granules migrated from the basal cytoplasm into apical portion of the cells. In conclusion, the present study demonstrated the change in subcellular localization of gastrin-like immunoreactivity in intestinal gastrin cells after stimulation with carbachol. Aggregation of immunoreactivity at the apical portion of the cells suggests that gastrin may be released into the intestinal lumen.

Effect of carbachol on the release of peptide YY from isolated vascularly and luminally perfused rat ileum.

Possible cholinergic control on the release of PYY from intestine into the lumen or blood vessel was studied by radioimmunoassay in the isolated perfused rat ileum. The basal release of PYY into the lumen was 43.1 +/- 8.9 pg/min, which was comparable with that into the vasculature (35.2 +/- 2.6 pg/min). The administration of 1 microM carbachol into the vascular perfusate resulted in a more than 40-fold increase of the luminal release but only a twofold increase of the vascular release. Carbachol-induced release of PYY into both lumen and vasculature was completely blocked by atropine, but not by hexamethonium. Tetrodotoxin abolished carbachol-induced release of PYY into lumen and vasculature. These data suggest that the ileal PYY release, into either lumen or vasculature, is under the control of postganglionic cholinergic neurons via muscarinic receptors.