Recent studies have shown that a number of novel G-protein coupled receptor (GPCR) ligands localize in brain tissue and perform a range of physiological functions, some of which have been studied and clarified, and others about which less is known. Here, we wish to describe the distribution and localization of GPCR ligands identified thus far and to examine their involvement in neuronal networks, particularly those networks related to feeding regulation. The review analyzes published reports of morphological and physiological data looking mainly at the functional significance of feeding-regulation factors, such as those described by our research group and others, and neuronal interactions among these GPCR ligands in the hypothalamus. Cross-talk among several GPCR ligand-containing neuron types in the hypothalamus plays a role in determining feeding states. We introduce structural and functional characteristics of novel GPCR ligands and summarize the known interactions between several GPCR ligand-containing neuron types and leptin-targeting neurons in the hypothalamus. Finally, we present a new scheme summarizing neuronal networks with regard to feeding regulation in the hypothalamus. Research in this area will play an important role in clarifying neurologically-based causes for appetite dysfunction and establishing therapies for people suffering from such conditions.

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Aquaporins (AQPs) are water channel proteins of cellular membranes serving in the permeation of water across the membrane. AQP families are found virtually in all types of life ranging from bacteria to plant and animal cells. In mammals, at least 13 isoforms of AQPs have been identified. They are classified into three subtypes: classical aquaporins, aquaglyceroporins, and superaquaporins. These AQPs are differentially expressed in a wide variety of cells and tissues in the body, and play important roles in water metabolism. In the kidney, at least 6 isoforms of AQPs, namely AQP1, AQP2, AQP3, AQP4, AQP6, and AQP7, are reported to be expressed. Water transfer occurs mainly in the proximal tubules and collecting ducts in the kidney. In the proximal tubules, AQP1 and AQP7 are expressed, among which AQP1 plays a major role in water reabsorption. In the collecting ducts, AQP2, AQP3, AQP4, and AQP6 are expressed. AQP3 and AQP4 are localized at the basolateral membrane. AQP2 is stored in the cytoplasmic vesicles and is translocated to the apical plasma membrane in response to antidiuretic hormone. Mutations of AQP2 lead to either loss of channel function or mistrafficking and result in nephrogenic diabetes insipidus, the inability to concentrate urine.

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Advanced biomolecular technologies have begun to provide beneficial means to detect a variety of cellular molecules for tumor diagnosis, as well as predictive and prognostic factors of cancers. This new understanding of the molecular basis of cancers has led to the development of molecular targeted approaches to cancer prevention and treatment. Molecules involved in the growth factor signaling system have become favored targets for molecular targeted therapies in translational research and clinical development. The current molecular targeting agents are classified into humanized or camera monoclonal antibodies and small-molecule tyrosine kinase inhibitors. Patients with HER2-overexpressing, metastatic breast cancer are beginning to benefit from the first commercially humanized monoclonal antibody (Herceptin), and the first small-molecule tyrosine kinase inhibitor (Gleevec/Glivec) has been introduced in Japan. Gleevec/Glivec has been demonstrated to have a long-term clinical benefit for most refractory patients with Philadelphia chromosome-positive chronic myelogenous leukemia as well as with KIT-expressing, unresectable and/or metastatic gastrointestinal stromal tumor. Accurate and reliable HER2 or KIT testing is performed to determine patient eligibility for Herceptin or Gleevec/Glivec therapy, respectively.
HER1 (EGFR) and HER2 are the most widely studied target molecules for therapeutic inhibitions. HER family targeting has been initiated as the basis of extensive and growing drug development programs in various companies. A key technology of targeted therapies is molecular diagnosis with target identification. Along with the progressive development of HER-targeted therapies, the need to standardize immunohistochemical diagnostic tests for each targeted therapy is ever more pressing. Although the standardization of immunohistochemistry using archival formalin-fixed specimens is a tremendous challenge that will require great effort and considerable research, the development of an ideal pharmDx test for target molecular identification will be achieved by the optimal combination of heat-induced antigen retrieval with calcium-chelating solutions, effective signal amplification, and automated staining approaches. The expression levels and phosphorylation/activation of various signaling molecules will be valuable in defining subpopulations of patients who may potentially respond to molecular targeted therapies.

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Silent pituitary somatotroph adenoma is an adenoma that expresses growth hormone (GH) and GH mRNA and has ultrastructural features common to somatotroph adenoma, without evidence of acromegaly. Forty-one silent somatotroph adenomas were reported in the literature, and their relatively common clinical symptoms include amenorrhea and/or galactorrhea. Random GH and insulin-like growth factor-1 levels are within normal in nearly half of the patients, whereas random PRL level is elevated in about 80% of patients. Generally, the GH immunostaining of silent somatotroph adenoma is weaker than that of classical somatotroph adenoma. Tissue culture studies indicate silent somatotroph adenoma has a very low basal secretory capacity of GH. Most of the silent somatotroph adenomas reported in the literature are sparsely granulated adenomas. The mechanism of silence may be attributed to unknown translational abnormalities of GH gene product, impaired secretion of GH, lack of biological activity of secreted GH, or it may represent an early stage of classical somatotroph adenoma. Meticulous endocrinological and histopathological examination may be helpful for the identification of silent somatotroph adenoma among patients with clinically non-functioning adenomas, especially female patients with amenorrhea and/or galactorrhea.

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To investigate the effects of low-dose X-irradiation on the expression of connexins (Cxs) in developing cerebral cortex, C57/BL6 pregnant mice were exposed to whole body irradiation of 0.1 Gy and 0.5 Gy, on embryonic day 14.5. Fetal telencephalons, at 1, 3, 12 and 24 hr after exposure, were studied immunohistochemically using anti-Cx43 and Cx26 antibodies, followed by quantitative image analyses. Cx43 expression was significantly reduced in all zones of developing cerebral cortex until 12 hr after exposure, and tended to recover to control level after 24 hr. Cx26 expression was also suppressed in the ventricular zone and cortical plate after irradiation, although it recovered much earlier. Our observations indicate that the expression of Cxs is affected by ionizing radiation during cortical histogenesis.

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To elucidate the molecular changes of motoneurons which innervate laryngeal muscles after transection of rat recurrent laryngeal nerve, we have devised a new method of DiI labeling combined with single-cell RT-PCR, by which nucleus ambiguus (NA) neurons projecting to the intrinsic laryngeal muscles were labeled retrogradely by injecting the fluorescent dye DiI into bilateral thyroarytenoid muscles; then either one of the recurrent laryngeal nerves was transected, and the fluorescence-bright single neurons were microdissected, followed by single-cell reverse transcription (RT)-PCR. Immunohistochemically, many DiI-labeled motoneurons in NA were immunoreactive to phosphorylated Stat3 in the nucleus on 1, 7, 10 and 14 days after axotomy. Thirty μm-thick frozen sections of brain stem were mounted onto membrane slide, and DiI-labeled single neurons in NA were microdissected under a fluorescence microscope. By real-time quantitative RT-PCR for single motoneurons, the expression levels of GAP-43 and nNOS genes were upregulated at 7 days and 1 day after axotomy, respectively. The expression levels of Stat3, Reg-2, and Bcl-2 genes were upregulated at 7 days, whereas that of Bax was downregulated at 1 and 7 days after axotomy as compared with control levels. With this novel method in which single-cell RT-PCR was combined with neurotracer DiI labeling we could demonstrate a part of the molecular cascades involving Stat3 in neurons projecting to laryngeal muscles after axotomy.

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Keratins (Ks) expressed in the absorptive cells of rabbit duodenal epithelium were studied immunohistochemically and immunoelectron microscopically. These keratins were classified into three groups based on their distribution: keratins forming an apex network just under the terminal web, those forming a Golgi-associated network surrounding the Golgi apparatus, and those forming an entire cytoplasmic network. The apex network was composed of K5/K18 keratin filaments. The Golgi-associated network at first consisted of K8/K14 keratin filaments alone. When the cell migrated out of the crypt to the mid-villus, actin and K7/K17 keratin filaments successively entered this network. The entire cytoplasmic network was detected at the villus base, and consisted of K7/K17 keratin filaments. Thereafter, K5/K18 keratin filaments entered this network during migration of the cell. The apex network may serve to maintain cell-cell contact, since its filaments are tightly anchored to desmosomes. The Golgi-associated network may be involved in the maintenance of the complex structure of the Golgi apparatus, since its filaments constitute a three-dimensional network around the organelle. The entire cytoplasmic network may play a major role in the maintenance of cell structure and the distribution of cell organelles.

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