Radial spokes of Chlamydomonas flagella: polypeptide composition and phosphorylation of stalk components

Polypeptides from flagella or axonemes of Chlamydomonas reinhardtii were analyzed by labeling cellular proteins by prolonged growth on 35S- containing media and using one- and two-dimensional electrophoretic techniques which can resolve greater than 170 axonemal components. By this approach, a paralyzed mutant that lacks axonemal radial spokes, pf14, has been shown to lack 17 polypeptides in the molecular weight range of 20,000 to 124,000 and in the isoelectric point range of 4.8- 7.1. Five of those polypeptides are also missing in the mutant pf-1 which lacks only radial spokeheads. The identification of the 17 polypeptides missing in pf-14 as components of radial spoke structures and the localization of the polypeptides lacking in pf-1 within the spokehead, are supported by experiments of chemical dissection of wild- type axonemes. Extraction procedures that solubilize outer and inner dynein arms preserve the structure of the radial spokes along with the 17 polypeptides in question. Six radial spoke polypeptides are solubilized in conditions that cause disassembly of radial spokeheads from the stalks and those components include the five polypeptides missing in pf-1. No Ca++- or Mg++-activated ATPase activities were found to be associated with solubilized preparations of wild-type radial spokeheads. In vivo pulse 32P incorporation experiments provide evidence that greater than 80 axonemal components are labeled by 32P and that five of the radial spoke stalk polypeptides are modified to different extents.     

Defective Fc-mediated phagocytosis in C3H/HeJ macrophages. II. Correction by cAMP agonists

Peritoneal macrophages from LPS hyporesponsive C3H/HeJ mice lose the capacity to bind and phagocytose opsonized sheep erythrocytes (EA) over a 48-hr culture period. This loss in Fc receptor capacity is markedly different from the progressive increase in phagocytic ability exhibited by cultured macrophages derived from LPS-responsive C3H/HeN mice. Since dibutyryl-cyclic adenosine monophosphate (DBcAMP) has previously been reported to modulate membrane receptor expression in lymphocytes and certain macrophage-like cell lines, we examined its effects on EA binding and phagocytosis by C3H/HeJ macrophages. DBcAMP not only reverses the binding defect in C3H/HeJ macrophages but also restores EA phagocytosis to the level of control C3H/HeN cultures. 8-Bromo-cAMP, as well as other agents known to elevate intracellular cAMP (i.e., isoproterenol plus isobutylmethylxanthine or prostaglandin E2) also corrected the phagocytic defect. Since the C3H/HeJ macrophage phagocytic defect can also be reversed by in vitro stimulation with a lymphokine-rich culture supernatant, we examined the effect of this treatment on intracellular cAMP levels. Lymphokine treatment produced a 60% increase in the levels of macrophage intracellular cAMP. These findings suggest that the C3H/HeJ differentiation defect may be secondary to some abnormality in a cAMP dependent pathway.     

Effect of diet on the weight of brown adipose tissue in rodents

Interscapular brown adipose tissue (BAT) weighed more in rats given access to a solution of sucrose in addition to a nutritionally complete basal diet than in rats eating only a basal diet. This incremental effect of drinking sucrose solution occurs across a variety of dietary conditions. In the first experiment, rats were fed diets containing either 9%, 18%, 27% or 36% casein. Rats given access to a sucrose solution had significantly larger brown fat pads than controls when the diets contained 9 or 18% casein, but not when diets contained either 27% or 36% casein. The second experiment examined the weight of brown adipose tissue as a function of the type of protein and the percentage of fat in the diet. Animals given a sucrose solution had significantly more BAT than animals not given sucrose. Neither the type of protein (casein or soy protein) nor the percentage of fat (14.5% or 36.4%) in the diet influenced the weight of BAT. Animals given access to either a sucrose solution or a glucose solution had significantly heavier BAT than animals given access to a fructose solution, granulated sucrose or water.     

An evaluation of tests using DNA repair-deficient bacteria for predicting genotoxicity and carcinogenicity. A report of the U.S. EPA's Gene-TOX Program

The detection of DNA-damaging agents by repair-deficient bacterial assays is based on the differential inhibition of growth of repair-proficient and repair-deficient bacterial pairs. The various methodologies used are described and recommendations are made for their improved use. In a survey of the literature through April 1979, 91 of 276 papers evaluated contained usable data, resulting in an analysis of 611 compounds that had been assayed in 1 or more of 55 pairs of repair-proficient and repair-deficient strains. The results indicate that (1) a liquid suspension assay is more sensitive than a spot (diffusion) test. In a review of the Escherichia coli polA assay, 45 compounds that gave "No Test" in the spot test were clearly positive or negative in the liquid suspension assay. (2) Of the 21 compounds analyzed by the E. coli polA assay and by other E. coli repair-deficient strains (e.g., rec, uvr, hcr, and exr derivatives of WP2 and AB1157), 10 were in complete agreement in all strains except uvrA strains. This indicates that strains other than polA+/polA- are useful for detecting DNA-damaging agents. However, in selecting strains for use in these assays, care should be taken to consider repair pathway specificity for particular compounds. (3) There was a 78% correspondence between results obtained with E. coli polA and Bacillus subtilis (H17/M45, 17A/45T) rec assay and between E. coli polA and Proteus mirabilis. (4) In a comparison of test results with carcinogenicity data, 44 of 71 (62%) carcinogenic compounds assayed by the polA system were positive, 10 (14%) were negative, and 17 (24%) gave No Test or doubtful results. 7 carcinogens were assayed by other E. coli strains and all were positive. 56 carcinogens were assayed in B. subtilis: 24 (43%) were positive, 9 (16%) were negative, and 23 (41%) gave No Test or doubtful results. Of the 7 carcinogens assayed in P. mirabilis, 6 (86%) were positive and 1 (14%) was negative. (5) The results were analyzed with respect to chemical classes. E. coli polA detected the highest percentage of hydroxylamines and alkyl epoxides. The B. subtilis rec assay detected the highest percentage of nitrosamines and sulfur and nitrogen oxides. It is concluded that some of these test systems are effective tools for the detection of DNA-damaging and potentially carcinogenic compounds, especially if the assay is done in liquid suspension and if more than 1 pair of tester strains is used. Advantages and disadvantages of the assay are discussed and suggestions are made for improvements in the system.     

Neutral sugars in lipopolysaccharides ofRhizobium trifolii and its non-nodulating mutant

Summary The nodulatingRhizobium trifolii strain 24 and its non-nodulating mutant 24 nod^–3 have been examined. The exopolysaccharides of both cultures studied contained mannose, galactose and glucose at similar molar ratios. On the other hand some quantitative differences have been found between the lipopolysaccharides in respect of the composition of neutral sugars. Glucose and rhamnose were the main constituents of the nodulating strain 24, whereas rhamnose and galactose in non-nodulating mutant 24 nod^–3 deprived of the plasmid pWZ2.     

Unit activity of the reticular and relay nuclei of the cat thalamus

Relations between neurons of the reticular and specific relay nuclei of the thalamus were studied in cats immobilized with tubocurarine. Under the influence of stimulation of the reticular nucleus (RN) unit activity in the thalamic relay nuclei was found to be considerably modulated. Cases of the appearance of IPSPs (possibly of monosynaptic nature), evoked by stimulation of RN, in neurons of the ventroposterolateral nucleus (VPLN) and lateral geniculate body (LGB) are described. During simultaneous recording of unit activity in RN and VPLN or LGB by means of two electrodes interaction of several types was found: inhibition of discharges of VPLN or LGB neurons accompanied by excitation of RN neurons: alternation of excitation-inhibition in neuron pairs in RN and VPLN or RN and LGB during low-frequency afferent or cortical stimulation (in this case excitation of RN neurons is associated with inhibition of VPLN or LGB neurons), and strengthening of the discharge of VPLN or LGB neurons during excitation of RN neurons. The possibility of the existence both of direct monosynaptic inhibition of activity of VPLN or LGB relay neurons under the influence of excitation of RN neurons and of their inhibition by activation of hypothetical interneurons of the relay nuclei themselves is accepted.I. S. Beritashvili Institute of Physiology, Academy of Sciences of the Georgian SSR, Tbilisi. Translated from Neirofiziologiya, Vol. 13, No. 1, pp. 24–31, January–February, 1981.     

Distribution of horseradish peroxidase-labeled neurons giving rise to descending fiber systems in the basal ganglia and hypothalamus in cats

The distribution of neurons giving rise to various descending fiber systems to brain-stem structures in the basal ganglia (including amygdaloid nuclei) and hypothalamus of the cat was studied by the retrograde axonal transport of horseradish peroxidase method. Neurons in the medial part of the central nucleus and of the magnocellular part of the basal nucleus of the amygdaloid group were shown to send axons to the dorsal hippocampus, substantia nigra, lateral part of the central gray matter, and the mesencephalalic reticular formation and also to the region of the locus coeruleus and the lateral medullary reticular formation at the level of the inferior olives. The predominant source of projections to the hypothalamus and brainstem structures is the central amygdaloid nucleus, which also sends projections to the nucleus of the tractus solitarius, the dorsal motor nucleus of the vagus nerve, and the superior cervical segments of the spinal cord. Uncrossed fiber systems descending from the basal ganglia terminate at the level of the pons, whereas uncrossed and crossed fiber systems descending from the dorsal and ventromedial hypothalamus can be traced into the spinal cord. The possible role of nuclei of the amygdaloid group, the hypothalamus, and their efferent projections in the regulation of somatic and vegetative functions and also of complex behavioral reactions is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 13, No. 1, pp. 14–23, January–February, 1981.     

The Biosynthesis of Transfer Ribonucleic Acid in the Developing Rat Brain and in Cultured Glial Cells

Abstract: The biosynthesis of tRNA was investigated in cultured astroglial cells and the 3-day-old rat brain in vivo. In the culture system astrocytes were grown for 19 days and were then exposed to [³H]guanosine for 1.5–7.5 h; 3-day-old rats were injected with [³H]guanosine and were killed 5–45 min later. [³H]tRNA was extracted, partially purified, and hydrolyzed to yield [³H]-guanine and [³H]methyl guanines. The latter were separated from the former by high performance liquid chromatography and their radioactivity determined as a function of the time of exposure to [³H]guanosine. The findings indicate that labeling of astrocyte tRNA continued for 7.5 h and was maximal, relative to total RNA labeling, at 3 h, while in the immature brain tRNAs were maximally labeled at 20 min after [³H]guanosine administration. The labeling pattern of the individual methyl guanines differed considerably between astrocyte and brain tRNAs. Thus, [³H]1-methylguanine represented up to 35% of the total [³H]methyl guanine radioactivity in astrocyte [³H]tRNA, while it became only negligibly labeled in brain [³H]tRNA. Conversely, brain [³H]tRNA contained more [³H]N²-methylguanine than did astrocyte [³H]tRNA. Approximately equal proportions of [³H]7-methylguanine were found in the [³H]tRNAs of both neural systems. The [³H]methylguanine composition of brain [³H]tRNA was followed through several stages of tRNA purification, including benzoylated DEAE-cellulose and reverse phase chromatography (RPC-5), and differences were found between the [³H]methylguanine composition of RPC-5 fractions containing, respectively, tRNA^lys and tRNA^phe. The overall results of this study suggest that developing brain cells biosynthesize their particular complement of tRNAs actively and in a cell-specific manner, as attested by the significant differences in the labeling rates of their methylated guanines. The notion is advanced that cell-specific tRNA modifications may be a prerequisite for the successful synthesis of cell-specific neural proteins.     

Globin-coding sequences in the nuclear 28S pre-mRNA and in the cytoplasmic RNA of pigeon erythroid cells

The steady-state content of globin-coding sequences in nuclear and cytoplasmic RNA of pigeon erythroid cells was estimated by hybridization in the excess of nuclear 28S RNA and cytoplasmic poly(A) + RNA with [3H]DNA, synthesized on globin mRNA. Sequences of 9S globin mRNA are found in 0.06% of molecules of non-ribosomal 28S nuclear RNA (pre-mRNA) of erythroblasts and in 0.5% of molecules of non-ribosomal 28S nuclear RNA of reticulocytes. The content of globin mRNA in erythroblast cytoplasm is, respectively lower than in that of reticulocytes.