Construction and expression of nonsense suppressor tRNAs which function in plant cells

An Arabidopsis thaliana L. DNA containing the tRNA(TrpUGG) gene was isolated and altered to encode the amber suppressor tRNA(TrpUAG) or the ochre suppressor tRNA(TrpUAA). These DNAs were electroporated into carrot protoplasts and tRNA expression was demonstrated by the translational suppression of amber and ochre nonsense mutations in the chloramphenicol acetyltransferase (CAT) reporter gene. DNAs encoding tRNA(TrpUAG) and tRNA(TrpUAA) nonsense suppressor tRNAs caused suppression of their cognate nonsense codons in CAT mRNAs, with the tRNA(TrpUAG) gene exhibiting the greater suppression under optimal conditions for expression of CAT. The development of these translational suppressors which function in plant cells facilitates the study of plant tRNA gene expression and will make possible the manipulation of plant protein structure and function.     

The GTPase stimulatory activities of the neurofibromatosis type 1 and the yeast IRA2 proteins are inhibited by arachidonic acid.

Three proteins, GTPase activating protein (GAP), neurofibromatosis 1 (NF1) and the yeast inhibitory regulator of the RAS-cAMP pathway (IRA2), have the ability to stimulate the GTPase activity of Ras proteins from higher animals or yeast. Previous studies indicate that certain lipids are able to inhibit this activity associated with the mammalian GAP protein. Inhibition of GAP would be expected to biologically activate Ras protein. In these studies arachidonic acid is shown also to inhibit the activity of the catalytic fragments of the other two proteins, mammalian NF1 and the yeast IRA2 proteins. In addition, phosphatidic acid (containing arachidonic and stearic acid) was inhibitory for the catalytic fragment of NF1 protein, but did not inhibit the catalytic fragments of GAP or IRA2 proteins. These observations emphasize the biochemical similarity of these proteins and provide support for the suggestion that lipids might play an important role in their biological control, and therefore also in the control of Ras activity and cellular proliferation.     

Regional assignment of the human uroporphyrinogen III synthase (UROS) gene to chromosome 10q25.2→q26.3

Summary Uroporphyrinogen III synthase [UROS; hydroxymethylbilane hydro-lyase (cyclizing), EC 4.2.1.75] is the fourth enzyme in the human heme biosynthetic pathway. The recent isolation of the cDNA encoding human UROS facilitated its chromosomal localization. Human UROS sequences were specifically amplified by the polymerase chain reaction (PCR) from genomic DNA of two independent panels of human-rodent somatic cell hybrids. There was 100% concordance for the presence of the human UROS PCR product and human chromosome 10. For each of the other chromosomes, there was 19%–53% discordance with human UROS. The chromosomal assignment was confirmed by Southern hybridization analysis of DNA from somatic cell hybrids with the full-length UROS cDNA. Using human-rodent hybrids containing different portions of human chromosome 10, we assigned the UROS gene to the region 10q25.2 q26.3.     

Phospholipids chiral at phosphorus: Fourier-transform infrared study on the gel-liquid crystalline transition of chiral thiophosphatidylcholine

Fourier-transform infrared spectroscopy (FT-IR) was used to study the structural properties of Rp, Sp, and Rp + Sp isomers of 1,2-dipalmitoyl-sn-glycero-3-thiophosphocholine (DPPsC), in comparison with those of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). For the vibrational modes of acyl chains, isomers of DPPsC show similar temperature and phase dependence to DPPC. However, the Rp isomer of DPPsC exhibits several unique properties: the CH2 symmetric stretching band is unusually weak, the CH2 asymmetric stretching band is unusually narrow, and the CH2 wagging bands do not disappear completely at temperatures above the main transition. These differences could imply a tighter packing and be responsible for the unique phase-transition property of (Rp)-DPPsC. For the vibrational modes of the thiophosphodiester group, the frequency of the P-O stretching mode of DPPsC suggests that the POS- triad exists predominantly in the mesomeric form. This is in contrast to the structure of nucleoside phosphorothioates where charge localization at sulfur has been demonstrated [Iyengar, R., Eckstein, F., & Frey, P. A. (1984) J. Am. Chem. Soc. 106, 8309-8310]. This suggests that the different biophysical properties between isomers of DPPsC are not due to different charge distribution in the POS- triad or different geometry of charge distribution on the membrane surface. Instead, factors such as size or hydration property of oxygen and sulfur, as well as the different configuration at phosphorus, could be responsible for the differences in the conformation and packing of acyl chains, as revealed by the different properties in the CH2 stretching and wagging modes of DPPsC.     

Retroperitoneal fibromatosis and acquired immunodeficiency syndrome in macaques: clinical and immunologic studies

A simian acquired immunodeficiency syndrome (SAIDS) associated with retroperitoneal fibromatosis (RF) has been observed in several species of macaque at the Washington Regional Primate Research Center. Clinical signs were recurrent diarrhea, weight loss, mesenteric lymphadenopathy, and opportunistic infections. Most affected macaques in the later stages of illness showed marked immunodeficiency. Response of peripheral blood mononuclear cells to mitogens was impaired significantly. There was sharply depressed primary and secondary antibody response to the T-cell dependent antigen, bacteriophage phi X174. Affected monkeys did not switch from IgM to IgG antibody following a secondary immunization, as did normal macaques. Twenty-four (67%) of 36 affected animals with progressive RF or deteriorated stages of illness had hypoproteinemia and hypoalbuminemia. Quantitative serum immunoglobulins of 23 cases showed that eight (35%) had hypogammaglobulinemia, six (26%) had hypergammaglobulinemia, and the remainder (39%) were within the normal range. Opportunistic infections were predominantly bacterial pathogens. Type D retrovirus appeared to be closely associated with RF-affected macaques (12/12 or 100%). The case fatality rate (including animals sacrificed after prolonged illness) was 98%. The leading cause of death was due directly to RF lesions in 43%, to enterocolitis in 36%, septicemia in 12%, amyloidosis in 5%, and malignant lymphoma (2%). Clinical, immunologic and pathologic changes reveal an acquired immunodeficiency syndrome that has many similarities to human AIDS. SAIDS and RF may be a useful model for studying human AIDS.     

Stimulation of facilitated [3H]uridine transport by thyroid hormone in GH1 cells. Evidence for regulation by the thyroid hormone nuclear receptor

We have previously shown that 3,5,3'-triiodo-L-thyronine (L-T3) stimulates cell growth and a 4- to 8-fold increase in growth hormone mRNA in GH1 cells. These effects appear to be mediated by a thyroid hormone nuclear receptor with an equilibrium dissociation constant for L-T3 of 0.2 nM and an abundance of about 10,000 receptors per cell nucleus. In this report, we show that L-T3 exerts a pleiotypic effect on GH1 cells to rapidly (within 2 h) stimulate [3H]uridine uptake to a maximal value of 2.5- to 3-fold after 24 h. This results from an increase in the number of functional uridine "transport sites" as shown by studies documenting an increase in the apparent Vmax with no change in the Km, 17 microM. Although the labeling of the cellular uridine pool and pools of all phosphorylated uridine derivatives was increased by L-T3, there was no change in the relative amounts of the individual pools in cells incubated with or without hormone. The intracellular concentration of [3H]uridine was estimated to be similar to that of the medium, suggesting that facilitated transport mediates [3H]uridine uptake. That this increase in [3H]uridine transport was nuclear receptor-mediated is supported by the excellent correspondence of the L-T3 dose-response curve for [3H]uridine uptake and that for L-T3 binding to receptor. Finally, inhibition of protein synthesis by cycloheximide and RNA synthesis by actinomycin D demonstrated that the L-T3 effect required continuing protein and RNA synthesis. These results are consistent with an effect of the L-T3-nuclear receptor complex to increase uridine uptake in GH1 cells by altering the expression of gene(s) essential for the transport process.     

Kinetic and spectroscopic studies of transhydrogenase activity and nucleotide site of lipoamide dehydrogenase

The kinetic behavior and spectroscopic characteristics of the nucleotide site(s) of lipoamide dehydrogenase have been investigated. Both subunits of the dimeric enzyme interact with NAD+. The binding of NAD+ is associated with a negative trough around 420-450 nm and a positive peak at 507 nm of the difference spectrum. The transhydrogenation between NADH and thionicotinamide nucleotide or acetylpyridine nucleotide is shown to proceed via a Ping Pong or an ordered Bi Bi mechanism, respectively, at pH above 7.0. Lowering pH or acetamidation lose the spectral characteristic of the positive peak of the enzyme-NAD+ complex with a concurrent change in the kinetic mechanism in the NADH+-acetylpyridine nucleotide transhydrogenation.     

Detailed profile of prolactin secretion in the immature female rat: evidence for the existence of an ultradian rhythm

The detailed profile of prolactin (PRL) secretion in 22-24 and 29-31 days old female rats was investigated by serial blood sampling through an intracardiac cannula at 15-min intervals for each of the 9 or 10-h periods beginning at 09.00 or 10.00 and 22.00 h. By analysis of the power spectrum and the least squares method the time series of PRL concentrations which were measured by RIA were found to have approximately a 3-h period ultradian rhythm in either sampling period of both the 22-24 and 29-31 days old rats. The peak times calculated based on the acrophase estimated through the calculation of periodicity were concentrated around 12.00, 15.00 and 18.00 h for the sampling period 10.00-19.00, and 24.00, 03.00 and 06.00 h for the sampling period 22.00-07.00 h. However, in more than half of the animals at 22-24 days of age, one secretory episode around 12.00 h, and two secretory episodes around 24.00 and 03.00 h had markedly small amplitudes, making the remaining secretory episodes distinct diurnal and nocturnal surges, respectively. In the animals at 29-31 days of age, the amplitudes of the PRL episodes occurring around 12.00 h were markedly small, making the remaining two episodes as diurnal surges, whereas the amplitudes of PRL secretory episodes during the period 22.00-07.00 h were analogous to each other. These findings indicate that the semicircadian rhythm of PRL secretion is established on the basis of PRL secretion with the 3.0-h period ultradian rhythm.