Sequence complexity and tissue distribution of chick lens crystallin mRNAs

Using hybridization reactions with a cDNA copy, the complexity of polysomal polyadenylated mRNA from the day-old chick lens was found to correspond to 5800–7200 sequences of average size, arranged in three abundance classes. Experiments with heterologous cDNAs suggest on a qualitative basis that many of the sequences expressed in 8-day embryonic neural retina and pigmented epithelium mRNAs are also present in lens mRNA. A cDNA fraction complementary to the most abundant lens mRNAs, representing an approximate minimum of four sequences, was used to assay the dosage of putative crystallin sequences in these and other embryonic tissues. Neural retina and pigmented epithelium cytoplasmic mRNAs have low concentrations of these sequences, which appear to be absent from mRNA prepared from headless bodies and muscle.     

The plasma pharmacokinetics and tissue distribution of dimethyl sulfoxide in mice

We defined the plasma and tissue concentrations and pharmacokinetics of dimethyl sulfoxide (DMSO) in 22–34 g male Swiss Webster mice injected i.v. with 15% DMSO at a dosage of 1.5 mg per g. Concentrations of DMSO in alkalinized, perchloric acid extracts of tissue and plasma were determined by gas-liquid chromatography. Plasma concentrations of DMSO declined in a biexponential fashion that was well described by the equation C_t = 2.36 exp(?0.449 t) + 1.28 exp(?0.00768 t), indicating a t $\text{1}\text{2}$ (alpha) of 1.5 min and t $\text{1}\text{2}$ (beta) of 90 min. DMSO was rapidly and extensively distributed through tissues and was not concentrated in any particular tissue, although at 1 min after injection, the brain contained the lowest concentration of DMSO of any tissue studied. By 8 hr after injection, there was little DMSO in plasma or any tissue. Intravenous injection of DMSO produced neuro-muscular disturbances, hemolysis, and hemoglobinuria in all animals. Intravenous injection of DMSO produced little increase in plasma osmolality and did not produce any histological evidence of central nervous system of renal tubular damage.     

Cold,freezing, and desiccation tolerance of the limpet Acmaea digitalis (Eschscholtz)

Cold, freezing, and desiccation tolerance were examined in the limpet, Acmaea digitalis (Eschscholtz). Fifty percent of the experimental population survived freezing at temperatures between ?10 ° and ?12 °C for at least 24 hr. In this temperature range, 60–80 % of the body water was frozen. The LD₅₀ for water loss by desiccation was 76.5% ± 1.3_δ. At 70–80% body water loss, solutes were concentrated between 350 and 500%. Some limpets failed to survive immersion in 450% seawater for 6 hr (100% scawater = salinity of 31‰), suggesting that much of the damage from freezing and desiccation was from an increased solute concentration. No glycerol could be detected in extracts of the foot and the visceral mass of A. digitalis during winter, confirming similar results in studies carried out on other intertidal molluscs.     

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.     

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.     

Assay of short-chain acyl coenzyme A intermediates in tissue extracts by high-pressure liquid chromatography

A high-pressure liquid chromatographic method for the measurement of short- and medium-chain-length acyl-CoA compounds is described. Compounds are separated on a reverse-phase μBondapak C₁₈ column with the order of elution based on differences in lipophilicity. The mobile phase consisted of variable mixtures of methanol and 50 mm KH₂PO₄, pH 5.3. Conditions are described that allow isocratic separation of groups of compounds of similar lipophilicity. With increasing methanol concentration, the more lipophilic compounds are eluted earlier. This has the effect of sharpening the peaks and improving quantitation. Detection of acyl-CoA intermediates is achieved using a uv detector and is based on the high absorbance of CoA-containing compounds at 254 nm. Neutralized perchloric acid extracts of tissues can thus be analyzed directly without further purification or derivatization. A mobile phase consisting of a 9:1 phosphate buffer-to-methanol mixture is used to separate CoASH, methylmalonyl-CoA, succinyl-CoA, β-hydroxy-β-methylglutaryl-CoA and acetyl-CoA. Increasing the methanol concentration to a 4:1 mixture allows separation of acetyl-CoA, propionyl-CoA, and isobutyryl-CoA, while with a 7:3 mixture of phosphate buffer to methanol, β-methylcrotonyl-CoA and isovaleryl-CoA are readily separated. Examples of results obtained using extracts from isolated hepatocytes, rat liver mitochondria, and perfused rat hearts incubated with α-ketoisocaproate, α-ketoisovalerate, or propionate are presented. In addition, methods and optimal conditions are presented for the analysis of malonyl-CoA, glutathione-CoA, dephospho-CoA, and oxidized CoA in tissue extracts.     

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

100 000 × 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 μM. ATP (1–100 μM) 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 K_m 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 tested. 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 γ-³² P between GTP and ATP could account for all the histone phosphorylation with [γ-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.     

Analysis of glial cell differentiation in peripheral nervous tissue: II. Neurons promote S100 synthesis by purified glial precursor cell populations

Isolated sensory neurons in vitro do not contain or synthesize S100, whereas glial cell precursor populations do. These precursor cells, when isolated from other cell types, produce low levels of S100 but never undergo the developmental transition to produce high levels of S100. When glial cell precursors are combined with isolated, live or paraformaldehyde-fixed sensory neurons, the precursor cells do undergo the second transition, and accumulate high levels of S100. Peroxidase-anti-peroxidase immunohistochemical staining for S100 confirms previous conclusions (B. Holton and J. A. Weston, 1982, Develop. Biol.89, 64–71) that only those glial cells which are closely apposed to neurons contain augmented levels of S100. This stimulation appears to be specific to neuronal/glial interactions since live or fixed fibroblasts, when cocultured with glial precursor cells, do not promote accumulation of S100 by the glial cells.     

Extrachromosomal circular DNAs from murine hemopoietic tissue cells

Extrachromosomal circular DNA complexes from cells of murine hemopoietic organs, bone marrow, thymus, spleen, and lymph nodes were examined by mica-press-adsorption method (H. Yamagishi, T. Kunisada, and T. Tsuda, 1982, Plasmid 8, 299-306). They showed wide size distribution, from 0.3 to 10 micron. The large-size DNAs of more than 1 micron (3.1 kb) in contour length were more abundant in bone marrow and thymus than they were in spleen and lymph nodes. The appearance of the large size DNAs was examined on splenocytes of athymic nude mice during ontogeny. The large-size DNAs first became detectable after 2 weeks of age and the amount increased thereafter until 9 weeks of age. It appears that large-size circular DNAs appear during differentiation from the hemopoietic stem cells into several descendent cells. Possible immunological implications for the appearance of extrachromosomal circular DNAs are discussed.     

Isolation and characterization of the cytosolic and mitochondrial superoxide dismutases of maize

The cytosolic and mitochondrial forms of Superoxide dismutase have been purified to homogeneity from an inbred line of maize. The cytosolic isozymes SOD-2 and SOD-4 are dimers with a molecular weight of 31,000–33,000, composed of apparently equal subunits, and are remarkably similar with respect to their ultraviolet absorption spectra, antigenic specificity, and sensitivity to cyanide, azide, hydrogen peroxide, and diethyldithiocarbamate. These and other data suggest that both isozymes belong to the family of copper and zinc-containing Superoxide dismutases. The mitochondrial isozyme, SOD-3, is unlike the cytosolic isozymes in every parameter studied and appears to be similar to the mitochondrial manganese-containing Superoxide dismutases purified from other eukaryotic organisms. It is a tetramer with a molecular weight of approximately 90,000, composed of apparently equal subunits, and is insensitive to both 1 mm cyanide and hydrogen peroxide.     

Calcitroic acid: biological activity and tissue distribution studies

Calcitroic acid was recently identified as a major metabolite of 1,25-dihydroxyvitamin D₃ (Esvelt, Schnoes, and DeLuca, Biochemistry 18, 3977, 1979). The metabolite was found to have little, although significant, activity in healing rickets, and causing bone mineral mobilization but elicited no significant elevation in intestinal calcium transport. The compound showed little affinity for either the serum 25-hydroxyvitamin D binding protein or the intestinal cytosol receptor for 1,25-dihydroxyvitamin D₃. Various tissues of the rat were examined for the presence of calcitroic acid following a 120-ng dose of 1,25-dihydroxy-[3α-³H]vitamin D₃. The metabolite was detected in liver, intestinal mucosa, kidneys, and blood with livers and mucosa containing the highest concentrations. In each of these tissues the calcitroic acid content increased during the period between 4 and 12 h after the dose. The presence of calcitroic acid in femurs was indicated but could not be confirmed. Bile duct cannulation reduced but did not abolish the intestinal calcitroic acid content. In addition to calcitroic acid, other polar metabolites of 1,25-dihydroxyvitamin D₃ were detected in these experiments.     

Coordinate behavior of orotate phosphoribosyltransferase and orotidylate decarboxylase in developing mouse liver and brain

A large body of data has accumulated in recent years supporting the view that orotate phosphoribosyltransferase and orotidylate decarboxylase exist as a bifunctional enzyme complex in adult mammalian tissues. This paper presents evidence that such a complex also occurs in mouse liver and brain, regardless of the developmental stage of the animal. Orotate phosphoribosyltransferase and orotidylate decarboxylase activities remained coordinate in fetal, neonatal, immature and adult liver and brain. In addition, these two enzymes routinely cosedimented during centrifugation of cell-free extracts in sucrose gradients. The sedimentation coefficient of the enzyme complex did not change significantly during mouse development. However, the liver complex exhibited a sedimentation coefficient (5.0 ± 0.2) that differed from that of the brain complex (4.3 ± 0.1)     

Radioimmunoassay for the detection and quantitation of bruceantin

A radioimmunoassay for a new anticancer drug, bruceantin, has been developed using [³H]acetylbruceantin and antibody induced by immunizing rabbits with succinylbruceantin-bovine serum albumin conjugates. [³H]Acetylbruceantin was synthesized by reacting bruceantin with [³H]acetyl anhydride. The assay is simple and reproducible. The standard curve was linear on a logit-log plot, and the lower limit of sensitivity of the assay was 1 ng/ml. Using this assay, drug levels were easily determined in tissues of experimental animals following bruceantin administration. The assay procedure does not require sample extraction for plasma, urine, and bile. Bruceantin in other tissues can be extracted quantitatively with ethanol before being measured by the radioimmunoassay.     

Cell surface morphology and adhesive properties of normal and virally transformed cells treated with tunicamycin, an inhibitor of protein glycosylation

Normal and virally transformed mouse (3T3) fibroblasts were treated with tunicamycin, a fungal antibiotic that specifically inhibits the synthesis of peptidyl asparaginyl-linked oligosaccharides. All cell lines exhibited changes in cell surface morphology, surface-associated proteins and adhesion to the culture plate in the presence of tunicamycin. Scanning electron microscopy (SEM) revealed that treated fibroblasts assumed a spherical shape and were partially detached from the substratum. In addition, the 3T3 cells showed numerous cell surface ruffles. Tunicamycin-treated cells exhibited no marked ultrastructural changes when compared with control cells. There were indications, however, that the rough endoplasmic reticulum was dilated and that there were fewer membrane-bound ribosomes in treated 3T3 cells. Surface iodination of pretrypsinized tunicamycin-treated cells, followed by analysis of the labeled proteins on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, showed a marked reduction in a cell surface protein, identical or similar to fibronectin. Both tunicamycin-treated 3T3 and transformed 3T3 cells demonstrated a reduction in plating efficiency as shown by attachment assays of viable cells. In addition, treated cells showed a reduction in adhesiveness and a delay in spreading. The latter changes were more pronounced in the virally transformed cell lines. These findings suggest that cell surface glycoproteins, including fibronectin, play a role in determining the surface morphology and adhesive properties of cells.     

The surface activity of PVP and other polymers and their antihemolytic capacity

The polymeric cryoprotective agents polyvinylpyrrolidone, dextran, and hydroxyethyl starch do not penetrate the cell membrane and are not present in high osmotic concentrations. Thus, they can exert little of the "antifreeze" behavior generally attributed to glycerol or dimethyl sulfoxide, and must protect cells from freezing injury by some action external to the cell surface. Surface energy measurements of droplets of hemoglobin solution immersed in solutions of cryoprotective polymers indicate that these polymers lower the surface energy of the solution below that of the hemoglobin droplets and form a stable interface. In injured cells, these polymers will therefore hide membrane defects by forming an interface across which hemoglobin cannot easily pass. When freezing is slow, the polymers have little if any true cryoprotective effect but interfere with hemoglobin release as an assay of injury.     

Isolation and cell-free translation of chick lens crystallin mRNA during normal development and transdifferentiation of neural retina

Messenger RNA has been isolated from day-old chick lens. Size characterization and heterologous cell-free translation demonstrate that the predominant species of mRNA present code for α-, β- and δ-crystallins. Total polysomal RNA and polysomal RNA which did not bind to oligo (dT)-cellulose translate in the cell-free system to give a crystallin profile qualitatively similar to that of poly(A)⁺ mRNA. RNA from postribosomal supernatant which binds to oligo(dT)-cellulose also translates to give crystallins, but the products are enriched for β-crystallins. Messenger RNAs isolated from 15-day embryo lens fiber and lens epithelium cells give products on translation which reflect the different protein compositions of these two cell types, as do mRNAs isolated from chick lenses at various developmental stages. Messenger RNAs were isolated from freshly excised 8-day embryo neural retina and from this tissue undergoing transdifferentiation into lens cells in cell culture. Cell-free translation demonstrates no detectable crystallin mRNAs in the freshly excised material, but by 42 days in cell culture, crystallin mRNAs are the most prominent species.     

Colchicine and cytochalasin B (CB) effects on random movement, spreading and adhesion of mouse macrophages

The role of microtubules and microfilaments in the control of random movement of mouse peritoneal macrophages was examined by studying the colchicine and cytochalasin B (CB) effects. Colchicine in the concentration range of 10^?8–10^?4 M enhances the random movement of these cells. Enhanced movement of macrophages is observed only at colchicine concentrations which cause inhibition of their spreading and adhesion. CB does not enhance random movement at any concentration; it inhibits movement at 10^?7 M and higher concentrations. Furthermore, though 10^?8 M CB alone has no effect on the migration of macrophages, when present along with colchicine, the two drugs act synergistically and enhance random movement to a greater extent than colchicine alone. These findings suggest a cooperative interaction between microtubules and microfilaments in the control of movement of macrophages. A model is presented to explain the nature of this interaction.     

5-Bromodeoxyuridine inhibition of differentiation. Kinetics of inhibition and reversal in myoblasts

This paper describes experiments on the kinetics of inhibition of muscle differentiation in vitro in the presence of 5-bromodeoxyuridine (BrdUrd) and the recovery phenomena that occur when such inhibited cells are permitted growth in normal medium. The studies consist of a quantitation of cell fusion in the presence of the analog and during recovery in its absence coupled with simultaneous studies on changes in buoyant density of cellular DNA. We find that if myoblasts are exposed to BrdUrd during the last doubling before cell fusion would normally occur, most cells do not differentiate, but as many as 18% of the cells can fuse in spite of the incorporation of BrdUrd into their nuclei. These nuclei contain approximately the amount of BrdUrd expected for a full round of DNA synthesis. Studies on the rate of recovery of inhibition of cell fusion following one generation in BrdUrd reveal that after one doubling of inhibited cells in the presence of normal medium. fusion reaches about 50% of the control value; after two doublings it reaches 75% of control value; and after 2.5 doublings of reversal, recovery is essentially complete. We find that both the degree of inhibition after approximately one round of BrdUrd incorporation and the rate of cell differentiation after two generations of reversal are consistent with a model which assumes that BrdUrd “sensitivity” resides on single pair of chromosomes and that inhibition occurs in a dominant fashion if approximately 30% or more of the thymidine is replaced by BrdUrd in the readout strand of either chromosome.