Close linkage of MEN2A with RBP3 locus in Japanese kindreds

Summary The gene responsible for multiple endocrine neoplasia type 2A (MEN2A) has recently been assigned to the pericentromeric region of chromsome 10 in European Caucasian kindreds by linkage analysis using a DNA marker, interstitial retinol-binding protein 3 (RBP3). We have found tight linkage between the MEN2A and RBP3 loci in Japanese MEN2A kindreds. The maximum lod score is 5.19 at a recombination fraction of 0.00. This result suggests that mutation of a certain gene close to RBP3 is responsible for MEN2A irrespective of ethnic backgrounds.     

Genotypes of alcohol dehydrogenase and aldehyde dehydrogenase loci in Japanese alcohol flushers and nonflushers

Summary A much higher incidence of alcohol flushing among Orientals in comparison to Caucasians, i.e., >50% vs 5%–10%, has been attributed to racial differences in alcohol-metabolizing enzymes. A large majority of Orientals are atypical in alcohol dehydrogenase-2 locus (ADH ₂ ), and their livers exhibit significantly higher ADH activity than the livers of most Caucasians. Approximately 50% of Orientals lack the mitochondrial aldehyde dehydrogenase (ALDH₂) activity, and elimination of acetaldehyde might be disturbed. We determined by means of hybridization of genomic DNA samples with allele specific oligonucleotide probes, genotypes of the ADH ₂ and ALDH ₂ loci in Japanese alcohol flushers and nonflushers. We found that all individuals with homozygous atypical ALDH ₂ ² /ALDH ₂ ² and most of those with heterozygous atypical ALDH ₁ ² /ALDH ₂ ² were alcohol flushers, while all subjects with homozygous usual ALDH ₁ ² /ALDH ₁ ² were nonflushers. Frequency of the atypical ADH ₂ ² was found to be higher in alcohol flushers than in nonflushers, but the statistical significance was not established in the sample size examined.     

Antimetastatic effect of endogenous tumor necrosis factor induced by the treatment of recombinant interferon γ followed by an analogue (GLA-60) to synthetic lipid A subunit

Summary We have investigated the effect of endogenous production of tumor necrosis factor (TNF) induced by the combination of recombinant interferon (rIFN) as a primer followed by GLA-60 as a trigger (rIFN/GLA-60) on murine lung metastases caused by B16-BL6 melanoma. In order to examine the therapeutic effect of endogenous TNF on tumor metastasis, the ability of multiple administrations of rIFN/GLA-60 to induce TNF production was also tested. The multiple administrations of rIFN/GLA-60 at intervals of 2 days were effective for the induction of endogenous TNF in mice but continuous multiple administrations of them for 2–4 days were not. In tumor-bearing mice, the production of endogenous TNF by rIFN/GLA-60 was less than that of normal mice, but treatment 3 days after the surgical excision of primary tumors showed the endogenous TNF production to be similar to that in normal mice. In the experimental lung metastasis model, intravenous administration of rIFN followed by intravenous or intranasal administration of GLA-60 showed potent inhibition of lung metastases of B16-BL6 melanoma, whereas the reverse sequence of administration (GLA-60/rIFN) or administration of a mixture of rIFN and GLA-60, which cannot induce the production of TNF, caused no inhibition of lung metastases. These results indicated that the regression of tumor metastases by rIFN/GLA-60 was mediated by the production of endogenous TNF in addition to the direct effects of both immunostimulants. Furthermore, the administration of rIFN and GLA-60 significantly inhibited the tumor metastases in spontaneous lung metastasis model. These results may provide a promising approach for the treatment of cancer metastasis as a result of its ability to induce endogenous TNF.     

Amino acid sequence of horseshoe crab, Tachypleus tridentatus, striated muscle troponin C

The amino acid sequence of troponin C obtained from horseshoe crab, Tachypleus tridentatus, striated muscle was determined by sequence analysis and alignments of chemically and enzymatically cleaved peptides. Troponin C is composed of 153 amino acid residues with a blocked N-terminus and contains no tryptophan or cysteine residue. The site I, one of the four Ca2+-binding sites, is considered to have lost its ability to bind Ca2+ owing to the replacements of certain amino acid residues.     

An ATP-driven Cl- pump in the brain

EDTA-treated microsomes prepared from rat brain mainly consisted of sealed membrane vesicles 200-500 nm in diameter and were rich in both Cl- -ATPase and Na+,K+-ATPase activities. Such Cl- -ATPase-rich membrane vesicles accumulated Cl- in an ATP-dependent and osmotically reactive manner in the presence of 1 nM ouabain. The Cl- uptake was maximally stimulated by ATP with a Km value of 1.5 mM; GTP, ITP, and UTP partially stimulated Cl- uptake, but CTP, beta, gamma-methylene ATP, ADP, and AMP did not. The ATP-dependent Cl- uptake was accelerated by an increase in the medium Cl- concentration with a Km value of 7.4 mM. Such stimulation of Cl- uptake by ATP was dependent on the pH of the medium, with an optimal pH of 7.4, and also on the temperature of the medium, with an optimal range of 37-42 degrees C. Ethacrynic acid dose dependently inhibited the ATP-dependent Cl- uptake with a concentration for half-maximal inhibition at 57 microM. N-ethylmaleimide (0.1 mM) completely inhibited and sodium vanadate (1 mM) partially inhibited the ATP-dependent Cl- uptake. The membrane vesicles did not accumulate H+ in the Cl- uptake assay medium. The ATP-dependent Cl- uptake profile agreed with that of Cl- -ATPase activity reported previously (Inagaki, C., Tanaka, T., Hara, M., and Ishiko, J. (1985) Biochem. Pharmacol. 34, 1705-1712), and this strongly supports the idea that Cl- -ATPase in the brain actively transports Cl-.     

Cloning and sequencing of the gene for Tetrahymena calcium-binding 25-kDa protein (TCBP-25)

With the intention of studying calcium-dependent ciliary reversal in Tetrahymena, we isolated a Tetrahymena calcium-binding protein of 10 kDa (TCBP-10) which was not calmodulin and reported its properties (Ohnishi, K., and Watanabe, Y. (1983) J. Biol. Chem. 258, 13978-13985). However, immunoblotting with an antiserum against TCBP-10 and sequencing of the cDNAs and partial genomic DNAs for this calcium-binding protein prove that this previously reported TCBP-10 is the degraded product of a 25-kDa calcium-binding protein. Thus, we correct the name of the protein from TCBP-10 to TCBP-25. From the analysis of the cDNA for TCBP-25, it is shown to be composed of 218 amino acid residues and its molecular weight is estimated to be 24,702. This protein is predicted to contain four EF-hand-type calcium binding domains and to be a member of the calmodulin family. Little sequence homology with other proteins was shown by a computer search, except in the EF-hand regions. The special feature of TCBP-25 is that the distance between calcium-binding domains II and III is extraordinarily long for a calmodulin family protein having four calcium-binding domains. The genomic DNA for TCBP-25 contains two introns situated at short distances before calcium-binding domains I and III, implying gene duplication in genealogy.     

Reversion of the apical hook of pea in the dark and its promotion by a red light pulse.

At the developmental stage at which the apical hook passed the 3rd and 4th nodes, dark-grown seedlings of pea ( Pisum sativum L. cv. Progress No.9) opened the hook upright and then formed a new hook above the node nearly in the opposite direction to the previous one. In cv. Alaska, in contrast, many (about 84%) seedlings closed the hook in the original direction after they partially (up to about 110°) opened it at the 3rd node, thus doing a wagging movement, while a small percentage (about 16%) of the seedlings reversed the hook direction. Exposure to red light of cv. Alaska seedlings for 10 min increased the percentage of the hook reversion up to 71% or more. The hook reversion was never observed except when the hook part passed the nodes, suggesting the involvement of the nodes in the phenomenon.     

Enhancement of the Division of Equisetum arvense Protoplasts in Culture by Activated Charcoal and Their Further Development

Protoplasts were isolated from subcultured gametophytes of Equisetumarvense by treatment with Driselase and then cultured in vitro.Addition of activated charcoal (AC) to the culture medium enhancedthe rate of cell division, as well as the survival of both protoplastsand regenerated protoplasts. However, subsequent division ofcells was not observed after one or two cycles of replicationin cultures supplemented with AC. When regenerated protoplastswere transferred to fresh medium without AC 3 to 5 weeks afterthe first plating, the transferred cells formed rhizoids anddeveloped into small, young gametophytes without the prior formationof cell clusters or calluses. Furthermore, sprophytic shootsdifferentiated from the protoplast-derived gametophytes whenthey were cultured on medium supplemented with 6-benzylaminopurine(BA). (Received April 5, 1990; Accepted July 30, 1990)     

Metabolism of Choline Chloride and Its Analogs in Wheat Seedlings

The incorporation rate of choline chloride and allylcholinebromide into wheat protoplasts were rapid compared with theincorporation rate of benzylcholine bromide. Choline chloridewas metabolized via two pathways: choline betaine and choline phosphorylcholine phos-phatidylcholine. Allylcholine bromidewas metabolized via only one pathway: allylcholine phosphorylallylcholine phosphatidylallylcholine, and benzylcholine bromide was notmetabolized at all. These results suggest that the stimulationof photosynthesis (Hyeon et al. 1988) by these compounds iscaused directly by these choline analogs and not by their metabolites. (Received June 29, 1989; Accepted October 20, 1989)