Role of uptake hydrogenase in providing reductant for nitrogenase in Rhizobium leguminosarum bacteroids

The role of uptake hydrogenase in providing reducing power to nitrogenase was investigated in Rhizobium leguminosarum bacteroids from nodules of Pisum sativum L. (cv. Homesteader). H₂ increased the rate of C₂H₂ reduction in the absence of added substrates. Malate also increased nitrogenase (C₂H₂) activity while decreasing the effect of H₂. At exogenous malate concentrations above 0.05 mM no effect of H₂ was seen. Malate appeared to be more important as a source of reductant than of ATP. When iodoacetate was used to minimize the contribution of endogenous substrates to nitrogenase activity in an isolate in which H₂ uptake was not coupled to ATP formation, H₂ increased the rate of C₂H₂ reduction by 77%. In the presence of iodoacetate, an ATP-generating system did not enhance C₂H₂ reduction, but when H₂ was also included, the rate of C₂H₂ reduction was increased by 280% over that with the ATP-generating system alone. The data suggest that, under conditions of substrate starvation, the uptake hydrogenase in R. leguminosarum could provide reductant as well as ATP in an isolate in which the H₂ uptake is coupled to ATP formation, to the nitrogenase complex.     

A 250-kilodalton cellular protein is induced by progestins in two human breast cancer cell lines MCF7 and T47D

We have studied the effect of R5020, a synthetic progestin, on the biosynthesis of cellular proteins extracted from the MCF7 and T47D human breast cancer cells, using gel electrophoresis. R5020 stimulates the synthesis, as measured after [35S]-methionine labelling, and the accumulation, as shown by silver staining, of a protein of molecular weight approximately equal to 250,000. The increase of the labelled 250-kilodalton protein was rapid (3 hours) and after 3 days this protein represented approximately equal to 6% of the total cellular proteins (approximately equal to 1 microgram/150,000 cells). The induction of the 250-kilodalton protein was obtained by physiologically active concentrations of several progestins and high concentrations of 5 alpha-dihydrotestosterone but not by estradiol or dexamethasone. It was inhibited by R486 , a progestin antagonist, but not by flutamide, an androgen antagonist. These results indicate a mediation by the progesterone receptor. The 250-kilodalton protein appears to be an excellent probe to study in cell culture the mechanism of action of progestin on human cells.     

Serum indole-3-acetic acid in control subjects and newly abstinent alcoholics after an oral loading with L-tryptophan: a preliminary study using liquid chromatography with amperometric detection

Described in this paper is a rapid, isocratic assay for serum indole-3-acetic acid (IAA). The sample preparation involves only protein precipitation using sulfosalicylic acid, and the sensitivity of amperometric detection is in the picogram range. The chromatographic analysis time is approximately 4 min. The devised method was used for a longitudinal study of IAA levels in serum samples from control subjects and newly abstinent alcoholics. Dietary variations were eliminated by administering a 2.0-g loading dose of L-Trp to all subjects investigated. The results are presented in the form of cumulative frequency polygons. Preliminary data indicate no differences in IAA levels between newly abstinent alcoholics and control subjects.     

A diphenylmethane derivative specific for the antiestrogen binding site found in rat liver microsomes

A new para-diphenylmethyl derivative, N,N-diethyl-2-[(4-phenylmethyl)-phenoxy]-ethanamine·HCl (N,N-DPPE) has been synthesized which binds with high affinity to the anti-estrogen binding site found in male rat liver microsomes. However, no evidence of significant interaction with the estrogen receptor can be observed at or below 10 μM in rat uterine cytosols; 10 nM N,N-DPPE fails to significantly induce progesterone receptor in MCF-7 cells. Tamoxifen also binds to anti-estrogen binding site but, unlike N,N-DPPE, binds significantly to estrogen receptor at much loeer concentrations and induces MCF-7 progesterone receptor. This property of high affinity for anti-estrogen binding site but not for estrogen receptor may make N,N-DPPE an important probe for the study of anti-estrogen binding site and its biological relevance.     

Association of leukotriene B4 with the cytoskeleton of rabbit neutrophils. Effect of chemotactic factor and phorbol esters

Following its addition to a suspension of rabbit neutrophils, leukotriene B4 is rapidly (less than 1 min) recovered from the cytoskeletal fraction (Triton X-100 insoluble pellet) of these cells. The association of leukotriene B4 with the cytoskeleton can be competed with by leukotriene B4 itself and by 20-OH leukotriene B4 but not by 20-COOH leukotriene B4. In addition, the preincubation of the cells with fMet-Leu-Phe or with phorbol 12-myristate 13-acetate, but not with 4 alpha-phorbol 12,13-didecanoate, results in a greatly decreased association of leukotriene B4 with the cytoskeleton. These results suggest that a specific association between the leukotriene B4 receptors and the cytoskeleton may be involved in signal transduction in the leukotriene B4 stimulated neutrophils.     

Prothrombin fragment 1-membrane interactions: a calcium-43 NMR study

The 43Ca NMR spectra are reported for solutions of prothrombin fragment 1 in the presence and absence of phospholipid. The calcium NMR spectrum permits distinction between three thermodynamic classes of calcium-binding interactions. The calcium ion in the lipid-free solutions was labile, with maximum residence times estimated for the average protein site in the range of 0.5-1 ms. The calcium spectrum was sensitive to the protein association and the addition of phospholipid, which appears to sharpen the calcium specificity for the protein sites. The calcium NMR spectra in the presence of phospholipid are similar to those in lipid-free solutions, which suggests that the calcium ion remains labile in the lipid-protein complex.     

Assessment of a model for intron RNA secondary structure relevant to RNA self-splicing--a review

A widespread class of introns is characterized by a particular RNA secondary structure, based upon four conserved nucleotide sequences. Among such "class I" introns are found the majority of introns in fungal mitochondrial genes and the self-splicing intron of the large ribosomal RNA of several species of Tetrahymena. A model of the RNA secondary structure, which must underlie the self-splicing activity, is here evaluated in the light of data on 16 further introns. The main body or "core structure" of the intron always consists of the base-paired regions P3 to P9 with the associated single-stranded loops, with P2 present also in most cases. Two minority sub-classes of core structure occur, one of which is typical of introns in fungal ribosomal RNA. Introns in which the core structure is close to the 5' splice site all have an internal guide sequence (IGS) which can pair with exon sequences adjacent to the 5' and 3' splice sites to align them precisely, as proposed by Davies et al. [Nature 300 (1982) 719-724]. In these cases, the internal guide model allows us to predict correctly the exact location of splice sites. All other introns probably use other mechanisms of alignment. This analysis provides strong support for the RNA splicing model which we have developed.     

The effect of inorganic phosphate on sodium fluxes in dog red blood cells

The effect of extracellular inorganic phosphate on Na⁺ movements in dog red blood cells has been studied. As the phosphate concentration is increased from 0 to 30 mM, Na⁺ efflux increases by 2- to 3-fold and Na⁺ influx increases approximately 2-fold. This enhancement of Na⁺ fluxes by phosphate can be prevented by the addition of iodoacetate (1 mM), an inhibitor of glycolysis, or 4-acetamido-4′-iso-thiocyantostilbene-2,2′-disulfonic acid (0.01 mM), which blocks anion transport, to the medium. The increases in Na⁺ movements are not caused by changes in cell volumes. These results suggest that phosphate must enter the cell to enhance Na⁺ fluxes and that the mechanism of action may be via a stimulatory effect on glycolysis.     

Prolactin increases the density of striatal dopamine receptors in normal and hypophysectomized male rats

Administration of prolactin to adult male rats, by s.c. injection, significantly increases the density of the striatal dopamine (DA) receptors, without altering the apparent affinity of the receptors for [³H]spiroperidol. Larger doses of prolactin are required to increase the density of the striatal DA receptors in hypophysectomized rates compared to normal rats. These results suggest that prolactin might be the common mediator of the increase in striatal DA receptor density produced by either estrogen or haloperidol administration. Monitoring and/or altering prolactin levels might be informative in neurologic or psychiatric disorders involving striatal DA neurotransmission.