Modulation of transient type K channel cloned from rat heart.

We have cloned a transient type K channel from rat heart (RH10) and coexpressed a metabotropic glutamate receptor (mGluR5) to study the functional modulation of RH10 coupled to the phosphatidylinositol (PI) hydrolysis. Stimulation of mGluR5 suppressed peak amplitude of RH10 current and affected voltage dependence of activation and inactivation of the channel.     

Isolation of thymidine-requiring variants from rat nerve-like cells

Thymidine auxotrophs (B3T) of rat nerve-like cells (B103) were isolated. B103 cells were preincubated in a thymidine medium and mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Afterward, cells were incubated in a medium supplemented with dialyzed fetal calf serum and then treated with 5-fluorodeoxyuridine for 24 h. Next, cells were grown in a thymidine medium for twenty days. Thymidine auxothrophs were thus obtained and one clone of them was designated B3T cells. The modal number of chromosomes in B3T cells was 85. Growth tests revealed the following interesting facts: (1) The medium containing pyrazofurin and uridine could not support the growth of B3T cells, but the addition of thymidine to such a medium enabled cells to grow. (2) Even deoxyuridine supported the growth of B3T cells when added to the medium of pyrazofurin and uridine. These findings suggest that the catalytic ability of thymidylate synthetase in B3T cells may have decreased, probably due to the decreased affinity of the enzyme molecules to the substrate (dUMP), and that thymidine kinase activity was high enough to support the growth of B3T cells. B3T cells have maintained the ability to differentiate and extend neurites in response to dibutyryl-cyclic AMP as also demonstrated in wild cells (B103). B3T cells will be available for genetical and molecular biological studies of neuronal cells.     

Carbachol and KCl-induced changes in intracellular free calcium concentration in isolated, fura-2 loaded smooth-muscle cells from the anterior byssus retractor muscle of Mytilus edulis

Intracellular free calcium concentration [( Ca2+]1) was measured in suspensions of fura-2 loaded smooth-muscle cells isolated from the anterior byssus retractor muscle of Mytilus edulis. Successive application of 5mM carbachol (CCh) and 100mM KCl to the cells transiently elevated [Ca2+]1 from the resting value of 124 +/- 4.5nM (mean +/- S.E., n = 14) to 295 +/- 15.3 and 383 +/- 20.5 nM, respectively. The response to CCh was concentration-dependent with an ED50 of 10(-5) M. Under the microscope, 67 +/- 3.0 and 83 +/- 1.3 % of fura-2 loaded cells contracted on the addition of 5mM CCh and 100mM KCl, respectively. In Ca2+ -free sea water, the CCh induced change in [Ca2+]1 was partially suppressed whereas that induced by KCl was completely abolished, suggesting an agonist-evoked release of stored Ca2+.     

Physiological role of peroxisomal beta-oxidation in liver of fasted rats

In the livers of fasted rats, the activity of peroxisomal palmitocyl-CoA oxidation (NADH production) was increased more rapidly and markedly than that of mitochondrial carnitine palmitoyltransferase, which is the rate limiting enzyme of mitochondrial beta-oxidation. The peroxisomal oxidizing activity was about twice that of the control throughout the period of fasting (1-7 days). carnitine acetyltransferase activity was increased to a similar extent in both peroxisomes and mitochondria. A possible physiological role of liver peroxisomes may thus be as an effective supply of NADH2, acetyl residues and short and medium-length fatty acyl-CoA in the cells on the enhancement of peroxisomal beta-oxidation of the animals under starvation; these substances thus produced may be transported into the mitochondria as energy sources.     

Effects of hemodynamic edema formation on peripheral vs. central airway mechanics

The mechanisms governing increased central (Rc) and peripheral airway resistance (Rp) during hemodynamic edema formation were studied in anesthetized dogs. Rc and Rp were measured by forced oscillation at 1 Hz by use of a retrograde catheter to partition resistance and a pleural capsule to detect alveolar pressure. After elevation of left atrial pressure to 30 cmH2O by inflation of the left atrial balloon, Rc gradually increased an average of 60% above control in approximately 100 min. Vagotomy had a small influence on the change. On the other hand, Rp with vagus nerves intact increased triphasically: first, it increased transiently by 160% above the control value within 15-20 min before returning to near base line. It then increased gradually for approximately 40 min and finally rose sharply up to five times the control value after approximately 100 min. With vagi cut, the initial phase disappeared, but the second gradual and final rapid phases were not affected. Several sequential mechanisms of increased Rp can be proposed: 1) transient bronchoconstriction mediated by vagal reflex; 2) gradual formation of peribronchial edema; and 3) a sharp increase in airway fluid and formation of bronchial froth. In addition, narrowing of the airways by vascular engorgement may have contributed to the increase of Rp throughout all stages.     

Anti-parasite activity of nucleoside analogues: the metabolism of carbocyclic inosine in promastigotes of Leishmania tropica and Leishmania donovani and its activity against amastigotes of Leishmania donovani in vitro

Carbocyclic inosine is a potent inhibitor for the growth of the promastigote form of Leishmania tropica and Leishmania donovani. In culture, the EC50 values of carbocyclic inosine are 8.3 X 10(-8) and 1.3 X 10(-7) M for the promastigotes of L. tropica and L. donovani, respectively. On the other hand, it is less toxic towards mouse mammary tumor FM3A cells: the EC50 value is 2.7 X 10(-4)M. Carbocyclic inosine is metabolized by Leishmania promastigotes to give carbocyclic adenosine-5'-triphosphate(aristeromycin-5'-triphosphate) and carbocyclic guanosine-5'-triphosphate. This metabolic conversion provides a mechanism for the parasite-selective toxicity of carbocyclic inosine. Carbocyclic inosine was found to be active against L. donovani amastigotes in an in vivo-like cultivation in vitro.     

Evidence for acrosin-like enzyme in sperm extract and its involvement in fertilization of the ascidian,halocynthia roretzi

The presence of a protease has been demonstrated in sperm of the solitary ascidian, Halocynthia roretzi, by using t-butyloxycarbonyl-L-Val-L-Pro-L-Arg-4-methylcoumaryl-7-amide (Boc-Val-Pro-Arg-MCA) and other arginyl or lysyl MCA derivatives as substrates. Several properties of the enzyme were investigated in a crude extract. The activity had a pH optimum near 8.0 and was enhanced by the addition of CaCl₂. The Km value of 87μM was determined for Boc-Val-Pro-Arg-MCA under the optimal conditions. An apparent molecular weight was estimated to be 35,000 by gel filtration. The enzyme was inhibited with diisopropyl fluorophosphate, leupeptin, antipain, p-aminobenzamidine, Val-Pro-Arg-CH₂Cl, and soybean trypsin inhibitor, but scarcely inhibited with chymostatin, elastatinal, p-chloromercuribenzoic acid, tosyl-Lys-CH₂Cl, and tosyl-Phe-CH₂Cl. Boc-Val-Pro-Arg-MCA, the most susceptible of the substrates examined, showed the most effective inhibition against fertilization of ascidian eggs. Thus, this enzyme in ascidian sperm extract has features closely similar to mammalian acrosin [EC 3.4.21.10], and we conclude that the enzyme is involved in fertilization as one of the lysins.