Tissue distribution of hepatocyte growth factor receptor and its exclusive down-regulation in a regenerating organ after injury.

Using 125I-labeled hepatocyte growth factor (HGF) as a ligand, we examined the tissue distribution of the HGF receptor in adult rats. Specific binding of 125I-HGF was detected in the plasma membranes of liver, spleen, kidney, lung, adrenal gland, pituitary, and thyroid. Scatchard analysis of HGF binding in liver, spleen, kidney, lung, and adrenal gland revealed the presence of a single class of high affinity receptor with a dissociation constant (Kd) of 20-30 pM. The maximum number of binding sites (Bmax) was determined to be 400-3,000 sites per ng of plasma membrane protein, the highest number being in the liver. Such a wide distribution of a high affinity HGF receptor indicates that HGF may be a multifunctional growth factor, targeting to a variety of organs, and not restricted to liver. After 70% partial hepatectomy, specific binding of 125I-HGF to membranes of the residual liver rapidly decreased, but there was no change in the kidney, lung, and spleen. On the other hand, after unilateral nephrectomy rapid down-regulation of the HGF receptor was clearly evident in the remaining kidney, but not in other organs including the liver. These findings suggest the presence of control mechanisms governing HGF receptor function only in a regenerating organ after injury.     

Effect of dehydration on thirst and drinking during immersion in men.

The mechanism for reduced voluntary water intake during water immersion was studied in eight men (19-25 yr of age) immersed to the neck while sitting for 3 h at 34.5 degrees C or in air at 28 degrees C when euhydrated (Eu-H2O and Eu-air, respectively) and hypohydrated (Hypo-H2O and Hypo-air) by 3.6% body weight loss. Thirst sensations (degree of thirst, mouth dryness and taste, drinking desirability, and stomach fullness) were similar at the beginning of Hypo-air and Hypo-H2O test periods. Initial drinking of tap water (15 degrees C) was 216 +/- 30 ml/7 min (P less than 0.05) with Hypo-air, decreased to 108 +/- 28 ml/7 min (P less than 0.05) with Hypo-H2O, and was 10-50 ml/10-30 min thereafter. Intake was less than 10 ml/10-30 min in Eu-air, and there was no drinking in Eu-H2O. Within the first 10 min of immersion, compared with Hypo-air findings, the significant reduction in drinking in the Hypo-H2O experiment was associated with unchanged plasma Na+, plasma osmolality, heart rates, and mean arterial pressures; the different responses were increased cardiac output, plasma volume, and atrial natriuretic peptides and decreased plasma renin activity and arginine vasopressin. Thus the extracellular pathway, as opposed to the osmotic pathway, appears to be the major mechanism for immersion-induced suppression of drinking.     

Enzymes of purine catabolism in soybean plants

Remarkable formation and utilization of allantoin is observedin soybean (Glycine max variety A62-1). To study this, variousenzymes involved in purine catabolism (i.e., xanthine oxidase,uricase and allantoinase) were measured in different regionsof soybean plants during development. Uricase, which catalyzesthe direct formation of allantoin from uric acid, was studiedin detail. The activities of these three enzymes were highest in the rootnodules, indicating that the nodules are the major site of allantoinmetabolism. Radicles only showed appreciable activity about80 hr after the seeds were planted. Allantoinase activity wasdetected in all regions tested, showing that allantoin translocatedfrom the nodules can be metabolized in the roots, stem and leaves.In the nodules, xanthine oxidase was localized in the nuclearfraction, while uricase was mainly restricted to the mitochondrialfraction and allantoinase to the soluble fraction. Uricase was partially purified from the nodules and radicles,respectively. The pH optimum of enzyme from the nodules was9.5, whereas that of enzyme from the radicles was 7.0. The enzymefrom the nodules did not require a cofactor, while that fromthe radicles showed an absolute requirement for a cofactor,which was a low molecular substance easily separable from theapoprotein. Thus, the uricase in nodules differs in chemicalproperties from that in the host plant. The results are discussedin relation to change in the allantoin level in soybean tissues. (Received November 1, 1974; )     

The promoter of rbcS in a C3 plant (rice) directs organ-specific,light-dependent expression in a C4 plant (maize), but does not confer bundle sheath cell-specific expression

The small subunit of ribulose-bisphosphate carboxylase (Rubisco), encoded by rbcS, is essential for photosynthesis in both C3 and C4 plants, even though the cell specificity of rbcS expression is different between C3 and C4 plants. The C3 rbcS is specifically expressed in mesophyll cells, while the C4 rbcS is expressed in bundle sheath cells, and not mesophyll cells. Two chimeric genes were constructed consisting of the structural gene encoding -glucuronidase (GUS) controlled by the two promoters from maize (C4) and rice (C3) rbcS genes. These constructs were introduced into a C4 plant, maize. Both chimeric genes were specifically expressed in photosynthetic organs, such as leaf blade, but not in non-photosynthetic organs. The expressions of the genes were also regulated by light. However, the rice promoter drove the GUS activity mainly in mesophyll cells and relatively low in bundle sheath cells, while the maize rbcS promoter induced the activity specifically in bundle sheath cells. These results suggest that the rice promoter contains some cis-acting elements responding in an organ-pecific and light-inducible regulation manner in maize but does not contain element(s) for bundle sheath cell-specific expression, while the maize promoter does contain such element(s). Based on this result, we discuss the similarities and differences between the rice (C3) and maize (C4) rbcS promoter in terms of the evolution of the C4 photosynthetic gene.     

A novel strategy to design highly specific PCR primers based on the stability and uniqueness of 3'-end subsequences

MOTIVATION: In contrast with conventional PCR using a pair of specific primers, some applications utilize a single unique primer in combination with a common primer, thereby relying solely on the former for specificity. These applications include rapid amplification of cDNA ends (RACE), adaptor-tagged competitive PCR (ATAC-PCR), PCR-mediated genome walking and so forth. Since the primers designed by conventional methods often fail to work in these applications, an improved strategy is required, particularly, for a large-scale analysis. RESULTS: Based on the structure of 'off-target' products in the ATAC-PCR, we reasoned that the practical determinant of the specificity of primers may not be the uniqueness of entire sequence but that of the shortest 3'-end subsequence that exceeds a threshold of duplex stability. We termed such a subsequence as a 'specificity-determining subsequence' (SDSS) and developed a simple algorithm to predict the performance of the primer: the algorithm identifies the SDSS of each primer and examines its uniqueness in the target genome. The primers designed using this algorithm worked much better than those designed using a conventional method in both ATAC-PCR and 5'-RACE experiments. Thus, the algorithm will be generally useful for improving various PCR-based applications.     

Rat NAD+-dependent 3alpha-hydroxysteroid dehydrogenase (AKR1C17): a member of the aldo-keto reductase family highly expressed in kidney cytosol

Mammalian 3α-hydroxysteroid dehydrogenases (3α-HSDs) have been divided into two types: Cytosolic NADP(H)-dependent 3α-HSDs belonging to the aldo-keto reductase family, and mitochondrial and microsomal NAD⁺-dependent 3α-HSDs belonging to the short-chain dehydrogenase/reductase family. In this study, we characterized a rat aldo-keto reductase (AKR1C17), whose functions are unknown. The recombinant AKR1C17 efficiently oxidized 3α-hydroxysteroids and bile acids using NAD⁺ as the preferred coenzyme at an optimal pH of 7.4-9.5, and was inhibited by ketamine and organic anions. The mRNA for AKR1C17 was detected specifically in rat kidney, where the enzyme was more highly expressed as a cytosolic protein than NADP(H)-dependent 3α-HSD (AKR1C9). Thus, AKR1C17 represents a novel NAD⁺-dependent type of cytosolic 3α-HSD with unique inhibitor sensitivity and tissue distribution. In addition, the replacement of Gln270 and Glu276 of AKR1C17 with the corresponding residues of NADP(H)-dependent 3α-HSD resulted in a switch in favor of NADP⁺ specificity, suggesting their key roles in coenzyme specificity.     

Characterization of na(+) and ca(2+) channels in zebrafish dorsal root ganglion neurons

Background

Dorsal root ganglia (DRG) somata from rodents have provided an excellent model system to study ion channel properties and modulation using electrophysiological investigation. As in other vertebrates, zebrafish (Danio rerio) DRG are organized segmentally and possess peripheral axons that bifurcate into each body segment. However, the electrical properties of zebrafish DRG sensory neurons, as compared with their mammalian counterparts, are relatively unexplored because a preparation suitable for electrophysiological studies has not been available.

Methodology/Principal Findings

We show enzymatically dissociated DRG neurons from juvenile zebrafish expressing Isl2b-promoter driven EGFP were easily identified with fluorescence microscopy and amenable to conventional whole-cell patch-clamp studies. Two kinetically distinct TTX-sensitive Na⁺ currents (rapidly- and slowly-inactivating) were discovered. Rapidly-inactivating I_Na were preferentially expressed in relatively large neurons, while slowly-inactivating I_Na was more prevalent in smaller DRG neurons. RT-PCR analysis suggests zscn1aa/ab, zscn8aa/ab, zscn4ab and zscn5Laa are possible candidates for these I_Na components. Voltage-gated Ca²⁺ currents (I_Ca) were primarily (87%) comprised of a high-voltage activated component arising from ω-conotoxin GVIA-sensitive Ca_V2.2 (N-type) Ca²⁺ channels. A few DRG neurons (8%) displayed a miniscule low-voltage-activated component. I_Ca in zebrafish DRG neurons were modulated by neurotransmitters via either voltage-dependent or -independent G-protein signaling pathway with large cell-to-cell response variability.

Conclusions/Significance

Our present results indicate that, as in higher vertebrates, zebrafish DRG neurons are heterogeneous being composed of functionally distinct subpopulations that may correlate with different sensory modalities. These findings provide the first comparison of zebrafish and rodent DRG neuron electrical properties and thus provide a basis for future studies.     

5-hydroxytryptamine synthesis in HL-1 cells and neonatal rat cardiocytes

Some reports showed that serotonergic system might have existed and that 5-hydroxytryptamine (5-HT) was detected in the hamster heart. The source of 5-HT in the heart, however, remains to be fully elucidated. So the present study was designed to define serotonergic system and to clarify which cell could produce 5-HT in the heart. As a result, 5-HT was detected in homogenates of HL-1 cardiomyocytes by high performance liquid chromatography with fluorescence detection, but not in those of neonatal rat non-cardiomyocytes (NMCs). And TPH and AADC mRNAs were expressed in HL-1 cardiomyocytes and neonatal rat cardiomyocytes (MCs), not in NMCs. mRNAs of 5-HT(2A) receptor were detected in both MCs and NMCs, and those of 5-HT(2B) receptor in NMCs. These findings definitively demonstrate that 5-HT is secreted from the myocytes of the heart and strongly implied that 5-HT might play a certain role in cardiac physiology.