Effect of parathyroid hormone on Na+-dependent phosphate transport and cAMP-dependent 32P phosphorylation in brush border vesicles from isolated perfused canine kidneys

Concentrative uptake of 32Pi induced by the dissipation of a Na+ gradient (overshoot) was demonstrated in brush border membrane vesicles obtained from isolated perfused canine kidneys. Na+-dependent 32Pi transport was decreased in brush border vesicles from isolated kidneys perfused with parathyroid hormone (PTH) for 2 h compared to uptake measured in vesicles from kidneys perfused without PTH. Cyclic AMP-dependent 32P phosphorylation of a 62,000 Mr protein band was demonstrable on autoradiograms of sodium dodecyl sulfate-polyacrylamide gels of membrane suspensions from kidneys perfused +/- PTH. Evidence that perfusion with PTH resulted in cAMP-dependent phosphorylation in isolated kidneys from parathyroidectomized dogs (decreased cAMP-dependent 32P phosphorylation of the 62,000-Mr band in brush border vesicles) was obtained after 2-h perfusion with PTH. Decreased 32P phosphorylation was not observed if membranes were allowed to dephosphorylate prior to 32P phosphorylation in vitro. We conclude that brush border vesicles from isolated perfused canine kidneys can be used to study the action of PTH on Na+-Pi cotransport in brush border membranes and on cAMP-dependent phosphorylation of the membrane. It is strongly suggested that PTH effects changes in Na+-dependent 32Pi transport in isolated brush border vesicles and changes in 32P phosphorylation of vesicles via a direct action on the renal cortical cell rather than as a consequence of extrarenal actions of the hormone.     

Calmodulin-stimulated protein kinase activity from rat pancreas

Previous work from our laboratory has demonstrated that neurohumoral stimulation of the exocrine pancreas is associated with the phosphorylation of the Mr 29,000 ribosomal protein S6. In a cell-free system using pancreatic postmicrosomal supernatant as the kinase donor, we found that the following co-factors stimulate the phosphorylation of the Mr 29,000 ribosomal protein: calcium with calmodulin, calcium with phosphatidyl serine, and cAMP. These findings suggest that the pancreas contains a calcium-calmodulin-dependent protein kinase (CaM-PK) that can phosphorylate the Mr 29,000 ribosomal protein. A CaM-PK activity was partially purified sequentially by ion exchange, gel filtration, and calmodulin-affinity chromatography. Phosphorylation of the Mr 29,000 ribosomal protein by the partially purified CaM-PK was dependent on the presence of both calcium and calmodulin and not on the other co- factors. The CaM-PK fraction contained a phosphoprotein of Mr 51,000 whose phosphorylation was also dependent on calcium and calmodulin. When 125I-calmodulin-binding proteins from the CaM-PK fraction were identified using electrophoretic transfers of SDS-polyacrylamide gels, a single Mr 51,000 protein was labeled. The preparation enriched in CaM- PK activity contained an Mr 51,000 protein that underwent phosphorylation in a calcium-calmodulin-dependent manner and an Mr 51,000 calmodulin-binding protein. It is therefore possible that the CaM-PK may comprise a calmodulin-binding phosphoprotein component of Mr 51,000.     

An analysis of the sensitivity of somatic cell hybrids to natural killer cell- and natural cytotoxic cell-mediated lysis

The analysis of the NK and NC sensitivity of somatic cell hybrids formed between parental cell lines that differ in their NK and NC sensitivity has shown the following. 1) The dominant expression of both NK and NC recognition determinants on target cells; 2) the dominant expression of two post-recognitive NC resistance mechanisms, one requiring protein synthesis and one being protein synthesis independent; and 3) the dominant expression of a post-recognitive NK resistance mechanism, which is protein synthesis independent. The post-recognitive protein synthesis-independent NC resistance mechanism confers no NK resistance and the post-recognitive NK resistance mechanism confers no NC resistance. Whether the post-recognitive protein synthesis-dependent NC resistance mechanism confers NK resistance remains open to question. The analysis of the hybrids indicates that transformed cells become sensitive to either NK- or NC-mediated lysis by losing their resistance to the lytic activity of these effector cells, and it appears that differentiation plays a role in determining whether NK or NC resistance will be lost upon transformation. A model is proposed in which the differentiation into a fibroblast associates the loss of NC resistance with transformation, whereas the differentiation into a lymphocyte associates the loss of NK resistance with transformation. Because the loss of NK resistance is not associated with the transformation of fibroblasts, they remain NK resistant, and because the transformation of lymphocytes is not associated with the loss of NC resistance, they remain NC resistant. This provides the basis for the target specificity exhibited by NK and NC effectors.     

Multiple splenic lymphoid cell subpopulations regulate H-2 antigen expression on teratocarcinoma cells in vivo

Undifferentiated murine 402AX teratocarcinoma cells do not express MHC antigens when passaged in vitro or in vivo in genetically susceptible host mice. When passaged in vivo in genetically resistant mice, however, the tumor cells become H-2b antigen positive regardless of the H-2 haplotype of the resistant host mouse. The present studies use monoclonal anti-H-2b antibodies to corroborate these earlier findings, which were performed with conventional antisera. Previous studies have established that host bone marrow plus lymphoid cells from resistant primed donors regulate tumor cell H-2b antigen expression. Using bone marrow and mature lymphoid cell reconstitution techniques, the present studies indicate that splenic Ig- cells from genetically resistant host mice are the most efficient lymphoid cell subpopulation in tumor cell H-2b antigen induction. Ig+ spleen cells also reconstitute the capacity to induce teratocarcinoma cell H-2 antigens but are less effective than Ig- spleen cells. Tumor cell H-2 antigen induction in C57BL/6 beige mice is impaired compared to C57BL/6 hosts, which suggests that host NK cells may also be involved in tumor cell H-2 antigen induction. Reconstitution of lethally irradiated resistant hosts for teratocarcinoma cell H-2 antigen expression requires bone marrow plus resistant primed lymphoid cell subpopulations; bone marrow alone is insufficient. These results indicate that multiple splenic lymphoid cell subpopulations requiring a radiosensitive host environment and/or factor for differentiation regulate teratocarcinoma 402AX H-2b antigen expression in vivo in genetically resistant mice.     

Involvement of Threonine Dehydratase in Biosynthesis of the α-Ketobutyrate Prosthetic Group of Urocanase

Seventeen mutants of Pseudomonas putida that were unable to grow on threonine as nitrogen source owing to a lack of threonine dehydratase were isolated, and all were found to be unable to synthesize active urocanase. Spontaneous revertants selected for urocanase production concomitantly regained threonine dehydratase. Mutants that were unable to utilize urocanate as carbon source were also isolated, and these were defective in urocanase formation but were normal in threonine dehydratase levels. Since alpha-ketobutyrate is the prosthetic group for urocanase, these results are consistent with the proposal that threonine dehydratase is necessary for urocanase prosthetic group biosynthesis. However, the lack of urocanase activity in threonine dehydratase-negative mutants was shown not to be the result of reduced levels of endogenous free alpha-ketobutyrate, nor to the participation of threonine dehydratase in the initiation of urocanase biosynthesis through the conversion of threonyl-tRNA(Thr) to alpha-ketobutyryl-tRNA(Thr). Other alternatives for the participation of threonine dehydratase in urocanase biosynthesis are discussed.     

CONSTANCY OF DNA CONTENT IN ADRENAL MEDULLA NUCLEI OF COLD-TREATED RATS

Reports of changes in DNA content of certain types of cells following exposure to conditions of stress has led to the suggestion that two kinds of DNA may be present. One is genetic DNA, and the other is called "metabolic" DNA. In a further attempt to investigate the possibility of this phenomenon, determinations of DNA content were made on Feulgen-stained nuclei of adrenal glands and kidneys in cold-treated rats. Feulgen-stained nuclei were measured by two-wavelength microspectrophotometry. Particular attention was given to the handling of the smears in hydrolysis and staining. Mean values of Feulgen-DNA contents in a total of 720 nuclei demonstrated (a) a constancy of DNA content within 2% in individual nuclei both in adrenal medulla and kidney cortex, (b) no more than an average of 2% difference in DNA content between control and experimental nuclei, and (c) no more than an average of 1.5% difference in DNA content between normal kidney cortex nuclei and normal adrenal medulla nuclei. These results confirm the view that the more precise the measurement, the more accurately the constancy rule is obeyed. Moreover, there is no support for the concept of a metabolic DNA in the rat adrenal medulla.     

CIRRHOSIS OF THE LIVER—Clinical Course in 2,377 Patients at San Francisco General Hospital

The records of 2,377 patients with Laennec''s cirrhosis were reviewed for the period 1947-1957. The chief presenting symptom was ascites in 46 per cent, bleeding in 23 per cent, coma in 18 per cent, jaundice in 9 per cent, and both jaundice and ascites in 4 per cent. Nearly half of the patients died during the period under study—one-third from hepatic failure, one-third from gastrointestinal bleeding, and one-third from other causes, most of which were related to alcoholism.Massive gastrointestinal bleeding occurred in 21 per cent of the patients at some time in their clinical course, and in the 10 per cent of these in whom ulcer was demonstrated, one-fifth died as a result of the hemorrhage. Of those presumed to be bleeding from esophageal varices, 64 per cent died at the first hemorrhage and 10 per cent at subsequent hemorrhages; 85 per cent of all those who bled from varices were dead at the end of one year, and 91 per cent were dead at the end of three years.The survival curve of a group of patients who bled once and were good operative risks but had received no operative treatment was compared to the survival curve for entire group who survived the first hemorrhage. The three-year survival in the good risk group was 47 per cent; for the group as a whole it was 30 per cent. The difference in mortality rate was primarily due to an increased number of deaths from hepatic failure in the combined group, whereas 60 per cent of the good risk group died of recurrent gastrointestinal hemorrhage.As 86 per cent of those who were to die of gastrointestinal bleeding did so at the first hemorrhage, it was concluded that any decided improvement in the salvage rate achievable by operation must come from some means of diagnostic forecast of the likelihood of bleeding, with resort to prophylactic operation in such cases.