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The kinetics of denaturation by guanidine hydrochloride (GuHCl) of a thermostable phosphoglycerate kinase (PGK) extracted from Thermus thermophilus and of yeast PGK at neutral pH were studied by circular dichroism. Denaturation by GuHCl proceeded as a first-order reaction. The activation free energy of the denaturation reactions (delta Gf not identical to ) in the absence of GuHCl was estimated to be 32.7 kcal/mol for T. thermophilus PGK and 27.9 kcal/mol for yeast PGK (at 25 degrees C). Measurements of the rate constants at various temperatures indicated that delta Gf not identical to has maximum values at 29 degrees C for T. thermophilus PGK and at 20 degrees C for yeast PGK, and that the temperature dependences of delta Gf not identical to, delta Hf not identical to, and delta Sf not identical to for T. thermophilus PGK are smaller than those of yeast PGK. Values of delta Sf not identical to for thermal denaturation for both PGK's are approximately 200 e.u.

The electrical properties of skeletal muscle membranes were investigated in genetically diabetic KK-CAy mice and alloxan-induced diabetic ddY mice. Using isolated phrenic nerve-diaphragm muscle or sciatic nerve-gastrocnemius muscle in situ preparations, nerve-stimulated twitch tensions (the maximal value) were obtained at lower voltage pulse in diabetic KK-CAy mice than in normal ddY mice. The diabetic state reduced resting membrane potentials (1.7-4.0 mV) and resting membrane conductance (0.37-0.44 mu siemen), decreased the amplitude (3.8-3.9 mV) and overshoot (4.5 mV) of directly induced-action potential, and prolonged action potential duration. In the diabetic state, resting membrane conductance was multiply-correlated with blood glucose level and resting membrane potential. In alloxan-induced diabetic mice, resting membrane potentials were significantly multiply-correlated with the weeks elapsed after alloxan injection and blood glucose level (p less than 0.01). Since the reduction of resting membrane potential correlated with the weeks, changes in resting membrane potential may be involved in the decrease in insulin-like growth factor action. The reduction of resting membrane conductance was correlated with the increase in blood glucose.

The effects of alpha-bungarotoxin (alpha-BuTX), D-tubocurarine (D-TC) and antibodies against muscle extract on acetylcholine (ACh) sensitivity were investigated in developing mouse myotubes in a nerve-muscle co-culture. Antibodies clearly suppressed the ACh potential amplitude in adult mouse diaphragm muscle, but antibodies in muscle pre-treated with D-TC (1 microgram/ml) weakly suppressed it. The addition of D-TC to muscle extract dose-dependently inhibited the formation of the immunoprecipitation lines. The exposure of developing myotubes to antibodies for 10 days in culture suppressed both resting potential and ACh potential, whereas co-existence of alpha-BuTX (1 microgram/ml) or D-TC (0.1 mg/ml) with antibodies suppressed ACh potential but did not affect resting potential compared with antibodies alone. The ACh potentials in myotubes cultured with alpha-BuTX and D-TC alone were also suppressed. The appearance (day 8 in culture) of this suppressive effect by alpha-BuTX was faster than that (day 11 in culture) of D-TC. These different effects depending on the time in culture may account for the conformational change of developing ACh receptors to alpha-BuTX and D-TC.

The rate of transmammary transmission of Stronglyloides ratti was examined in albino rats in terms of the route of subcutaneous (s.c.) migration from the infection site (the skin) to the cranium. Inoculation sites nearer the cranium resulted in less frequent transmammary infection. The maximum number of adult worms was recovered from the sucklings when the mother was inoculated in her hindquarter and sucklings were allowed to feed for 30-36 h after inoculation (AI). Few worms were recovered from sucklings when they were allowed to nurse during periods of less than 24 h AI or greater than 42 h AI. In lactating mothers, larval infection of the mammary glands was commonly observed, and these larvae showed an increased esophagus length. In nonlactating mothers, most larvae completed their migration to the cranium within 36 h AI.

A new monoterpene, paeoniflorigenone (PFG) (100-900 micrograms/ml), which was isolated from paeony roots and identified chemically, suppressed both indirectly and directly stimulated muscle twitchings of frog sciatic nerve-sartorius muscle preparation, and it indirectly stimulated muscle twitchings of phrenic nerve-diaphragm muscle preparations. The suppression effect by PFG (300 micrograms/ml) on twitching was not reversed by neostigmine (60 micrograms/ml) and was restored by washing out of PFG. PFG (150 micrograms/ml) depolarized the diaphragm muscle membranes by 10 mV and did not change the electrotonic potentials. PFG (100 micrograms/ml) inhibited weakly acetylcholine (5 micrograms/ml)-induced slow contractions. These results demonstrated that PFG is a depolarizing neuromuscular blocking agent, being similar to succinylcholine, except that PFG did not produce any contraction, but succinylcholine did.

The release of Schistosoma japonicum cercariae from Leytean Oncomelania quadrasi snails was observed under laboratory conditions. Two patterns of emergence were noted. The initial, nonperiodic emergence occurred immediately after submerging the snails in water and was followed by a periodic, diurnal emergence which peaked in the afternoon. The daily cercarial output of the periodic emergence appeared to be affected by exogenous light intensity. Furthermore, there was a cessation or reduction in cercarial output every 3rd or 4th day.

The 3-phosphoglycerate kinase (EC 2.7.2.3) of yeast which contains two tryptophyl and eight tyrosyl residues per molecule, displayed an unusualy fluorescence emission spectrum with a maximum at 308 nm when excited at 280 nm. The emission peak shifted to 329 nm when excited at 295 nm. We could confirm that it was due to the efficient quenching of tryptophyl fluorescence as well as to the incomplete energy transfer from tyrosyl to tryptophyl residues. The average fluorescence quantum yield of this protein was 0.076 (excitation at 280 nm) and that of tryptophyl residues was 0.046 (excitation at 295 nm). As the pH of the solution was lowered, the fluorescence intensity of phosphoglycerate kinase at 329 nm dramatically increased between pH 5 and 4, while the position of the peak remained unchanged. When denatured in 4 M guanidine hydrochloride, the protein showed two emission peaks, one at 343 nm and the other at 303 nm.

Clones carrying cDNA sequences for the alpha-subunit of the Na,K-ATPase from HeLa cells have been isolated. Nucleotide sequence analysis of the cloned cDNA has revealed the primary structure of this polypeptide, which consists of 1,023 amino acids. The alpha-subunit of the human Na,K-ATPase exhibited 87% homology with its Torpedo counterpart and 98% homology with its sheep counterpart. The six putative transmembrane segments M1-M6 showed higher conservation than the total segments. Total genomic Southern hybridization indicated the existence of at most two copies, possibly only one, of the gene encoding the Na,K-ATPase alpha-subunit in the human genome.

The long-term maintenance of Schistosoma mansoni in intermediate snail hosts is described. Snails were infected with one or five miracidia to obtain the initial parasitized liver tissue. The serial implantations were conducted. When the materials for serial implantation were obtained 20 to 60 days after the hosts had begun to release cercariae, the proportion of snails becoming infected was higher than when materials obtained earlier were used. It made no difference whether the initial infection was with one or five miracidia. Our findings suggest the possibility of cloning of unisexual infections in experimental infections.

The Na,K-adenosine triphosphatase (ATPase) alpha 2 subunit gene was found to display restriction fragment length polymorphisms (RFLPs) between the genomes of normotensive and hypertensive rats when digested with the restriction enzymes Bgl II and Hind III. In normotensive rats, we tested the spontaneously hypertensive rat (SHR) and its substrain, the stroke-prone spontaneously hypertensive rat (SHR-SP). Rat (SD) complementary (c) DNA encoding the alpha 2 subunit of Na,K-ATPase was used as a probe. When the probe was dissected these RFLPs were found to occur in the vicinity of the genomic locus encoding the middle part of the messenger (m) RNA for the alpha 2 subunit of Na,K-ATPase. A Northern blot analysis indicated that these RFLPs did not influence the alpha 2 subunit with regard to either size or amount of mRNA.

The hypersensitivity of the neuromuscular junctions of diabetic mice to succinylcholine (SuCh), but not to d-tubocurarine (d-TC), was investigated using a cross culture preparation of diabetic skeletal muscle or spinal cord extract with normal tissues. Whether the hypersensitivity is due to the muscle cells themselves was examined using adult muscle of diabetic KK-CAy, prediabetic KK-CAy and normal ddY mice cocultured with embryonic spinal cord of normal ddY mice. The cultured neuromuscular junctions between diabetic KK-CAy muscle and normal ddY spinal cord was hypersensitive to SuCh, but not to d-TC. In contrast, such junctions between prediabetic KK-CAy muscle and normal ddY spinal cord were not hypersensitive to either drug. The involvement of neuronal factors in hypersensitivity to SuCh in diabetic KK-CAy neuromuscular junctions was examined using adult spinal cord extract (SCE) from diabetic KK-CAy and from normal ddY mice. We followed the time course of change in sensitivity of the acetylcholine (ACh) receptors in normal ddY embryonic myotubes to SuCh and d-TC. Both diabetic SCE and normal SCE reduced the sensitivity of myotubes to ACh; the reduction of ACh potential amplitudes by the former was less than that by the latter. Myotubes cultured with diabetic SCE was hypersensitive to both 1.51 microM SuCh and 0.134 microM d-TC. These results suggest that the hypersensitivity of the neuromuscular junctions in diabetic KK-CAy mice to SuCh but not to d-TC is mainly attributable to the diabetic muscle cells themselves.

In summary, we present a list of phage clones we have obtained from rat genomic libraries (from lamba SC1 to lambda SC31, lambda WC1 and lambda WC40) together with cDNA clones we have obtained from a rat brain cDNA library (PRCM1,5,3 and 4). pRCM5 corresponds to 4.0 kb mRNA species observed primarily in skeletal muscle. These clones can be classified into three groups. They belong to three bona fide calmodulin genes with five to six exons called CaM I, CaM II and CaM III and four intronless retropseudogenes, one derived from CaM I and three derived from CaM II. We have not obtained retropseudogenes for CaM III so far. These three bona fide genes are transcribed into multiple sized mRNA species in a tissue-specific manner, that is, CaM I is ubiquitous, CaM II is transcribed mainly in brain and CaM III is transcribed primarily in brain and skeletal muscle. Four retropseudogenes do not appear to be transcribed. They are probably relics of inactivated genes. The physiological meanings of multiple calmodulin mRNA species and mechanisms of transcriptional regulation of these three bona fide genes will be the main subjects of our future experiments.

Sodium- and potassium-dependent ATPase [(Na+ + K+)ATPase], which is responsible for the active transport of Na+ and K+, is distributed universally among animal cell membranes and consists of two types of subunits, alpha and beta (refs 1-4). The larger alpha-subunit with a relative molecular mass (Mr) of 84,000-120,000 is thought to have the catalytic role. We have now cloned and sequenced DNA complementary to the Torpedo californica electroplax messenger RNA encoding the alpha-subunit of (Na+ + K+)ATPase and have deduced the complete amino-acid sequence of the polypeptide. Some structural features of the alpha-subunit molecule related to the function of this active-transport protein are discussed.

Differential expression during rat development of three genes for calmodulin (CaM I-III) was examined in amnion, decidua, embryo, liver, placenta, parietal and visceral yolk sacs and uterus. CaMI expression was constant except for increasing activity in VYS during gestation. CaMII expression increased in all tissues except for a decrease in embryo. CaMIII did not change dramatically. Differential expression was also found in chemically or virally induced rat tumors, and in metastatic lung nodules of mouse mammary carcinoma. CaMII was the major gene expressed in all these neoplastic tissues.

Sulphation of the carcinogen 5-hydroxymethyl-chrysene (5-HCR) to the active metabolite 5-HCR sulphate occurred at significant rates in all of hepatic cytosols prepared from the male and female experimental animals, rats, mice, guinea-pigs and hamsters. The 5-HCR-sulphating activity was also found in kidney cytosols of all the experimental animals used, while their activities were much less than those of hepatic cytosols. In the male mice, the enzyme activity of testis was higher than any other examined tissue. Small intestine and adrenal of male and female guinea-pigs had relatively high enzyme activities. Small enzyme activities were also found in a variety of extrahepatic tissues of some of these animals. Marked species and sex differences (female much greater than male in the rat and mouse) were observed in the hepatic enzyme activity. In the female rat liver which showed the highest 5-HCR-sulphating activity among the examined tissues of all the animals, a typical hydroxysteroid sulphotransferase inhibitor, dehydroepiandrosterone (DHA) sulphate (1 mM), potently and competitively inhibited the sulphation of 5-HCR as well as that of DHA, a typical substrate for hydroxysteroid sulphotransferases. On the contrary, the phenol sulphotransferase inhibitors, pentachlorophenol and 2,6-dichloro-4-nitrophenol, had only a little effect on these enzyme activities even at a concentration of 50 microM that showed a potent inhibition of the phenol sulphotransferase activity. These results suggest that 5-HCR be sulphated in the female rat liver by hydroxysteroid sulphotransferases, but not by phenol sulphotransferases.

The effects of light, agitation, and salinity on the hatching pattern of Schistosoma haematobium eggs were examined. Whereas all three factors influenced the hatching pattern, only salinity affected the hatching rate. Eggs began to hatch 5 min after dilution and reached a peak 10-15 min after dilution when urination, dilution of urine, and observation of hatching were conducted under ordinary laboratory conditions (25 degrees C in diffuse sunlight). Complete darkness delayed hatching. Agitation of urine by aspiration into a syringe accelerated egg hatching, the miracidia reaching peak numbers 5 min earlier.

(1) A glycolytic enzyme, phosphoglycerate kinase [EC 2.7.2.3], was purified from cells of an extreme thermophile, Thermus thermophilus strain HB8. The enzyme was resistant to heat, and no loss of activity was observed after incubation for 10--20 min at 79 degrees C. (2) Catalytic properties such as pH optimum (pH 6--8.5), kinetic parameters (Km=0.28 mM for ATP, 1.79 mM for glycerate 3-phosphate), substrate specificity and inhibitors of the enzyme were investigated and compared with those of phosphoglycerate kinase from other sources. (3) The enzyme protein consists of a single polypeptide chain of molecular weight 44,600. The isoelectric point is 5.0 The amino acid composition of the enzyme was studied. The contents of ordered secondary structures were estimated to be 29% alpha-helix and 11% pleated sheet from the circular dichroic spectrum of the enzyme protein. (4) The fluorescence spectrum of the enzyme protein showed an emission maximum at 320 nm when excited at 280 nm. The quantum yield was 0.19. Tryptophyl fluorescence was not quenched, in contrast to the fluorescence reported for yeast phosphoglycerate kinase.

The myopathy in skeletal muscles of genetically diabetic male KK-CAy mice or alloxan-induced diabetic mice was investigated. In these diabetic mice, nerve-stimulated twitch tensions of in situ sciatic nerve-gastrocnemius muscle preparations were inhibited by intraarterially administered succinylcholine (SuCh) to a greater extent than in normal (non-diabetic) ones. Despite the high blood glucose level, at one week after alloxan administration, no hypersensitivity to SuCh was induced in mice, but mice at 2 weeks and 4 weeks after alloxan showed greater sensitivities. In isolated diaphragm muscles of diabetic KK-CAy mice, the acetylcholine (ACh, iontophoretically applied) potential amplitude was greater than in KK-CAy prediabetic muscles. SuCh in diabetic KK-CAy muscles inhibited ACh potentials to a greater extent than in normal ddY muscles. Hill coefficients obtained from the inhibition curve by SuCh of the nerve-stimulation response were decreased by the diabetic state. The sensitivities to d-tubocurarine and alpha-bungarotoxin were the same in both kinds of muscles. Both extrajunctional ACh receptors in denervated muscles of normal ddY and diabetic KK-CAy mice revealed the lower sensitivity to SuCh than junctional receptors in non-denervated normal muscles. In conclusion, diabetic muscles showed the hypersensitivity restricted to SuCh. These phenomena are neither due to glycosylation nor to denervation supersensitivity.

Two distinct processed calmodulin genes of rat (lambda SC8 and lambda SC9) were identified, cloned and their DNA sequences determined. The existence of direct repeats of 19 base-pairs for lambda SC8 or 9 base-pairs for lambda SC9 at both ends of the coding plus non-coding regions suggested a possible involvement of a mRNA-mediated process of insertion. Total genomic Southern hybridization suggested the existence of at least three different calmodulin-related genes in the rat genome. The other gene was the bona fide calmodulin gene (lambda SC4) which was split into at least five exons. lambda SC9 contained insertions of one nucleotide and two 17 base-pair direct repeats in the coding region. These insertions cause frameshift mutations probably preventing it from encoding a functional calmodulin. It also carried an insertion of a rat middle repetitive sequence, identifier sequence (IDS: Sutcliffe et al., 1982) in the 3'-non-coding region. Otherwise, it consisted of an almost identical DNA sequence to that of the bona fide calmodulin gene (lambda SC4), including the 3'-non-coding region down to the poly(A) recognition signal, A-A-T-A-A-A. On the other hand, lambda SC8 did not possess frameshift mutations in the coding region, and hence was capable of encoding a functional protein. In fact, a probe specific to the lambda SC8 sequence identified a band in Northern blotting whose size was 300 nucleotides smaller than that of authentic calmodulin mRNA. Comparison of the nucleotide sequences showed that only the coding regions of these two processed genes were homologous, indicating that the divergence of these two processed genes from the common ancestor calmodulin was an ancient event.

The changes in worm burden, distribution, length, and fecundity after and during single and repeated inoculations of 10, 50, or 500 larvae of Strongyloides ratti were examined in rats. Worm burden after a single inoculation of a higher larval dose reduced rapidly. Repeated inoculations of lower larval doses at weekly intervals led to a delayed peak and slower reduction of worm burden; the repeated inoculations of 10 larvae did not induce worm expulsion for at least 7 wk. In repeated inoculations at 3-wk intervals, a primary inoculation of 500 larvae induced strong resistance to reinfection at week 3, whereas no resistance was induced until week 6 in rats receiving repeated inoculations of 10 or 50 larvae. Similar dose-dependent reductions in worm length and fecundity were observed in single and repeated inoculations, and the reductions began earlier than worm expulsion. Intestinal migration of worms from the upper small intestine to the large intestine was observed during the course of single and repeated inoculations. Earlier and clearer migration was observed in rats receiving higher doses. These findings indicate that in S. ratti infection, the changes of worm burden, distribution, length, and fecundity are dependent on the inoculated larval dose.

The mechanisms involved in inhibitory effects of isofloxythepin, a newly synthesized dibenzothiepin neuroleptic, on post-decapitation convulsions were studied in rats. Isofloxythepin (0.05-2.0 mg/kg s.c.) inhibited post-decapitation convulsions in a dose-dependent manner as shown by the decrease in the incidence and the shortening of the duration of convulsions. The convulsions were also inhibited by oxyprothepin, zotepine or chlorpromazine but not by haloperidol. Prazosin and bunazosin, both alpha 1-adrenoceptor antagonists, suppressed the post-decapitation convulsions but a non-selective alpha 2-adrenoceptor agonist, tolazoline, was without effect. The convulsions were inhibited dose dependently by clonidine, an alpha 2-adrenoceptor agonist, but were prolonged in duration by yohimbine, an alpha 2-adrenoceptor antagonist. Yohimbine antagonized the inhibitory effects of isofloxythepin, prazosin and clonidine. The noradrenaline-induced contraction of rat vas deferens was inhibited by isofloxythepin, prazosin or chlorpromazine. Isofloxythepin bound to alpha 1-receptors as did chlorpromazine in the rat brain cortex. The results imply that post-decapitation convulsions seem to be inhibited by a block of postsynaptic alpha 1-adrenoceptors, enhanced by a block of presynaptic alpha 2-adrenoceptors and reduced by isofloxythepin via the blocking of postsynaptic alpha 1-adrenoceptors. The convulsions thus could serve as a good model for studying the actions of drugs on the central nervous system alpha-adrenoceptors.

Elsewhere, we have reported the structure of a rat calmodulin gene and two distinct rat calmodulin cDNAs, pRCM1 and pRCM3. Here, I report the cloning and sequencing of the third calmodulin cDNA (pRCM4) and two additional rat calmodulin genes. The original calmodulin gene is named CaM I (pRCM1) and the newly discovered calmodulin genes are named CaM II (pRCM3) and CaM III (pRCM4). CaM II spans about 10 x 10(3) base-pairs and consisted of five exons, while CaM III spans about 7.2 x 10(3) base-pairs and consisted of six exons. One of the introns (intron 3) observed in CaM I and CaM III is lost in CaM II. Otherwise, the intron/exon organization of these genes is exactly the same. In all calmodulin genes, the first intron separates the initiation codon (ATG) from the coding region of the protein. Northern blotting showed that CaM I is transcribed primarily into 1.7 x 10(3) base-pair mRNA in various tissues examined and 4.0 x 10(3) base-pair mRNA mainly in skeletal muscle, CaM II is transcribed into 1.4 x 10(3) base-pair mRNA almost exclusively in brain and CaM III is transcribed predominantly into 2.3 x 10(3) base-pair mRNA and faintly into 1.0 x 10(3) base-pair mRNA mainly in skeletal muscle and brain. DNA sequences in the promoter-regulator regions of these genes are partly homologous but essentially distinct and possess a number of direct repeats, palindromes and feasible stem-loop structures. Together with these, I report here the structures of the third and fourth calmodulin retropseudogenes.