Mitogen Synergism in low-responding CBA/CaJ mice.

Although neither phytohemagglutinin (PHA) nor concanavalin A (Con A) stimulated blood cultures in vitro from low-responding CBA/CaJ mice effectively, a mixture of PHA and Con A over a range of concentrations stimulated a response from CBA/CaJ mouse blood that was greater than the sum of the responses produced by using PHA or Con A individually. This synergistic effect was expressed as the percentage by which the responses to the PHA and Con A mixture exceeded the sum of the responses to PHA alone and Con A alone. When the mitogen concentrations that gave maximum responses individually were used, the synergistic effect averaged 319% in cultures of blood from low-responding CBA/CaJ mice. Apparently simultaneous exposure to PHA and Con A stimulates DNA synthesis in white blood cells of CBA/CaJ mice that fail to respond to either mitogen alone.     

Characterization of prostaglandin production in tissue culture of rat renal medullary cells

Renal medullary cells from the rat were used to establish a cell culture line. The morphologic characteristics of these cells were similar to renal medullary interstitial cells. They produced substantial amounts of PGE when provided with arachidonic acid or fetal calf serum. PGE production was inhibited 80–90% by indomethacin or meclofenamate. PGE release by the cells was sensitive to and stimulated by changing the culture media. Stable levels of PGE in the media could be achieved if media changes were avoided during the experimental period.     

Increased PRPP synthetase activity in cultured rat hepatoma cells containing mutations in the hypoxanthine-guanine phosphoribosyltransferase gene.

Nine independently derived clones of mutagenized rat hepatoma cells selected for resistance to 6-mercaptopurine (6-MP) or 6-thioguanine (6-ThioG) have been isolated. Each has severely reduced catalytic activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and seven of them possess significantly increased activities of phosphoribosylpyrophosphate (PRPP) synthetase. The degrees of elevations of PRPP synthetase activities do not correlate with the degrees of deficiencies of HPRT activities. The cells from one of these clones, 1020/12, posses 40% of the normal HPRT catalytic activity and overproduce purines. We have extensively examined the cells from this clone. Immunotration studies of 1020/12 cells indicate that there is a mutation in the structural gene for HPRT. Although they possess increased specific catalytic activities of the enzyme. PRPP synthetase, the catalytic parameters, heat stability, and isoelectric pH of PRPP synthetase from 1020/12 cells are indistinguishable from those of the enzyme from wild-type cells. The cause of purine overproduction by 1020/12 cells appears to be the elevated PRPP synthetase activity, rather than a PRPP "sparing" effect stemming from reduced HPRT activity. Support for this idea is provided by the observation that the complete loss of HPRT activity in a clone derived from 1020/12 cells does not further enhance the levels of PRPP synthetase or purine overproduction. We propose that the elevated levels of PRPP synthetase activity in these HPRT deficient cells result from a mutational event in the structural gene for HPRT, and that this causes the disruption of a previously undescribed regulatory function of this gene on the expression of the PRPP synthetase gene.     

Inactivation of porcine heart cytoplasmic malate dehydrogenase by pyridoxal 5'-phosphate.

Pyridoxal 5'-phosphate (pyridoxal-5'-P) has been found to act as a bifunctional reagent during the inactivation of porcine heart cytoplasmic malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37). The biphasic kinetics and X-azolidine-like structure formed were similar to those observed for mitochondrial malate dehydrogenase (Wimmer, M.J., Mo, T., Sawyers, D.L., and Harrison, J.H. (1975) J. Biol. Chem. 250, 710-715). In the cytoplasmic enzyme, however, irreversible inactivation representing X-azolidine formation was found to be the dominant characteristic of the interaction with pyridoxal-5'-P. Spectral evidence indicated that at total inactivation 2 mol of pyridoxal-5'-P were incorporated per mol of enzyme or one pyridoxal-5'-P per enzymatic active site. The presence of NADH protected the enzyme from inactivation suggesting interaction of pyridoxal-5'-P at or near the enzymatic active centers of this enzyme. Fluorometric titrations indicated that pyridoxal-5'-P-inactivated enzyme failed to bind NADH or at least failed to bind NADH in the same fashion as native enzyme.     

Radioimmunoassay studies of intestinal calcium-binding protein in the pig. I. Identification of intestinal calcium-binding protein in blood and response to a low calcium diet.

We have developed a radioimmunoassay for porcine intestinal calcium-binding protein (CaBP) and have used it to detect CaBP in pig plasma. Plasma CaBP is identical to intestinal CaBP on the basis of immunological activity, molecular size, and molecular charge properties. The plasma CaBP concentration was greater in the portal blood than in mixed venous blood, suggesting that blood CaBP originates in the gut. Two of four 15-week-old littermate pigs were placed on a low calcium diet (0.15% calcium, 0.65% phosphorus) and two on a control diet (0.65% calcium, 0.65% phosphorus). After 2 weeks, the entire small intestine was removed and divided into nine 1.8-m segments. CaBP was assayed in both plasma and intestinal mucosa. When the two pigs on a low calcium diet were compared with two control pigs, there was a general increase in immunoreactive CaBP in both plasma and intestinal mucosa. However, there was no increment in immunoreactive CaBP in the first 1.8-m segment of small intestine. Seventy-one percent of the increment in CaBP occurred distal to the first two segments. The largest fractional low calcium diet effect occurred in the ileum. The mean CaBP concentration for the total small intestine increased by a factor of 1.9. The plasma CaBP concentration increased by a factor of 2.6. In these pigs, plasma CaBP was a more reliable indicator of change in CaBP status than was the measurement in the proximal gut segment which contained the duodenum. The assay of CaBP in blood is convenient and may obviate the sampling errors inherent in intestinal biopsy.     

Appearance of lymphocyte surface markers and functional responses in neonatal and young rabbit spleens

We examined spleen cells from newborn to 1-month-old rabbits for easily detectable surface immunoglobulin, complement receptors, and for in vitro proliferative responsiveness to anti-immunoglobulin antisera and several mitogens. From birth through the first month of life about 15% of the cells from rabbit spleens had easily detectable surface immunoglobulin while about 45% had C3 receptors. In adults as many as 77% of the spleen cells had easily detectable surface Ig but the proportion with C3 receptors remained about 45%. The proliferative response to anti-allotype antisera was present at birth, and was at adult levels by 1 month of age. The proliferative response to pokeweed mitogen was low when cells were obtained during the first week of life but was comparable in magnitude to the response of adult cells by 2 weeks of age. In vitro responsiveness to concanavalin A was present at low levels at birth and increased sharply during the first week. We did not observe significant stimulation of spleen cells from neonatal to 4-week-old rabbits by lipopolysaccharide from Salmonella typhosa. Our data suggest that lymphocyte surface markers and functional responses appear asynchronously in spleen cells of developing rabbits.     

ORDINAL AFFINITIES OF THE AEGIALORNITHIDAE

The Aegialornithidae of the Eocene-Oligocene, previously regarded as swifts, have been transferred by Brodkorb (1971) to the nightjar order Caprimulgiformes. The fossil forelimb bones representing this early family have now been compared with those of recent species of the true swifts and tree swifts (Apodi in Apodiformes) and with nightjars, Oilbirds and frogmouths (Caprimulgiformes). The corresponding bones in swifts differ consistently from those of nightjars in the greater development of various prominences for muscle attachment. In all critical characters the fossil bones resemble those of swifts, and it is concluded that the Aegialornithidae should be reinstated as a family of the Apodiformes.     

Chromosome Studies in Wild Populations of D. MELANOGASTER

Chromosome studies of wild D. melanogaster populations from Missouri, Mississippi, Louisiana and Texas uncovered 58 inversions. Six were common and cosmopolitan; 52 were new, rare and generally endemic. In one of two Missouri populations tested, structurally heterozygous females carried significantly more sperm at capture than did the homozygotes. In both populations comparisons of wild sperms with the females carrying them indicated significant positive assortative mating and an excess production of homozygotes among the F₁ progeny. Wild females structurally heterozygous in up to three major autosomal arms showed no associated nondisjunctional egg lethality; those heterozygous in all four arms produced from 0% to 24% dead eggs, suggesting the presence of intrapopulational gene modifiers of meiosis. Texas populations supported on windfall citrus fruit showed a slight but significant difference in inversion frequencies between flies breeding on oranges and those breeding on grapefruit. Within these populations inversions were not distributed at random among individuals; rather there was an observed excess of individuals carrying intermediate numbers, and a deficiency of those carrying very few or very many inversions. While there was no significant linkage disequilibrium associated with this central tendency, there was a significant interchromosomal interaction: flies carrying inversions in chromosome 2 tended not to carry them in chromosome 3, and vice versa.     

Survival of marrow allografts inW/W v anemic mice: Effect of disparity at theEa-2 locus

Erythrocyte producing tissues of genetically anemicW/W ^v mice were completely populated and the anemic mice were permanently cured by marrow cell grafts from donors that differed at theEa-2 locus. Circulating erythrocytes contained ≧ 80% donor Ea-2 antigen and ≧ 90% donor hemoglobin. Population occurred without immunosuppressive treatment. No sign of graft-versus-host reaction was observed, although donors were congenic with one parent strain of the F₁ hybrid recipients. When partially congenic donors carrying the T6 chromosome marker were used, the immune systems of curedW-anemic mice were populated to an appreciable extent by donor cells. The dependence of cure upon route of injection and the persistence of functioning donor type cells across the strong immunological barrier of the Ea-2 system are discussed.     

The relation between lipid mobility and the specific hormone binding of thyroid membranes.

1. The specific binding of thyroid-stimulating hormone to isolated human thyroid membranes was examined under a variety of conditions. 2. In phosphate-saline buffer (in the presence of 0.14 M-NaCl) on increasing the temperature the binding of the hormone is increased, the plots of bound/free hormone against temperature showing a distinct break around 30 degrees C. 3. Detailed analysis showed that the increased binding is associated with an increase in the number of binding sites. 4. The motional characteristics of three membrane-bound fluorescent probes, 2-(9-anthroyl)palmitic acid, 12-(9-anthryl)stearic acid and N-1-naphthyl-N-phenylamine, were also examined as a function of temperature by measuring both fluorescence polarizations and lifetimes. 5. The results indicated that the 'fluidity' of membrane lipids also increased with temperature. The temperature-dependence of this property also shows a change at about 30 degrees C. 6. Bivalent cations decreased both membrane fluidity and hormone binding. 7. Similar correlations were found between the binding of adrenocorticotrophic hormone and the fluidity of the plasma membranes obtained from adrenal-cortical cells, with the discontinuity occurring in this case at 23 degrees C. 8. The possibility of lipid mobility being important in controlling hormone-receptor function is discussed.