Tetrahymena contain two distinct and unusual high mobility group (HMG)- like proteins

Previous studies have described the existence of high mobility group (HMG)-like proteins in macronuclei of the ciliated protozoan, Tetrahymena thermophila (Hamana, K., and K. Iwai, 1979, J. Biochem. [Tokyo], 69:1097-1111; Levy-Wilson, B., M. S. Denker, and E. Ito, 1983, Biochemistry, 22:1715-1721). In this report, two of these proteins, LG- 1 and LG-2, have been further characterized. Polyclonal antibodies raised against LG-1 and LG-2 fail to cross react with each other or any other macronuclear polypeptide in immunoblotting analyses. As well, LG- 1 and LG-2 antibodies do not react with calf thymus, chicken, or yeast HMG proteins. Consistent with these results, a 47 amino-terminal sequence of LG-1 has been determined that shows limited homology to both calf thymus HMGs 1 and 2 and HMGs 14 and 17. Two internal sequences of V8 protease-generated peptides from LG-2 have been determined, and these do not share any homology to the LG-1 sequence or any other sequenced HMG proteins. Comparison of the partial sequences of LG-1 and LG-2 with the complete amino acid sequence of the Tetrahymena histone H1 (Wu, M., C. D. Allis, R. Richman, R. G. Cook, and M. A. Gorovsky, 1986, Proc. Natl. Acad. Sci. USA, 83:8674-8678) rules out the possibility that LG-1 and LG-2 are proteolytically derived from H1, the other major macronuclear perchloric acid-soluble protein. Interestingly, however, both LG-1 and LG-2 are efficiently extracted from macronuclei by elutive intercalation (Schroter, H., G. Maier, H. Ponsting, and A. Nordheim, 1985, Embo (Eur. Mol. Biol. Organ.) J., 4:3867-3872), suggesting that both may share yet undetermined properties with HMGs 14 and 17 of higher eukaryotes. Examination of the pattern of LG-1 and LG-2 synthesis during the sexual phase of the life cycle, conjugation, demonstrates that the synthesis of LG-1 and LG-2 is coordinately increased from basal levels during the differentiation of new macronuclei (7-13 h), suggesting that both of these proteins play a role in determining a macronuclear phenotype. However, a specific induction of LG-2 synthesis is detected in early stages of conjugation (meiotic prophase, 1-4 h), leading to maximal synthesis of LG-2 at 3 h. Interestingly, the early induction of LG-2 synthesis closely parallels the hyperphosphorylation of histone H1. Taken together, these data suggest that LG-1 and LG-2 are not strongly related to each other or to higher eukaryotic HMG proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

tRNA recognition site of Escherichia coli methionyl-tRNA synthetase

We have previously shown that anticodon bases are essential for specific recognition of tRNA substrates by Escherichia coli methionyl-tRNA synthetase (MetRS) [Schulman, L. H., & Pelka, H. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6755-6759] and that the enzyme tightly binds to C34 at the wobble position of E. coli initiator methionine tRNA (tRNAfMet) [Pelka, H., & Schulman, L. H. (1986) Biochemistry 25, 4450-4456]. We have also previously demonstrated that an affinity labeling derivative of tRNAfMet can be quantitatively cross-linked to the tRNA binding site of MetRS [Valenzuela, D., & Schulman, L. H. (1986) Biochemistry 25, 4555-4561]. Here, we have determined the site in MetRS which is cross-linked to the anticodon of tRNAfMet, as well as the location of four additional cross-links. Only a single peptide, containing Lys465, is covalently coupled to C34, indicating that the recognition site for the anticodon is close to this sequence in the three-dimensional structure of MetRS. The D loop at one corner of the tRNA molecule is cross-linked to three peptides, containing Lys402, Lys439, and Lys596. The 5' terminus of the tRNA is cross-linked to Lys640, near the carboxy terminus of the enzyme. Since the 3' end of tRNAfMet is positioned close to the active site in the N-terminal domain [Hountondji, C., Blanquet, S., & Lederer, F. (1985) Biochemistry 24, 1175-1180], this result indicates that the carboxy ends of the two polypeptide chains of native dimeric MetRS are folded back toward the N-terminal domain of each subunit.     

In vivo and in vitro phosphorylation of murine lymphocyte differentiation antigen CD5

Ly-1, the murine lymphocyte differentiation antigen CD5, is phosphorylated constitutively in vivo. This phosphorylation is enhanced by phorbol 12-myristate 13-acetate (PMA) treatment, but not by concanavalin A, Ca2+ ionophore or dibutyryl cAMP. Prolonged PMA treatment abolished PMA-induced Ly-1 phosphorylation but not constitutive phosphorylation, suggesting that protein kinase C (PKC) is responsible for this enhanced phosphorylation, but not the basal phosphorylation of Ly-1. Ly-1 is phosphorylated by PKC added to membranes, further supporting a role for protein kinase C in the in vivo phosphorylation of Ly-1.     

"Rates" of birth defects

The proportion of children born with a particular defect is not a "birth defect rate" but, rather, a prevalence proportion. The implications of confusing a rate and a proportion are discussed in terms of the interpretation of birth defect data. It is recommended that "prevalence proportion" or "prevalence" be used to report the frequency of various defects rather than the often-used "prevalence rate."     

Role of the Escherichia coli glnALG operon in regulation of ammonium transport.

Escherichia coli expresses a specific ammonium (methylammonium) transport system (Amt) when cultured with glutamate or glutamine as the nitrogen source. Over 95% of this Amt activity is repressed by growth of wild-type cells on media containing ammonia. The control of Amt expression was studied with strains containing specific mutations in the glnALG operon. GlnA- (glutamine synthetase deficient) mutants, which contain polar mutations on glnL and glnG genes and therefore have the Reg- phenotype (fail to turn on nitrogen-regulated operons such as histidase), expressed less than 10% of the Amt activity observed for the parental strain. Similarly, low levels of Amt were found in GlnG mutants having the GlnA+ Reg- phenotype. However, GlnA- RegC mutants (a phenotype constitutive for histidase) contained over 70% of the parental Amt activity. At steady-state levels, GlnA- RegC mutants accumulated chemically unaltered [14C]methylammonium against a 60- to 80-fold concentration gradient, whereas the labeled substrate was trapped within parental cells as gamma-glutamylmethylamide. GlnL Reg- mutants (normal glutamine synthetase regulation) had less than 4% of the Amt activity observed for the parental strain. However, the Amt activity of GlnL RegC mutants was slightly higher than that of the parental strain and was not repressed during growth of cells in media containing ammonia. These findings demonstrate that glutamine synthetase is not required for Amt in E. coli. The loss of Amt in certain GlnA- strains is due to polar effects on glnL and glnG genes, whose products are involved in expression of nitrogen-regulated genes, including that for Amt.     

Ca2+/calmodulin-dependent microtubule-associated protein 2 kinase: broad substrate specificity and multifunctional potential in diverse tissues

In previous studies, we described a soluble Ca2+/calmodulin-dependent protein kinase which is the major Ca2+/calmodulin-dependent microtubule-associated protein 2 (MAP-2) kinase in rat brain [Schulman, H. (1984) J. Cell Biol. 99, 11-19; Kuret, J. A., & Schulman, H. (1984) Biochemistry 23, 5495-5504]. We now demonstrate that this protein kinase has broad substrate specificity. Consistent with a multifunctional role in cellular physiology, we show that in vitro the enzyme can phosphorylate numerous substrates of both neuronal and nonneuronal origin including vimentin, ribosomal protein S6, synapsin I, glycogen synthase, and myosin light chains. We have used MAP-2 to purify the enzyme from rat lung and show that the brain and lung kinases have nearly indistinguishable physical and biochemical properties. A Ca2+/calmodulin-dependent protein kinase was also detected in rat heart, rat spleen, and in the ring ganglia of the marine mollusk Aplysia californica. Partially purified MAP-2 kinase from each of these three sources displayed endogenous phosphorylation of a 54 000-dalton protein. Phosphopeptide analysis reveals a striking homology between this phosphoprotein and the 53 000-dalton autophosphorylated subunit of the major rat brain Ca2+/calmodulin-dependent protein kinase. The enzymes phosphorylated MAP-2, synapsin I, and vimentin at peptides that are identical with those phosphorylated by the rat brain kinase. This enzyme may be a multifunctional Ca2+/calmodulin-dependent protein kinase with a widespread distribution in nature which mediates some of the effects of Ca2+ on microtubules, intermediate filaments, and other cellular constituents in brain and other tissues.     

Tissue Selenium in Pigs with Dietetic Microangiopathy (MAP, “Mulberry Heart”)

Mean selenium contents of liver, heart muscle and skeletal muscle of pigs with dietetic microangiopathy (MAP), but without waxy muscle degeneration (MD) and hepatosis diaetetica (HD), were 1113, 850 and 265 ng/g d.s., respectively. These values were not lower than corresponding values of control pigs without MAP, MD and HD.     

The reticulocyte-mediated release of iron and bicarbonate from transferrin: Effect of metabolic inhibitors

The effect of three groups of metabolic inhibitors on the incorporation of Fe and release of bicarbonate from transferrin by rabbit reticulocytes was measured. Inhibitors which affect reticulocyte Fe and transferrin uptake to the same extent (sodium arsenite, N-ethylmaleimide and iodoacetamide); those which inhibit reticulocyte Fe uptake to a greater extent than transferrin uptake (NaN₃, NaF, NaCN, rotenone, oligomycin, 2,4-dinitrophenol and cycloheximide); and compounds which after reticulocyte heme synthesis (CoCl₂, isonicotinic acid hydrazide and hemin) were used. In each case the effect on Fe incorporation and bicarbonate release was the sameThus, additional evidence has been obtained for the idea that the reticulocyte-mediated release of Fe and bicarbonate from transferrin are tightly coupled. The results are consistent with the hypothesis that an enzymatic attack on transferrin-bound bicarbonate is involved in the removal of Fe from transferrin by erythroid cells.     

Mid-trimester abortion with 15 (S) methyl prostaglandin F 2 alpha

The efficacy of intramuscular administration of 15 methyl (15S) prostaglandin F2alpha (PGF2a) in midtrimester pregnancy termination was evaluated in 16 healthy patients (mean age, 23.3; mean parity, 1.4; mean number of menstrual weeks, 16.1) by measuring dose response; oxytocin conversion; abortion time; side effects; intrauterine dynamics and progesterone withdrawal. Labor was monitored using extraovular balloon placed transvaginally; transcervically; and connected to a Physiograph machine. Patients not aborting within 48 hours after the first dose were considered failures. Blood samples were collected at 0, 3, and 6 hours and at abortion time for plasma progesterone measurement. Average dose given was 789 +or- 60 micrograms. Only 9 of 10 patients aborted within the prescribed 48 hours: 7 were complete abortions, and 2 were incomplete and required suction curettage. Mean induction to abortion time was 20.2 +or- 2.7 hours. Nausea, vomiting and diarrhea were the main side effects. The findings suggest that 15 methyl PGF2a in the dosages and routes prescribed is not as efficient as PGF2a. It is also suggested that prostaglandin affects the myometrium at 2 levels: 1) a membrane effect, and 2) a more fundamental intracellular regulatory effect which is necessary to initiate labor.