Effects of vanadate on Na+,K+-ATPase and on the force of contraction in guinea-pig hearts.

Vanadate has been shown to be a potent inhibitor of isolated Na⁺,K⁺-ATPase. Since the inhibition of this enzyme system has been implicated in a mechanism for the positive inotropic action of cardiac glycosides, the cardiac actions of vanadate were examined in connection with its action on Na⁺,K⁺-ATPase. Vanadate inhibited isolated Na⁺,K⁺-ATPase obtained from various tissues. The differences in the vanadate sensitivity due to enzyme source were relatively small. K⁺-stimulated phosphatase activity was more sensitive than Na⁺,K⁺-stimulated ATP hydrolysis. The compounds was more potent than phosphate in supporting [³H] oubain binding in the presence of Mg²⁺, indicating a higher affinity of the enzyme for vanadate. It, however, failed to inhibit oubain sensitive ⁸⁶Rb uptake in electrically stimulated atrial muscle of guinea-pig hearts in concentrations which would inhibit isolated Na⁺,K⁺-ATPase. These latter concentrations of vanadate also failed to produce positive inotropic effects in electrically stimulated left atrial preparations of guinea-pig hearts. Higher concentrations produced marked negative inotropic effects associated with a shortening of the action potential duration. These results indicate that vanadate is a potent inhibitor of isolated Na⁺,K⁺-ATPase, but cannot inhibit the enzyme in intact myocardial cells or produce positive inotropic effects when applied extracellularly. Inhibitory sites on the enzyme are probably located at the internal surface of the cell membrane which are normally inaccessible to vanadate in intact tissue.     

Studies on the cell wall of Chlorella II. Mode of increase of glucosamine in the cell wall during the synchronous growth of Chlorella ellipsoidea

The dry weight and glucosamine content of the alkali-insoluble"rigid wall" of Chlorella ellipsoidea were measured in synchronouslygrowing and dividing cells. The content of glucosamine, the major component of the rigidwall, was measured by the Elson-Morgan and the ninhydrin reactions.The results revealed, in agreement with previous observations,that the amount of the rigid wall, measured in terms of wholedry weight or glucosamine content, remains almost constant duringthe growing phase and increases only in the reproduction phase. (Received February 19, 1979; )     

Synthetic ColE1 plasmids carrying genes for penicillin-binding proteins in Escherichia coli

Clarke and Carbon's collection of 2000 Escherichia coli strains which harbor ColE1 plasmids carrying small random segments of the E. coli chromosome was screened for the correction of mutational defects in penicillin-binding proteins (PBPs): ponA (PBP-1a), ponB (PBP-1b), dacB (PBP-4), and pfv (PBP-5). We found plasmids carrying chromosomal segments containing ponA⁺-aroB⁺ (pLC29-47), ponB⁺-tonA⁺ (pLC4-43, pLC4-44, and pLC19-19), and argG⁺-dacB⁺ (pLC10-46 and pLC18-38). Characters of these plasmids were analyzed. Two other plasmids (pLC26-6 and pLC4-14) previously found to correct ftsI mutation (Y. Nishimura, Y. Takeda, A. Nishimura, H. Suzuki, M. Inouye, and Y. Hirota (1977)Plasmid1, 67–77) were also investigated further. Restriction maps of chromosomal DNAs carried by pLC29-47, pLC4-44, pLC19-19, pLC18-38, pLC26-6, and pLC4-14 were constructed. The regions of ponB-tonA on pLC4-44 and pLC19-19, and of leuA-ftsI-murE and F on pLC26-6 were located on the restriction maps. Although both pLC26-6 and pLC4-14 corrected a thermosensitive mutation, ftsI, which causes a defect in cell division due to abnormal PBP-3, only pLC26-6 led to restoration of PBP-3 production by an ftsI mutant, while pLC4-14 did not. Restriction and heteroduplex analyses of pLC26-6 and pLC4-14 have shown the absence of nucleotide sequence homology between them. The plasmids, pLC29-47 carrying ponA⁺ and pLC4-43, pLC4-44, and pLC19-19 carrying ponB⁺ led the host cell to overproduce the respective PBP.     

Immune Response to Toxoplasma gondii

Peripheral blood leukocytes (PBL) from patients with toxoplasmosis were shown to be highly responsive to in vitro stimulation with Toxoplasma gondii extract as measured by incorporation of [³H]methylated thymidine. Analysis of Toxoplasma-specific proliferative cells in PBL by using monoclonal antibodies specific for human T cell subsets revealed that the Toxoplasma-specific proliferation response of PBL from the patients was mediated by Leu 1, Leu 3a positive cells, that is, helper/inducer T cells. Tests for the Toxoplasma-specific proliferation response may provide a readily available method for the diagnosis of congenital toxoplasmosis, especially during the newborn period.     

Association of neutral α-glucosidase with cytoplasmic granules of peritoneal macrophages

A neutral α-glucosidase (EC 3.2.1.20) activity was shown to be associated with granules which are sedimentable at 10 000 g after differential centrifugation of mouse peritoneal macrophage homogenates. When the post-nuclear supernatant was centrifuged in a sucrose density gradient, high activities for neutral α-glucosidase and β-glucuronidase (EC 3.2.1.31) were detected in the bottom fractions because of aggregation of the granules. Neutral α-glucosidase-containing granules were completely disaggregated by the addition of 20 units/ml of heparin and 10 mM Tris-HCl (pH 7.2), which caused only a partial disaggregation of β-glucuronidase-containing granules. The addition of a high concentration of heparin, Tris buffer, or KCl to the gradient gave the same patterns of disaggregation of the granules. Under the condition in which about 50% of the total β-glucuronidase activity was released into the medium, depending on phagocytosis, very little α-glucosidase was released. These observations suggested that neutral α-glucosidase may localize in non-lysosomal granules.     

Foot protein isoforms are expressed at different times during embryonic chick skeletal muscle development

We have investigated the time course of expression of the alpha and beta triad junctional foot proteins in embryonic chick pectoral muscle. The level of [3H]ryanodine binding in muscle homogenates is low until day E20 of embryonic development, then increases dramatically at the time of hatching reaching adult levels by day N7 posthatch. The alpha and beta foot protein isoforms increase in abundance concomitantly with [3H]ryanodine binding. Using foot protein isoform-specific antibodies, the alpha foot protein is detected in a majority of fibers in day E10 muscle, while the beta isoform is first observed at low levels in a few fibers in day E15 muscle. A high molecular weight polypeptide, distinct from the alpha and beta proteins, is recognized by antifoot protein antibodies. This polypeptide is observed in day E8 muscle and declines in abundance with continued development. It appears to exist as a monomer and does not bind [3H]ryanodine. In contrast, the alpha isoform present in day E10 muscle and the beta isoform in day E20 muscle are oligomeric and bind [3H]ryanodine suggesting that they may exist as functional calcium channels in differentiating muscle. Comparison of the intracellular distributions of the alpha foot protein, f-actin, the heavy chain of myosin and titin in day E10 muscle indicates that the alpha foot protein is expressed during myofibril assembly and Z line formation. The differential expression of the foot protein isoforms in developing muscle, and their continued expression in mature muscle, is consistent with these proteins making different functional contributions. In addition, the expression of the alpha isoform during the time of organization of a differentiated muscle morphology suggests that foot proteins may participate in events involved in muscle differentiation.     

Early Determination of Developmental Fate in Presumptive Intestinal Endoderm of the Chicken Embryo

The endodermal epithelia of esophagus, proventriculus and gizzard of 6-day chicken embryos can form glands and express embryonic chicken pepsinogen (ECPg), when they are subjected to the influence of proventricular mesenchyme, while intestinal epithelium of the same age cannot respond to the inductive influence of proventricular mesenchyme. We attempted in this paper to know whether this regional difference of epithelia to respond to mesenchymal influence originates very early in development or it is established gradually in the course of development of digestive tract.
The young presumptive intestinal endoderm taken from embryos having 15–20 somites was associated and cultivated with 6-day proventricular mesenchyme. The presumptive intestinal endoderm never expressed ECPg although it formed gland-like structures. In the control explants composed of presumptive stomach endoderm and proventricular mesenchyme, glands were formed and gland cells expressed ECPg detected by immunocytochemistry and in situ hybridization.
These results indicate that the developmental fate of presumptive intestinal endoderm is determined rather strictly at very early developmental stage, and suggest that the segregation of at least two cell lineages occurs early in the development; one which can express ECPg under the influence of proventricular mesenchyme, and another one which cannot express ECPg and differentiates mainly into intestinal epithelium.     

Expression of the GABAB receptor in Xenopus oocytes and inhibition of the response by activation of protein kinase C.

The functional GABAB receptor was expressed in Xenopus oocytes by injecting mRNA obtained from the cerebellum of the rat. Application of GABA in the presence of bicuculline induced a hyperpolarization under current-clamp conditions and an outward current under voltage-clamp conditions. Baclofen mimicked the effect of GABA in the presence of bicuculline, and the effect of baclofen was antagonized by phaclofen. The GABA-induced outward current was slightly inhibited by treatment with GDP-beta-S and was completely inhibited by treatment with GTP-gamma-S. The activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate (TPA), but not 4 alpha-phorbol-12,13-didecanoate, suppressed the GABAB receptor-mediated hyperpolarization, and the effect of TPA was antagonized by sphingosine. Thus, activation of protein kinase C inhibits the expressed GABAB receptor-mediated response.