Protein kinases in brown adipose tissue of developing rats. I. GTP as nucleotide substrate for histone phosphorylation.

100 000 times g soluble extracts from interscapular brown adipose tissue catalyzed the transfer of the terminal phosphoryl group from GTP to histone. Maximal velocity was achieved only with both cyclic AMP and ATP present. The cyclic AMP dose-response curve was the same as for the ATP-utilizing enzyme, with maximum stimulation at 0.5 muM. ATP (1--100muM) increased the rate of histone phosphorylation with GTP as the radioactive substrate. Higher concentrations had a dilution effect similar to that of GTP on the ATP-utilizing enzyme. Similar effects were observed with ADP and AMP. The apparent Km values for histone were the same with both GTP and ATP as nucleotide substrates. The effects of pH, purified beef muscle kinase inhibitor and of NaCl were also the same. Maximum velocities of histone phosphorylation from ATP and those from GTP were almost the same in brown fat of all age groups testes, Separated on histone-Sepharose, the GTP-utilizing activity was absolutely dependent on the re-addition of the ATP-utilizing enzyme (a linear relationship with a slope of approx. 0.95). An extremely active nucleotide phosphotransferase activity was found in the same subcellular fraction. The rate of equilibration of the gamma-32-P between GTP and ATP could account for all the histone phosphorylation with [gamma-32-P] GTP. It is concluded that, in spite of the presence of nucleotide phosphotransferase and ATP-protein kinase activities, a direct transfer from GTP to a protein substrate cannot be excluded. Also, histone may not be the natural protein acceptor for GTP-linked phosphorylation.     

Protein phosphorylation in intact lymphocytes stimulated by Concanavalin A

The phosphorylation of proteins in intact mouse spleen lymphocytes was monitored following mitogenic activation. Little change in the autoradiographic patterns of phosphorylated protein fractionated by polyacrylamide gel electrophoresis occurred during the first 8 h after Concanavalin A (conA) treatment. The intensity of ³²P incorporation into two proteins of 135 000 and 150 000 mol. wt began to increase, relative to control cells, 10 h after conA treatment and was maximal at 50 h. This increased phosphorylation followed the rise in RNA synthesis but preceded the onset of DNA synthesis. In addition to this temporal link between enhanced phosphorylation of these proteins and the initiation of DNA synthesis, various agents which inhibited the onset of S phase also blocked the phosphorylation of both proteins. Such treatments included the displacement of conA from its surface receptors by α-methyl-mannoside (αMM), the omission of serum from the culture medium, and the presence of indomethacin. The similar time courses of phosphorylation and responses to various proliferation inhibitors supports the idea that the 135 000 and 150 000 mol. wt proteins have a common physiological function. These proteins may be involved in the progression of stimulated lymphocytes toward S phase, and their phosphorylation may be an important regulatory event in this sequence.     

Bacteria-derived human growth hormone lacks lipolytic activity in rat adipose tissue

Bacteria-derived human growth hormone (hGH) shows little $\text{in}\text{vitro}$ lipolytic activity in adipose tissue from fed rats. In adipose tissue from fasted rats no lipolytic activity is observed. However, bacteria-derived hGH increased serum free fatty acids after intraperitoneal administration to hypophysectomized rats to the same extent as purified pituitary hGH. The dose response of the bacteria-derived hGH tested for $\text{in}\text{vitro}$ insulin-like activity was very similar to the pituitary extracted material. Thus bacteria-derived hGH behaves in a manner indistinguishable from highly purified preparations of pituitary hGH.     

Human brown adipose cells in culture

Brown adipose tissue (BAT), obtained from the axillary and perirenal regions of newborns 24-48 h after death, was digested with collagenase and the free cells were cultured. Only the cultures of cells from tissue obtained later than 24 h post mortem were successful. These cells grew slowly to reach confluence. Their typical mitochondria gradually disappeared, being replaced by untypical mitochondria. After confluence, the cells accumulated large amounts of lipid in non-coalescent multivacuolar depots. This model can be useful for the study of the metabolic and morphological features of human brown fat cells.     

Immunofluorescent and autoradiographic analysis of the relationship between the presence of histone H5 and DNA synthesis in developing chick erythroid cells

Simultaneous detection of histone H5 by indirect immunofluorescence and of [³H]thymidine incorporation by autoradiography on the same preparations of developing erythroid cells have been used to precisely define the extent of correlation between the loss of nuclear activity and the presence of histone H5. It was found that from day 3–12 of embryonic life there are two successive waves of double-labelled cells. At some stages, as many as 30% of the cells which incorporate [³H]thymidine also contain histone H5. Thus, the simple presence of H5 cannot be sufficient to cause nuclear inactivation. A kinetic analysis of the appearance and disappearance of [³H]thymidine-labelled cells, containing histone H5, and cells which are positive for both markers is presented. The result is consistent with the interpretation that the appearance of H5 in the first wave of double labelled cells occurs just before the erythroid cells become metabolically inactive. These observations modify the concept that histone H5 functions uniquely or solely as a template repressor.     

The effect of 3-mercaptopicolinic acid on phosphoenolpyruvate carboxykinase (GTP) in the rat and guinea pig.

In vitro, 3-mercaptopicolinic acid inhibited phosphoenolpyruvate carboxykinase activity in supernatant fractions of liver, kidney cortex, and adipose tissue obtained from fasted rats. 3-Mercaptopicolinic acid also inhibited enzymatic activity in the mitochondrial and supernatant fractions of liver obtained from fasted guinea pigs. In the fasted rat, the oral administration of 3-mercaptopicolinic acid increased liver carboxykinase activity even though the blood glucose concentrations decreased. Kidney cortex carboxykinase decreased while adipose tissue enzyme was unchanged. In the fasted guinea pig, the oral administration of 3-mercaptopicolinic acid lowered blood glucose concentrations but had no effect on liver mitochondrial or supernatant carboxykinase activity. The elevation in rat liver enzymatic activity appears to be due to protein synthesis, since the concurrent administration of cycloheximide prevents the increase in enzyme activity. 3-Mercaptopicolinic acid appears to be noncompetitive with respect to Mn²⁺.     

Effects of polyamines on in vitro phosphorylation and acetylation of histones of the cerebral cortex of rats of various ages

$\text{In}\text{vitro}$ phosphorylation and acetylation of histones and their modulation by spermine and spermidine were studied using slices of cerebral cortex of female rats of various ages. Phosphorylation and acetylation of individual histones decrease with increasing age. Spermine and spermidine have stimulatory effects on both the modifications of specific histones in immature rats. These effects decrease with increasing age. Such changes in covalent modifications of histones may alter gene expression and contribute to the aging process.     

Circadian periodicity of tissue glutathione and its relationship with lipid peroxidation in rats

Circadian fluctuations in tissue glutathione (GSH) concentrations and lipid peroxidation in male Sprague-Dawley rats were investigated. Blood and all the organs studied exhibited distinct circadian variation both in GSH concentrations and peroxidation of polyunsaturated fatty acids. There was a great variation among organs in the periodicity and amplitude of the fluctuations in GSH concentrations. Liver displayed the highest variation (approximately 50%) followed by stomach (approximately 37%), heart (approximately 25%) and kidney (approximately 19%). The changes in other organs were significant but of less magnitude. Implications of such variations and caution in interpretation of experimental results in response to the exposure of animals to xenobiotics are discussed.     

Arsenazo I and tetramethylmurexide as optical calcium indicators

Calcium-binding stoichiometry, dissociation equilibrium constants at zero ionic strength (K⁰), and molar extinction difference coefficients (Δ?_λ) at the wavelength λ of the metallochromic indicators arsenazo I (ArsI) and tetramethylmurexide (TMX) were reevaluated with a computerized method based on mass conservation and thermodynamic consistency checks. This new method is shown to provide a more critical assessment of the assumed calcium-dye complexing model than is afforded by the commonly used reciprocal-plot method. The analyses of spectrophotometric Ca titrations confirm that both dyes form only 1:1 complexes in aqueous solution. For TMX, K⁰ = 1.3 × 10^?3m and Δ?₄₈₀ = 1.5 × 10⁴m^?1 cm^?1; for ArsI, K⁰ = 5.8 × 10^?3m and Δ?₅₆₂ = 1.8 × 10⁴m^?1 cm^?1 at pH 7.0 and T = 293°K. The discriminatory power of the analytical method is demonstrated by comparison of these results with those found for a different dye, arsenazo III, which complexes Ca in 1:1, 1:2, and 2:1 forms.     

Size and sequence homology of masked maternal and embryonic histone messenger RNAs.

The messenger RNAs for five classes of histone proteins are shown by competitive RNA-DNA hybridization to be stored in the unfertilized egg of the sea urchin, Lytechinus pictus. The masked mRNAs for f2b, f2a2, f3 and f2al histones migrate in polyacrylamide slab gels with the same mobility as the histone mRNAs that are synthesized after fertilization and are found engaged in protein synthesis on polysomes. The masked maternal and embryonic mRNAs for histone f2a1 are identical in mobility when analyzed in a gel system capable of resolving differences estimated as small as 4–5 nucleotides in length. We conclude that these histone mRNAs synthesized during oogenesis and inactive prior to fertilization are not activated during embryogeny by alteration in their molecular size.     

Heterogeneous nuclear RNA (HnRNA) size in developing frog embryos.

Almost one-fifth of cells disaggregated from 16-cell mouse embryos neither divide nor die. On the basis of a number of criteria, it is concluded that these cells differentiate to trophoblast. Thus, neither cell-cell interaction nor cell division is required for execution of the programme for trophoblast differentiation from the 16-cell stage.     

A sensitive and specific enzyme assay for elastase activity using α-[3H]elastin as substrate

A method for the determination of elastase activity is described which uses soluble α-[³H]elastin as substrate. Soluble α-elastin was shown to have the same substrate specificity as natural insoluble elastin. At a substrate concentration of 1 mg/ml, approximately three times half-saturating substrate concentration, the assay is rapid, 1 h, sensitive, 10 ng/ml elastase, and linear up to an enzyme concentration of 250 ng/ml. The addition of 1000 μ/ml Trasylol or 10^?4mN-α-tosyl-l-lysyl chloromethane and 10^?4m tosyl-l-phenylalanyl chloromethane allowed the specific measurement of elastase activity in the presence of trypsin and chymotrypsin activity.     

Mitochondrial matrix granules in soft tissues. II. Isolation and initial characterization of a calcium-precipitable, soluble lipoprotein subfraction from brown fat and liver mitochondria

Degradation of ¹²⁵I-labelled HDL ([¹²⁵I]HDL) was measured in isolated rat hepatocytes that had been preincubated with [¹²⁵I]HDL and then reincubated in fresh medium without [¹²⁵I]HDL. About 5 % of the [¹²⁵I]HDL associated with the cells in advance were degraded per hour at 37 °C. This in vitro degradation was inhibited about 50% by lysosomal inhibitors such as chloroquine, ammonia and leupeptin. Depolymerization of microtubuli by colchicine inhibited the degradation of [¹²⁵I]HDL to about 65–75 % of the control cells. Cytochalasin B (CB), a destabilizer of microfilaments, had a less marked effect on the degradation in vitro. Degradation of [¹²⁵I]HDL associated with cells in vivo after intravenous injection was also studied in isolated cells. About 8.5% of the [¹²⁵I]HDL associated with the cells in vivo were degraded per hour in the isolated cells. The effects of ammonia, chloroquine, leupeptin and colchicine on HDL degradation were similar for [¹²⁵I]HDL taken up in vivo and in vitro. Subcellular fractionation by centrifugation in sucrose gradients indicated that [¹²⁵I]HDL associated with hepatocytes in vivo are primarily accumulated in lysosomes. [¹²⁵I]HDL associated with the cells in vitro are located in organelles whose distribution coincides with that of 5′-nucleotidase. These organelles may be endocytic vesicles. It is concluded that the internalization of [¹²⁵I]HDL in rat hepatocytes is relatively slow. The intracellular degradation of the apoproteins of HDL is at least partly lysosomal.     

The metaphase chromosome ultrastructure : I. Acute angle metal deposition technique as an appropriate use of shadow casting in chromosome structure investigation

The ultrastructure of the metaphase chromosome, isolated by spreading and dried by the critical point method, has been studied by the technique of rotary shadow casting at very acute angles, equivalent to deposition of metal on the specimens at mere grazing incidence. Acute angle deposition visualizes a three-dimensional image of the chromosome periphery and the substructure of the basic chromosome fibre. This paper describes the technical details of acute angle deposition as a particular use of shadow casting. The technique seems to be adequate for the study of the three-dimensional aspects of relatively large subjects and represents an alternative to scanning electron microscopy when the details under study are in the 150 Å range.     

The uptake of cyclic AMP by human erythrocytes and its effect on membrane phosphorylation.

The effects of time and cyclic AMP concentration on cyclic AMP uptake and membrane phosphorylation were studied using intact human erythrocytes. The rate of uptake of cyclic [³H]AMP was nearly linear with respect to cyclic AMP concentration. The amount taken up was small compared to the extracellular cyclic AMP concentration, but was sufficient to significantly increase the intracellular cyclic AMP concentration. Incubation with cyclic AMP resulted in increased incorporation of ³²P_i into several phosphorylated membrane peptides of the intact erythrocytes. Although cyclic AMP altered the distribution of radioactivity among the membrane components, the total amount of incorporation was not increased. The effect of cyclic AMP on phosphorylation of membrane peptides was observed with extracellular cyclic AMP concentrations as low as 1 μm and was most pronounced in incubations of 1 to 4 h. These results indicate that cyclic AMP can enter erythrocytes in sufficient amounts to alter the activity of cyclic AMP-dependent protein kinases, or to alter the rate of turnover of certain phosphorylated membrane peptides.     

Changing rates of histone mRNA synthesis and turnover in Drosophila embryos

The rates of synthesis and turnover of histone mRNA in Drosophila embryos were determined by hybridization of in vivo and in vitro labeled embryonic RNA to Drosophila histone DNA of the recombinant plasmid cDm500. There is a large store of maternal histone mRNA, equivalent to at least 7 X 10(7) copies of each of the five classes of histone mRNA per embryo. Embryonic synthesis of histone mRNA begins at 90 min after oviposition, making the histone genes among the first to be transcribed by embryonic nuclei. Embryonic histone mRNA accumulates rapidly during the blastoderm and gastrula stages. The peak in the rate of histone mRNA synthesis per embryo coincides with the peak in the rate of DNA synthesis per embryo, which occurs at 6 hr after oviposition. After 6 hr, as the rate of DNA synthesis per embryo decreases, the rate of histone mRNA synthesis and the total mass of histone mRNA per embryo both drop sharply. The rate of histone mRNA synthesis per gene falls more than 60 fold in the first 13 hr after oviposition, from 1.3 -2.5 copies per gene-min at 2 hr to 0.02-0.03 copies per gene-min at 13 hr. From measurements of the mass of histone mRNA per embryo and of the rate of accumulation of newly synthesized histone mRNA at a number of stages of early embryogenesis we determined that the cytoplasmic half-life of histone mRNA decreases approximately 7 fold during early Drosophila development, from 2.3 hr at blastoderm to 20 min by the end of gastrulation. Thus the level of expression of histone genes in Drosophila development is controlled not only by the size of the maternal mRNA pool and changes in the rate of histone mRNA synthesis, but also by changes in the rate of histone mRNA turnover.     

Collagen in developing chick muscle in vivo and in vitro

Because of the known role of collagen in chick skeletal muscle differentiation the collagen synthesized by embryonic chick muscle was studied. The major collagen synthesized by this muscle was found to be type I collagen. In addition, the effectiveness of types I, II, III and IV collagens in promoting myoblast fusion in vitro was compared. These collagens were found to be equally effective as in vitro substrates.     

Pyridine nucleotide transhydrogenases of parasitic helminths.

Mitochondria from the parasitic helminth, Hymenolepis diminuta, catalyzed both NADPH:NAD⁺ and NADH:NADP⁺ transhydrogenase reactions which were demonstrable employing the appropriate acetylpyridine nucleotide derivative as the hydride ion acceptor. Thionicotinamide NAD⁺ would not serve as the oxidant in the former reaction. Under the assay conditions employed, neither reaction was energy linked, and the NADPH:NAD⁺ system was approximately five times more active than the NADH:NADP⁺ system. The NADH:NADP⁺ reaction was inhibited by phosphate and imidazole buffers, EDTA, and adenyl nucleotides, while the NADPH:NAD⁺ reaction was inhibited only slightly by imidazole and unaffected by EDTA and adenyl nucleotides. Enzyme coupling techniques revealed that both transhydrogenase systems functioned when the appropriate physiological pyridine nucleotide was the hydride ion acceptor. An NADH:NAD⁺ transhydrogenase system, which was unaffected by EDTA, or adenyl nucleotides, also was demonstrable in the mitochondria of H. diminuta. Saturation kinetics indicated that the NADH:NAD⁺ reaction was the product of an independent enzyme system. Mitochondria derived from another parasitic helminth, Ascaris lumbricoides, catalyzed only a single transhydrogenase reaction, i.e., the NADH:NAD⁺ activity. Transhydrogenase systems from both parasites were essentially membrane bound and localized on the inner mitochondrial membrane. Physiologically, the NADPH:NAD⁺ transhydrogenase of H. diminuta may serve to couple the intramitochondrial metabolism of malate (via an NADP linked “malic” enzyme) to the anaerobic NADH-dependent ATP-generating fumarate reductase system. In A. lumbricoides, where the intramitochondrial metabolism of malate depends on an NAD-linked “malic” enzyme which is localized primarily in the intermembrane space, the NADH:NAD⁺ transhydrogenase activity may serve physiologically in the translocation of hydride ions across the inner membrane to the anaerobic energy-generating fumarate reductase system.