Characterization of a novel acidic protein of 38 kDa, A0, in yeast ribosomes which immunologically cross-reacts with the 13 kDa acidic ribosomal proteins, A1/A2

A new ribosomal protein of 38 kDa, named A0, was detected in yeast ribosomes on immunoblotting. The antibody used here was that against A1/A2, 13 kDa acidic ribosomal proteins which cross-reacted with A0. Although A0 and A1/A2 share common antigenic determinants, they differ in the following biochemical properties. While A1/A2 could be extracted from ribosomes with ethanol and ammonium sulfate, A0 could not. A0 gave two protein spots in a less acidic region than for A1/A2 on two-dimensional gel electrophoresis. The heterogeneity observed for A0 was ascribable to phosphorylation because one spot disappeared after treatment of the ribosomes with phosphatase. The syntheses of A0 and A1/A2 are directed by different mRNA species, as judged with a cell-free translation system, ruling out the possibility that A0 is a precursor of A1/A2. Although a mammalian ribosomal protein equivalent to A0 has been shown to be associated with 13 kDa acidic proteins in the cytoplasm, essentially no A0 was detected on immunoblotting in the yeast cytosol, while a small but detectable amount of A1/A2 was present. The possibility that A0 is a eukaryotic equivalent of L10 of Escherichia coli is discussed.     

Purification and properties of haloalkane dehalogenase from Corynebacterium sp. strain m15-3

A haloalkane dehalogenase was purified to electrophoretic homogeneity from cell extracts of a 1-chlorobutane-utilizing strain, m15-3, which was identified as a Corynebacterium sp. The enzyme hydrolyzed C2 to C12 mono- and dihalogenated alkanes, some haloalcohols, and haloacids. The Km value of the enzyme for 1-chlorobutane was 0.18 mM. Its molecular weight was estimated to be 36,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 33,000 by gel filtration. The isoelectric point was pH 4.5. The optimum pH for enzyme activity was found to be 9.4, and the optimum temperature was 30 to 35 degrees C. The enzyme was stable for 1 h at temperatures ranging from 4 to 30 degrees C but was progressively less stable at 40 and 50 degrees C.     

Activation of sea urchin sperm flagellar dynein ATPase activity by salt-extracted axonemes

When 21S dynein ATPase [EC 3.6.1.3] from sea urchin sperm flagellar axonemes was mixed with the salt-extracted axonemes, the ATPase activity was much higher than the sum of ATPase activities in the two fractions, as reported previously (Gibbons, I.R. & Fronk, E. (1979) J. Biol. Chem. 254, 187-196). This high ATPase level was for the first time demonstrated to be due to the activation of the 21S dynein ATPase activity by the axonemes. The mode of the activation was studied to get an insight into the mechanism of dynein-microtubule interaction. The salt-extracted axonemes caused a 7- to 8-fold activation of the 21S dynein ATPase activity at an axoneme : dynein weight ratio of about 14 : 1. The activation was maximal at a low ionic strength (no KCl) at pH 7.9-8.3. Under these conditions, 21S dynein rebound to the salt-extracted axonemes. The maximal binding ratio of 21S dynein to the axonemes was the same as that observed in the maximal activation of 21S dynein ATPase. The sliding between the outer doublet microtubules in the trypsin-treated 21S dynein-rebound axonemes took place upon the addition of 0.05-0.1 mM ATP in the absence of KCl. During the sliding, the rate of ATP hydrolysis was at the same level as that of the 21S dynein activated by the salt-extracted axonemes. However, it decreased to the level of 21S dynein alone after the sliding. These results suggested that an interaction of the axoneme-rebound 21S dynein with B-subfibers of the adjacent outer doublet microtubules in the axoneme causes the activation of the ATPase activity.     

B cell growth-promoting activity of recombinant human interleukin 4

Human interleukin 4 (IL-4), also known as B cell stimulatory factor 1, is a T cell-derived glycoprotein consisting of 129 amino acids for which a cDNA has been recently isolated. IL-4 displays little or no B cell growth factor (BCGF) activity in the standard anti-IgM costimulatory assay using suboptimal concentrations of soluble anti-IgM antibody whereas the low m.w. BCGF is very active. When insolubilized anti-IgM was used as the costimulating agent, both IL-4 and the low m.w. BCGF were found to promote B cell proliferation. Human IL-4 is able to induce the proliferation of B lymphocytes preactivated for either 1 day with insolubilized anti-IgM antibody or for 3 days with Staphylococcus aureus strain Cowan I. However, IL-4 is poorly mitogenic for B cells preactivated for 1 day with the Staphylococcus strain whereas the low m.w. BCGF strongly enhances the proliferation of these B cells. These two findings demonstrate that the preactivation signal necessary to induce human B cells to proliferate in response to IL-4 is critical. The increased tritiated thymidine ([3H]dThd) uptake in preactivated B cell cultures with IL-4 reflects cel proliferation because cell cycle analysis demonstrates that IL-4 induces activated B cells to enter the S and G2/M phases of the cell cycle and the addition of IL-4 to preactivated B cell cultures permits the recovery of three- to fourfold more B cells after 4 days of culture. IL-4 and the low m.w. BCGF act in concert to induce the proliferation of anti-IgM-preactivated B cells as demonstrated by [3H]dThd uptake and cell cycle analysis. In striking contrast to the demonstrated antagonistic effect of interferon-gamma on the IL-4-induced expression of the low affinity receptor for IgE (Fc epsilon RL/CD23), on B cells, it was found that interferon-gamma enhanced the IL-4-induced proliferation of anti-IgM-preactivated B cells. Finally, it was found that IL-4 had to be present continuously during the culture period to exert an optimal growth-promoting effect on B cell blasts. As a conclusion, IL-4 is able to induce the proliferation of an appropriately activated subpopulation of human B cells.     

Effect of Exogenous Gangliosides on Amino Acid Uptake and Na+,K+-ATPase Activity in Superior Cervical and Nodose Ganglia of Rats

The effects of some gangliosides on active uptake of nonmetabolizable alpha-aminoisobutyric acid (AIB) and Na+, K+-ATPase and Ca2+, Mg2+-ATPase activities in superior cervical ganglia (SCG) and nodose ganglia (NG) excised from adult rats were examined during aerobic incubation at 37 degrees C for 2 h. In NG, amino acid uptake was greatly accelerated with the addition of galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylgluc osyl ceramide (GM1) (85%) and also with N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide (GM2) or [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetyl- neuraminyl]-galactosylglucosyl ceramide (GD1a) (43% each) compared with a nonaddition control at a 5 nM concentration. Under identical conditions, Na+, K+-ATPase activity was strongly stimulated with GM1 (180%) and GD1a (93%), whereas Ca2+, Mg2+-ATPase activity showed no change. In SCG, on the other hand, AIB uptake was apparently inhibited (-27%) by addition of GM1, with a slight decrease in Na+, K+-ATPase but no change in Ca2+, Mg2+-ATPase activity in the tissue. Both asialo-GM1, in which N-acetylneuraminic acid is deficient, and Forssman glycolipid, which is not present in nervous tissue, failed to produce any significant increase in both SCG and NG not only in amino acid uptake, but also in Na+, K+-ATPase activity. A kinetic study of active AIB uptake showed that GM1 ganglioside produced an increase in Km with no change in Vmax in SCG, whereas it caused a decrease in Km with a slight increase in Vmax in NG. Treatment of NG and SCG with neuraminidase from Vibrio cholerae, an enzyme that split off sialic acid from polysialoganglioside, leaving GM1 intact, caused little inhibition of the amino acid uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular transport of albumin through the secretory apparatus of rat liver parenchymal cells. An immunocytochemical study

The fine structural localization of albumin in rat liver parenchymal cells was determined by an improved immunocytochemical method and serial sectioning. Albumin in the secretory apparatus of the parenchymal cells was present in segments of the rough endoplasmic reticulum, interrupted with negative segments, in transport vesicles, Golgi saccules, finely anastomosed tubules and vesicles on the trans side of the Golgi complex, and in secretion granules. Horizontally sectioned Golgi saccules contained lipoprotein particles on one side and albumin on the other side. After transport, the vesicles that contained albumin fused with the so-called rigid lamellae on the trans-side of the Golgi complex. Ultrathin serial sections revealed no true structural continuity between the endoplasmic reticulum and the cis-aspect of the Golgi complex. We concluded that secretory proteins are transported from the endoplasmic reticulum to the Golgi complex by transport vesicles that bud from the endoplasmic reticulum and fuse with the Golgi saccules. These vesicles fuse regularly with the Golgi saccules on the cis-side and occasionally with tubular elements on the trans-aspect that may belong to the so-called GERL.     

High affinity binding of human interleukin 4 to cell lines

Purified human recombinant interleukin 4 (IL-4) was radio iodinated to high specific radioactivity without loss of biological activity. 125I-IL-4 bound specifically to the Burkitt lymphoma Jijoye cells and other cell lines. Jijoye cells showed a high affinity for 125I-IL-4 (Kd approximately equal to 7 10(-11) M) and displayed 1200-1400 specific receptors per cell at 4 degrees C or 37 degrees C. The equilibrium dissociation constant (Kd) corresponds to the IL-4 concentration which induces 50% maximal expression of the low affinity IgE receptor (Fc epsilon RL/CD23) on Jijoye cells. At 4 degrees C the rate constant of association K1 is 1.7 x 10(6) M-1 s-1 and the rate contant of dissociation k -1 is 1.3 x 10(-4) s-1 (t 1/2 = 91 min.) No human recombinant lymphokines other than IL-4 were able to compete for the binding of 125I-IL-4 to its receptor.     

Characterization of a polysaccharide component of lipopolysaccharide from Pseudomonas aeruginosa IID 1008 (ATCC 27584) as D-rhamnan

Structural studies were carried out on a rhamnose-rich polysaccharide isolated from the O-polysaccharide fraction of lipopolysaccharide in Pseudomonas aeruginosa IID 1008 (ATCC 27584) after destruction of the major O-specific chain by alkaline treatment. The isolated polysaccharide contained rhamnose, 3-O-methyl-6-deoxyhexose, glucose, xylose, alanine, galactosamine and phosphorus in a molar ratio of 67:6.9:4.3:2.1:1.1:1.0:4.1. Data from analysis involving Smith degradation, methylation, 1H-NMR spectroscopy and optical rotation measurement showed that the polysaccharide was built up of three moieties, a rhamnan chain composed of about 70 D-rhamnose residues, the core chain and an oligosaccharide chain comprising 3-O-methyl-6-deoxyhexose, xylose, rhamnose and probably glucose. The repeating unit of the rhamnan chain was indicated to have the following structure:----3)D-Rha(alpha 1----3)D-Rha(alpha 1----2)D-Rha(alpha 1----. This structure is identical with that proposed previously for the repeating unit of the side chain of lipopolysaccharide from plant pathogenic bacteria Pseudomonas syringae pv. morsprunorum C28 [Smith, A.R.W., Zamze, S.E., Munro, S.M., Carter, K. J. and Hignett, R.C. (1985) Eur. J. Biochem. 149, 73-78].     

The complete sequence of the rice (Oryza sativa) chloroplast genome: intermolecular recombination between distinct tRNA genes accounts for a major plastid DNA inversion during the evolution of the cereals

Summary The entire chloroplast genome of the monocot rice (Oryza sativa) has been sequenced and comprises 134525 bp. Predicted genes have been identified along with open reading frames (ORFs) conserved between rice and the previously sequenced chloroplast genomes, a dicot, tobacco (Nicotiana tabacum), and a liverwort (Marchantia polymorpha). The same complement of 30 tRNA and 4 rRNA genes has been conserved between rice and tobacco. Most ORFs extensively conserved betweenN. tabacum andM. polymorpha are also conserved intact in rice. However, several such ORFs are entirely absent in rice, or present only in severely truncated form. Structural changes are also apparent in the genome relative to tobacco. The inverted repeats, characteristic of chloroplast genome structure, have expanded outward to include several genes present only once per genome in tobacco and liverwort and the large single copy region has undergone a series of inversions which predate the divergence of the cereals. A chimeric tRNA pseudogene overlaps an apparent endpoint of the largest inversion, and a model invoking illegitimate recombination between tRNA genes is proposed which accounts simultaneously for the origin of this pseudogene, the large inversion and the creation of repeated sequences near the inversion endpoints.