Identification of two alpha-subunit species of GTP-binding proteins, Galpha15 and Galphaq, expressed in rat taste buds.

We cloned cDNAs for two G protein alpha-subunits belonging to the Galphaq family, each capable of activating PLCbeta, from rat tongue. One is a Galphaq in the narrow sense, and the other, termed rat Galpha15, is a rat counterpart of mouse Galpha15, sharing an amino acid sequence similarity of 94%. RT-PCR and Northern blot analysis demonstrated that rat Galpha15 and Galphaq were distinctly expressed in tongue epithelia containing taste buds. Immunostaining also showed that rat Galpha15, together with the Galphaq, was localized mainly in taste buds. These studies suggest the possibility that these two Galpha proteins function for taste signal transduction in sensory cells.

Shh and Ptc are associated with taste bud maintenance in the adult mouse.

In mammals, taste receptor cells are organized into taste buds on tongue. Taste buds are trophically maintained by taste neurons and under continuous renewal, even in adults. We found that the receptor for Sonic hedgehog (Shh), Patched1 (Ptc), was expressed around taste buds where cells were proliferating, and that Shh was expressed within basal cells of taste buds. Denervation caused the loss of Shh and Ptc expression before the degeneration of taste buds.

Addition of carbamazepine to long-term treatment with neuroleptics may induce neuroleptic malignant syndrome.

BACKGROUND: Carbamazepine is an anticonvulsant, but also has an anti-manic effect, and recently it has been increasingly used in combination with neuroleptics. Nevertheless, there have been very few reports on the involvement of carbamazepine in the occurrence of neuroleptic malignant syndrome (NMS). METHODS: A case of NMS occurring after addition of carbamazepine to long-term neuroleptic administration is described. RESULTS: The patient had been treated with neuroleptics for about 30 years, and NMS developed when carbamazepine (400 mg/day) was added. CONCLUSIONS: This case suggests that clinicians should consider the risk of NMS when carbamazepine is administered to patients undergoing long-term treatment with neuroleptics.

Multiple genes for G protein-coupled receptors and their expression in lingual epithelia.

Using the polymerase chain reaction (PCR), we identified a gene family including more than 60 members which encoded similar G protein-coupled seven-transmembrane receptors. Sequence analyses of six representatives out of the 60 PCR clones showed that they had significant structural similarity to olfactory and optic receptors. Their expression is restricted in the surface of lingual epithelia.

A water channel closely related to rat brain aquaporin 4 is expressed in acid- and pepsinogen-secretory cells of human stomach.

We isolated a cDNA clone encoding a water channel protein, aquaporin ( AQP), from human stomach. The encoded protein consisted of 323 amino acid residues, containing six putative transmembrane domains. The protein was designated human aquaporin 4 (hAQP4) because of its 94% sequence similarity to rat brain AQP4. Expression of hAQP4 cRNA in Xenopus oocytes resulted in a significant increase in osmotic water permeability, indicating that this protein functions as a water channel. Northern blot analysis demonstrated a strong signal of hAQP4 mRNA in brain, lung, and skeletal muscle as well as in stomach. Immunohistochemical experiments with human stomach tissues showed that hAQP4 as a protein is expressed mainly in cells located in the glandular portion of the fundic mucosa. These include chief cells which secrete pepsinogen and parietal cells which secrete hydrochloric acid. These results strongly indicate that hAQP4 is a principal factor involved in the osmotic regulation of pepsinogen and acid secretion in the stomach.

Identification of taste-tissue-specific cDNA clones from a subtraction cDNA library of rat circumvallate and foliate papillae.

To identify genes specifically expressed in taste tissues, we constructed a subtraction cDNA library of epithelium of rat circumvallate and foliate papillae and carried out differential screening of this library. Dot blot analysis showed 46 out of 88 clones obtained by this method to be expressed in the epithelium of papillae. The cDNA inserts in these clones were sequenced and analyzed for similarity to entries the GenBank database. About 54.3% of the clones were known sequences, including the sequences of ebnerin, cytokeratin 18, and Na+,K+-ATPase, that were shown by in situ hybridization to be expressed in the circumvallate papillae. About 41.3% of the papillae-specific clones had no significant similarity to known sequences and are candidates for novel taste bud-specific marker genes.

Molecular cloning and taste bud-specific expression of a novel cyclic nucleotide-gated channel.

Cyclic nucleotide-gated (CNG) channels serve as downstream targets of signaling pathways in vertebrate photoreceptor cells and olfactory sensory neurons. For taste signaling as well, a great deal of information is available predicting the presence of a CNG channel, but no report has been presented on its molecular entity. Here we report on molecular cloning and functional expression of a taste bud-specific CNG channel tentatively named CNGgust. Reverse transcriptase polymerase chain reaction (RT-PCR) primers were synthesized according to some amino acid sequences generally conserved in many CNG channels. RT-PCR was conducted using rat circumvallate papillary mRNA-derived cDNA as a template to obtain positive clones. A corresponding genomic DNA clone was then obtained by screening from a genomic DNA library. Dissecting the entire structure of this gene, we found that the encoding protein had an amino acid sequence similarity of 80% to each of retina and olfactory CNG channels. It was also found by immunostaining with a specific antibody that this gustatory CNG channel (CNGgust) is localized in the tongue and also expressed specifically on the pore side of each taste bud in the circumvallate papillae. Electrophysiological experiments demonstrated that CNGgust resided in a functional state. All these data suggest that CNGgust may be involved in taste signal transduction in sensory cells.

A gustatory cyclic nucleotide-gated channels CNGgust, is expressed in the retina.

Cyclic nucleotide-gated (CNG) channels are essential proteins that contribute to the intracellular signal transduction of the senses of sight and smell. Recently, we found a novel CNG channel (CNGgust) in rat taste buds, and demonstrated its possible involvement in taste signal transduction. In the present study, we used RT-PCR and immunostaining to prove that this gustatory CNG channel is expressed in the outer segments of rat cone photoreceptor cells. The study strongly suggests that the senses of taste and sight share, at least in part, a common signal transduction pathway.

Antibiotic no. 6016, a polyether antibiotic.

A new polyether antibiotic, No. 6016, was isolated from the culture of Streptomyces albus strain No. 6016. The antibiotic was obtained as colorless prisms having a molecular formula of C46H77O16Na, m.p. 192 approximately 195 degrees C (dec.), and has only end absorption in ultraviolet region. The infrared and NMR spectra of the antibiotic suggest the presence of carbonyl and methoxyl groups. The antibiotic No. 6016 exhibits activity against Gram-positive bacteria including mycobacteria and is effective in the treatment of coccidiosis of fowl.

Ferensimycins A and B. Two polyether antibiotics. Taxonomy, fermentation, isolation, characterization and structural studies.

Ferensimycin A* (I), C34H59O10Na, mp 133 approximately 135 degrees C, and ferensimycin B** (II), C35H61O10Na, mp 143 approximately 145 degrees C, were isolated as their sodium salts from the fermentation broth of Streptomyces sp. No. 5057, a strain similar to Streptomyces myxogenes Shomura et al. The physicochemical data of I and II showed that they are both closely related congeners of lysocellin (III). Ferensimycins A and B exhibit activity against Gram-positive bacteria and are effective in the treatment of coccidiosis of fowl.

[A detection method for recombinant DNA from genetically modified maize CBH351].

A method using polymerase chain reaction (PCR) was designed for the detection of genetically modified maize CBH351, which has not authorized as safe for use in foods and feeds in Japan yet. We analyzed a recombinant DNA (r-DNA) sequence introduced into CBH351 maize and designed specific primer pairs to amplify a segment including part of the r-DNA. The PCR products obtained by using the designed primer pairs are specific for CBH351 and should prevent false positive results caused by other maizes and other main cereal crops. The r-DNA introduced into CBH351 could be detected from maize samples containing 0.05-0.1% CBH351 maize. This sensitivity is theoretically equivalent to a level of several genome copies and so this technique is a very efficient means to detect CBH351 maize.

A multiplex PCR method of detecting recombinant DNAs from five lines of genetically modified maize.

Seven lines of genetically modified (GM) maize have been authorized in Japan as foods and feeds imported from the USA. We improved a multiplex PCR method described in the previous report in order to distinguish the five lines of GM maize. Genomic DNA was extracted from GM maize with a silica spin column kit, which could reduce experimental time and improve safety in the laboratory and potentially in the environment. We sequenced recombinant DNA (r-DNA) introduced into GM maize, and re-designed new primer pairs to increase the specificity of PCR to distinguish five lines of GM maize by multiplex PCR. A primer pair for the maize intrinsic zein gene (Ze1) was also designed to confirm the presence of amplifiable maize DNA. The lengths of PCR products using these six primer pairs were different. The Ze1 and the r-DNAs from the five lines of GM maize were qualitatively detected in one tube. The specific PCR bands were distinguishable from each other on the basis of the expected length. The r-DNA could be detected from maize samples containing 0.5% of each of the five lines of GM maize. The sensitivity would be acceptable to secure the verification of non-GMO materials and to monitor the reliability of the labeling system.

Development of a method to quantitatively monitor the effect of inhibition of nitric oxide synthase on tumour vascular activity using dynamic contrast-enhanced computed tomography.

This study was undertaken to develop a method to quantitatively monitor the effect of inhibition of nitric oxide synthase (NOS) on tumour vascular activity using dynamic contrast-enhanced computed tomography (DCE-CT). The DCE-CT studies were performed in 13 anaesthetized rats bearing tumours. To investigate the effect of NOS inhibition, N-nitro-L-arginine (L-NNA) was intravenously administered in eight rats, while only the vehicle was administered in five rats. The contrast enhancement (CE) images were generated by subtracting the CT images before and after the administration of contrast agent. The tumour blood volume (TBV) images were also generated. The CE significantly decreased after L-NNA administration, while there were no significant changes when only the vehicle was administered. There was a good correlation between CE and TBV, suggesting that CE mainly reflects TBV. In conclusion, the present method appears to be useful for monitoring the effect of NOS inhibition on tumour vascular activity.