New mutations fts-36, lts-33, and ftsW clustered in the mra region of the Escherichia coli chromosome induce thermosensitive cell growth and division.

Three new mutants of Escherichia coli showing thermosensitive cell growth and division were isolated, and the mutations were mapped to the mra region at 2 min on the E. coli chromosome map distal to leuA. Two mutations were mapped closely upstream of ftsI (also called pbpB), in a region of 600 bases; the fts-36 mutant showed thermosensitive growth and formed filamentous cells at 42 degrees C, whereas the lts-33 mutant lysed at 42 degrees C without forming filamentous cells. The mutation in the third new thermosensitive, filament-forming mutant, named ftsW, was mapped between murF and murG. By isolation of these three mutants, about 90% of the 17-kilobase region from fts-36-lts-33 to envA could be filled with genes for cell division and growth, and the genes could be aligned.     

Synchronized endocytosis studied in the oocyte of a temperature-sensitive mutant of Drosophila melanogaster

Summary This study demonstrates that endocytosis in the oocyte of Drosophila melanogaster is reversibly blocked at the stage of pit formation by the temperature-sensitive, single-gene mutant, shibire ^ts1. Uptake of horseradish peroxidase conjugated with wheat-germ agglutinin was observed to be normal in mutant oocytes at 19°C, but was blocked at 29°C. After 10 min at 29°C, there was a build-up of coated pits along invaginations of the plasma membrane. Also, the endosomal compartment consisting of tubules, bulbs, and small yolk spheres, disappeared. Lowering the temperature to 19°C after 10 min at 29°C released a synchronized wave of endocytosis into a cytoplasm cleared of uptake-related organelles. By observing this synchronized wave after exposure to 19°C for varying durations, we determined that endocytosis proceeds as follows: coated pits/vesiclestubulessmall yolk spheresmature yolk spheres. The observations suggest that these organelles transform one into another within this sequence.     

Studies on DNA markers (D4S10 and D4S43/S127) genetically linked to Huntington's disease in Japanese families

Summary This is the first full report on the genetic linkage between Japanese Huntington's disease and the DNA markers D4S10 and D4S43/S127. With use of the HindIII, BglI, and EcoRI polymorphisms detected at D4S10, and the combination of all these polymorphisms to give composite haplotypes, nine Japanese Huntington's disease families were found to be informative. Three recombinants for D4S10 were detected in these families, giving a maximum lod score of 1.662 at a of 0.10. Similarly, when we used the MspI and PvuII polymorphisms detected by D4S43/S127, five families gave informative results. No recombinant was detected in these families, giving a maximum lod score of 3.348 at a of 0.00. These results clearly support the view that the Japanese Huntington's disease gene may be identical with the Western gene, in spite of the lower prevalence rate in Japan.     

Effect of 24,25-dihydroxyvitamin D3 on 1,25-dihydroxyvitamin D3 metabolism in calcium-deficient rats.

The effect of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] on 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] metabolism was examined in rats fed on a low-calcium diet. These rats exhibit hypocalcaemia, high urinary cyclic AMP excretion, a markedly elevated serum 1,25(OH)2D concentration and low serum concentrations of both 24,25(OH)2D and 25(OH)D. When the rats are treated orally with 1, 5 or 10 micrograms of 24,25(OH)2D3/100 g every day, there is a dramatic decrease in serum 1,25(OH)2D concentration in a dose-dependent manner concomitant with an increase in serum 24,25(OH)2D concentration. Serum calcium concentration and urinary cyclic AMP excretion are not significantly affected by the 24,25(OH)2D3 treatment, which suggests that parathyroid function is not affected by the 24,25(OH)2D3 treatment. The 25(OH)D3 1 alpha-hydroxylase activity measured in kidney homogenates is markedly elevated in rats on a low-calcium diet but is not affected by any doses of 24,25(OH)2D3. In contrast, recovery of intravenously injected [3H]1,25(OH)2D3 in the serum is decreased in 24,25(OH)2D3-treated rats. Furthermore, when [3H]1,25(OH)2D3 is incubated in vitro with kidney or intestinal homogenates of 24,25(OH)2D3-treated rats there is a decrease in the recovery of radioactivity in the total lipid extract as well as in the 1,25(OH)2D3 fraction along with an increase in the recovery of radioactivity in the water-soluble phase. These results are consistent with the possibility that 24,25(OH)2D3 has an effect on 1,25(OH)2D3 metabolism, namely that of enhancing the degradation of 1,25(OH)2D3. However, because a considerable proportion of the injected 24,25(OH)2D3 is expected to be converted into 1,24,25(OH)3D3 by renal 1 alpha-hydroxylase in 24,25(OH)2D3-treated rats, at least a part of the decrease in serum 1,25(OH)2D concentration may be due to a competitive inhibition by 24,25(OH)2D3 of the synthesis of 1,25(OH)2D3 from 25(OH)D3. Thus the physiological importance of the role of 24,25(OH)2D3 in regulating the serum 1,25(OH)2D concentration as well as the mechanism and metabolic pathway of degradation of 1,25(OH)2D3 remain to be clarified.     

Electrogenic responses elicited by transmembrane depolarizing current in aerated body wall muscles ofDrosophila melanogaster larvae

Summary Electrical excitability of the longitudinal ventrolateral body wall muscle of the third instar larva ofDrosophila melanogaster was demonstrated. This is in contrast to previous papers which have reported that this muscle is electrically inexcitable. It was found that an air supply to the muscle through the tracheoles is essential for maintaining its excitability. In an aerated preparation, the muscle maintained a resting potential of around –80 mV for more than 1.5 h, while a nonaerated muscle depolarized to about –30 mV within 30 min. Muscles with resting potentials larger than –70 mV showed graded regenerative potentials with a double-peaked configuration in response to transmembrane depolarizing current. A tetrodotoxin- (TTX-)sensitive, voltage-dependent inward sodium current, and a tetraethylammonium-(TEA-)sensitive, voltage-dependent outward potassium current were found to be responsible for the first peak of the electrogenic response of this muscle. The rising phase of the second peak was caused by a cobalt/manganese-sensitive, voltage-dependent inward calcium current that had a threshold level near –40 mV. Elimination by TEA or barium of the delayed rectification following the first peak caused the second peak to be triggered at a lower threshold. The second peak was profoundly elongated by barium, and this effect was antagonized by external calcium. Thus, the falling phase of the second peak was most likely driven by a calcium-dependent, outward potassium current.     

Mulcos: a vector for amplification and simultaneous expression of two foreign genes in mammalian cells

A method was developed for amplification and expression of foreign genes in mammalian cells. This procedure exploits the fact that an SfiI cleavage site, GGCCGCCT/CGGCC (the recognition sequences are underlined), is present at the SV40 replication origin and the cleaved ends, CCT-3' and AGG-3', are not rotationally equivalent. Thus DNA fragments flanked by the SfiI sites can be ligated in head-to-tail tandem arrays and cloned in cosmids; the resulting construct is called a mulcos. The cosmid vector we have used, pCHD2L, contains the single SfiI site as well as HmBR and dhfr genes, selectable markers in mammalian cells. Cassette plasmid pmoRH contains two expression units, each of which consists of SV40 early promoter, EcoRI or HindIII cloning site, small T splicing region, and poly(A) signal, and the two units as a whole are flanked by the SfiI sites. A set of alpha- and beta-chain cDNAs of a human major histocompatibility class-II antigen were inserted into the EcoRI and HindIII sites, respectively. The purified SfiI fragment, containing both expression units, was then ligated with SfiI-linearized cosmid vector pCHD2L at a molar ratio of 20:1. A mulcos containing eight pairs of the alpha- and beta-chain expression units was isolated by in vitro packaging in phage lambda heads and subsequent transfection into Escherichia coli. Drug-selected cells transfected with the mulcos contained significantly higher copy numbers of the expression units and higher expression levels than those obtained using conventional plasmids. More than 85% of these cells expressed class-II antigen on their cell surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal Antibodies Specific for NADH-Ferricyanide Reductase Domain of Spinach Leaf Nitrate Reductase

Two monoclonal antibodies, 17(3)9 and 36(79)4, were preparedagainst nitrate reductase from Spinacia oleracea L. leaves.An enzyme-linked immunosorbent assay showed that 17(3)9, butnot 36(79)4, reacted more strongly to heat-denatured than nativeantigen. These antibodies inhibited NADH-nitrate reductase aswell as its various partial activities including reduced methylvilogen-nitrate reductase, reduced flavin mononucleotide-nitratereductase and NADH-cytochrome c reductase activities, but notNADH-ferricyanide reductase activity. Immunoblotting after electrophoreticseparation of nitrate reductase fragments obtained by Staphyrococcusaureus V8 protease digestion of native enzyme revealed thatthe two monoclonal antibodies bind to different epitopes locatedon the 28 kDa of the NADH-ferricyanide reductase domain. (Received October 2, 1987; Accepted June 9, 1988)     

Antidepressants and seizure-interactions at the GABA-receptor chloride-ionophore complex

Convulsive seizures are a potential side effect of antidepressant drug treatment and can be produced by all classes of antidepressants. It is also known that some convulsant and anticonvulsant drug actions are mediated by the GABA-receptor chloride-ionophore complex. Drugs acting at this complex appear to induce convulsions by inhibiting chloride conductance through the associated chloride channel. Using the method of GABA-stimulated 36Cl-uptake by rat cerebral cortical vesicles, we show that some antidepressant drugs (imipramine, amitryptyline, and mianserine) can inhibit the GABA-receptor chloride uptake, and that the degree of chloride channel inhibition by these drugs correlates with the frequency of convulsive seizures induced by them.