Characterization of entomopox virions of the army cutworm,Euxoa auxiliaris (Lepidoptera: Noctuidae)

Virions were released from virus-containing inclusions (VCI) of an entomopoxvirus of the army cutworm, Euxoa auxiliaris, with carbonate-thioglycolate solution. Knoblike projections present on the surface of the viral envelope were removed by digestion with trypsin. Trypsin-treated virions were homogeneous in both sucrose and CsCl gradients. The virions were similar to vertebrate poxviruses in morphology, contained 1.13 ± 0.3% DNA and had a buoyant density of 1.261 ± 0.003 gm/cm³ in CsCl. The virion preparations were infective and possessed RNA polymerase activity. Of eight species of Lepidoptera tested, only the species from which the virus was originally isolated proved susceptible to infection.     

In vitro synthesis of proteoglycans associated with medullary bone in Japanese quail

A short-term incubation system was used to study proteoglycan synthesis during the early stages of medullary bone formation in estrogen-treated male Japanese quail. The proteoglycans were separated by chromatography on a DEAE Bio-Gel A column eluted with a 400-ml 0-1 M NaCl gradient. The profile from uninjected control birds showed a single peak, whereas profiles from estrogen-treated birds showed development of another peak. Incorporation of [35S]sulfate into the estrogen-induced proteoglycan increased most dramatically between 25 and 37 h after hormone treatment. The estrogen-induced proteoglycan has a Kav = 0.65 on Sepharose CL-4B, an average buoyant density of 1.50 g/ml, and contains keratan sulfate as its constituent glycosaminoglycan. The second proteoglycan has a Kav = 0.52 on Sepharose CL-4B, an average buoyant density of greater than or equal to 1.7 g/ml, and has chondroitin sulfate as it major glycosaminoglycan. It may also contain some heparin or heparan sulfate. The results support the usefulness of the incubation system for studying the dynamics of bone matrix production.     

Glutamine synthetase from Salmonella typhimurium: manganese(II), substrate, and inhibitor interaction with the unadenylylated enzyme.

Unadenylylated glutamine synthetase (EC 6.3.1.2) was isolated and purified to homogeneity from Salmonella typhimurium. The enzyme molecule is a symmetrical aggregate of 12 subunits arranged in two hexagonal layers, as is evident from electron micrographs. The subunit molecular weight of the enzyme was found to be approximately 50,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate when compared to Escherichia coli glutamine synthetase and other protein standards. A long tube of glutamine synthetase was formed as a single-stranded coil resulting from incubation of the enzyme in a low ionic strength buffer. A study of Mn(II) binding to the unadenylylated enzyme at 25 °C was conducted as a function of pH. At pH 7.1 two classes of metal ion sites per subunit were found with K_D values of 3.7 × 10^?6 and 1.7 × 10^?4m, while at pH 6.8 these values were 1.1 × 10^?5 and 1.0 × 10^?4m, respectively. Only one set of binding sites was observed at pH 6.2 with a K_D value of 1.0 × 10^?4m. The metal ion binding sites were further investigated by monitoring proton relaxation rates (prr) and the epr spectrum of enzyme-bound Mn(II). The longitudinal prr of water protons at pH 7.1 indicate that protons interacting with enzyme-Mn(II) at the “tight” site (K_D = 3.7 × 10^?6) are de-enhanced (?_b1 = 0.42) and result from water protons beyond the inner coordination sphere. The second Mn(II) site has a value of ?_b2 = 35 for the binary enhancement, suggesting that this site probably has two to three rapidly exchanging water molecules in its coordination sphere. The epr spectrum of enzyme-bound Mn(II) at the “tight” site is isotropic and is dramatically sharpened by adding the substrate analog methionine sulfoximine. Subsequent addition of ATP or the ATP analog, AMP-PCP (adenylyl methylene diphosphate) produced anisotropic spectra that were similar, suggesting that both ATP and AMP-PCP bind similarly on the enzyme surface. However, a marked change in the Mn(II) environment from anisotropic to near cubic results from the addition of ADP to the quaternary enzyme-Mn(II)-sulfoximine- (AMP-PCP) complex, indicating that ADP displaces AMP-PCP. No change in the anisotropic spectrum due to the enzyme-Mn(II)-sulfoximine-ATP complex is seen by the addition of ADP. This experimental result supports the experimental findings of Ronzio and Meister [Proc. Nat. Acad. Sci. USA59, 164 (1968)], who established that ATP phosphorylates methionine sulfoximine, thereby producing an inactive enzyme. The allosteric effectors, AMP and Trp, have little effect on the epr spectrum of the complex formed from Mn(II), enzyme, sulfoximine, and ADP, suggesting the absence of direct coordination of AMP or Trp to the bound Mn(II). The prr and epr results reported herein with glutamine synthetase from S. typhimurium when compared to those seen for the enzyme from E. coli [Villafranca et al., Biochemistry15, 544 (1976)] demonstrate some similarities but also many substantial differences between the enzymes from these two bacterial sources.     

Experimental laboratory infection of mosquito larvae with fungi of the genus Coelomomyces. II. Experiments with Coelomomyces punctatus in Anopheles quadrimaculatus

Experiments on the infection of Anopheles quadrimaculatus with Coelomomyces punctatus indicate that planonts released from sporangia are not infective for mosquito larvae but most likely infect the copepod Cyclops vernalis. Exposure of early-instar larvae to up to 5 × 10³ planonts per larva for as long as 48 hr resulted in no larval infections. Motile planonts were no longer detectable after 48 hr. However, incubation of early-instar larvae in media to which planonts, algae, and copepods had been added several days previously resulted in larval infection. Infection did not occur during the first 6 days after planont introduction. On day 7 and for several days thereafter, copepods were detected in the media which had an extensive mycelium developing in the hemocoel. This mycelium cleaved into thousands of posteriorly uniflagellate planonts. The presence of planonts at the time of mosquito infection, in conjunction with the above results, suggests that Cyclops vernalis is an alternate host for Coelomomyces punctatus and that the latter has a life cycle similar to that proposed for C. psorophorae involving a mosquito and a copepod as obligate alternate hosts. In established infection containers, dilution of the media with water significantly increased levels of infection 6 days later. All larval instars were susceptible to C. punctatus.     

Purification and characterization of human factor II

Human plasma Factor II has been purified approximately 800-fold by a combination of barium citrate adsorption, ion-exchange chromatography and preparative polyacrylamide gel electrophoresis. The procedure is relatively simple and results in excellent yields of purified Factor II essentially free of Factor X activity. The purified factor behaved as a single component by analytical polyacrylamide gel disc electrophoresis at pH 8.9. No Factor V, VII or IX activity was detected in the purified Factor II. Its molecular weight was 7200±3000 as determined by analytical ultracentrifugation, electrophoresis in the presence of sodium dodecyl sulfate and gel filtration on Bio-Gel P-200. An apparent molecular weight of 90 000–100 000 was observed on calibrated columns of Sephadex G-100, G-150, and G-200. The specific activity of human factor II was approximately 1300 N.I.H. units/mg as determined by the two-stage assay and 7 Ortho units/mg by the one stage assay. The purified protein contained by weight 2.8% neutral hexose, 2.3% sialic acids and 3.1% hexosamines.     

Effect of a bifunctional imidoester on dissociation of 70S ribosomes in Escherichia coli

Treatment of the 70S ribosome from $\text{Escherichia coli}$ with diethyl malonimidate dihydrochloride, a bifunctional imidoester, was found to result in the formation of crosslinkage between the two subunits. The 70S complex thus obtained no longer dissociates into 50S and 30S particles at 0.5mM Mg²⁺ concentration, but do so at lower concentrations (0.1mM), suggesting the release of protein(s) involved in the inter-particle cross-linkage from one or both ribosomal subunits.     

A partial characterization of the cyclic nucleotide phosphodiesterases of Drosophila melanogaster

The cyclic nucleotide phosphodiesterases in crude homogenate, soluble material, and particulate preparations of adult Drosophila melanogaster flies, hydrolyze cyclic AMP with nonlinear kinetics. Cyclic GMP is hydrolyzed by the phosphodiesterases in crude homogenate and soluble material with linear kinetics. Physical separation techniques of gel filtration, velocity sedimentation, and ion-exchange chromatography reveal that Drosophila soluble fraction contains two major forms of cyclic nucleotide phosphodiesterase. Form I hydrolyzes both cyclic AMP and cyclic GMP. Inhibition experiments suggest that the hydrolysis of both cyclic nucleotides by Form I occurs at a single active site. The K_m's for hydrolysis of both substrates are about 4 μm. This form has a molecular weight of about 168,000 as estimated by gel nitration. Form II cyclic nucleotide phosphodiesterase is specific for cyclic AMP as substrate. Gel filtration indicates that this form has a molecular weight of about 68,000. The K_m for cyclic AMP is about 2 μm.     

On the computation of the tertiary structure of globular proteins. III. Inter-residue distances and computed structures

An analysis of geometrical models for computing the tertiary structure of globular proteins from the primary structure is presented. The roles of initial configuration, input information on inter-residue distances and the errors in this information are delineated. It is shown that for local information like that on secondary structure, the calculated structure is very sensitive to errors and to the initial configuration. Thus, such information is far from adequate for predicting the tertiary structure. On the other hand, global information on all the inter-residue distances is quite insensitive to errors. A semi-empirical method is presented to estimate these distances and the calculated structures are given for two proteins—pancreatic trypsin inhibitor and parvalbumin. These structures have good resemblances to those determined by X-ray diffraction. A strategy for further refinement of the method is indicated.     

2,3,7,8-Tetrachlorodibenzo-P-dioxin: potent anticarcinogenic activity in CD-1 mice.

Topical pretreatment with non-toxic doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin, a contaminant formed in the commercial synthesis of the herbicide 2,4,5-trichlorophen-oxyacetic acid, strongly inhibited the initiation of skin tumors by 7,12-dimethylbenz(a)-anthracene and benzo(a)pyrene in female CD-1 mice. 2,3,7,8-tetrachlorodibenzo-p-dioxin also produced marked induction of epidermal monooxygenase enzymes functional in the conversion of 7,12-dimethylbenz(a)anthracene to a variety of hydroxylated products. The time course of anticarcinogenic effects resulting from pretreatment with the dioxin correlated with the magnitude of induction as well as with a singnificant reduction in the quantity of 7,12-dimethylbenz(a)anthracene metabolites covalently bound $\text{in vivo}$ to epidermal DNA and RNA but not protein.     

Radioiodination and characterization of the plasma membrane of sea urchin sperm

Two methods were used to radioiodinate sea urchin sperm: lactoperoxidase-glucose oxidase and Iodo-Gen. Following iodination the sperm are viable, they undergo the acrosome reaction, and they fertilize eggs. Of the radioactivity associated with the labeled sperm, 28–50% is presumed to be free ¹²⁵I^?, 37–47% is incorporated in lipid, and 8–15% is in trypsin-digestible material believed to be protein. Digestion of the labeled, living sperm with trypsin removes 95.6–99.5% of the macromolecular label (the cells are alive after digestion) suggesting that almost all the protein label is on the external surface of the cell. Thin-layer chromatography of the lipid fraction shows that the major membrane phospholipids and cholesterol are labeled. SDS-PAGE analysis shows the protein-incorporated ¹²⁵I is distributed among four glycoproteins of >250K, 84K, 64K, and 52K dalton apparent molecular weight. Twenty-eight percent of the total protein (trypsin-digestible) label is in the 84K component and 46% in the 64K band. Although both molecules contain much of the label, they are relatively minor components of the TX-100 extract of sperm. The methods outlined will be useful in determining the role of sperm surface components in fertilization.     

Purification and characterization of calmodulin from porcine renal medulla

A calcium sensitive phosphodiesterase (PDE) activated by an endogenous calmodulin was identified in the cytosolic fraction of porcine renal medulla. The PDE and calmodulin were separated from each other by DEAE-cellulose column chromatography. Calmodulin was purified from a heat-treated supernatant by column chromatography with DEAE-cellulose and hydroxylapatite. The purified renal calmodulin has a molecular weight of 17,500, is heatstable, and has a pI of 4.2. Activation of the renal PDE by calmodulin was immediate and stoichiometric. The renal calmodulin and PDE cross react with bovine brain calmodulin and PDE, indicating a lack of tissue and species specificity. Thus, renal calmodulin is very similar to bovine brain calmodulin. However, renal calmodulin did not affect detergent-solubilized or membrane-bound renal adenylate cyclase or the antidiuretic hormone-stimulated activity of the enzyme. These results suggest that calmodulin may function in the renal medulla to regulate cAMP levels by stimulation of PDE but not adenylate cyclase. However, the ubiquitous distribution of calmodulin in eukaryotic cells and its effects on a number of other enzymes allow the possibility that calmodulin may have a role in renal function other than cAMP metabolism.     

Enucleation eliminates a differentiation-specific surface marker from normal and leukemic murine erythroid cells

Mammalian erythropoiesis includes a step in which the nucleus is extruded through the cell membrane. We have investigated the relationship between concanavalinA (conA) plasma membrane receptors, which are known to leave the incipient reticulocyte during enucleation, and regions of the plasma membrane which bind merocyanine 540, a differentiation-specific marker of hematopoietic cells. The distribution of these two fluorescent probes was examined on living cells from the spleens of neonatal mice and on erythroleukemia cells induced to enucleate in culture. In both cases, the region of the membrane extruded with the nucleus preferentially binds conA and merocyanine 540, whereas the plasma membrane which is left behind retains the capacity to bind another lectin, wheat germ agglutinin (WGA). The implications of these findings are discussed with respect to the mechanism by which markers are eliminated from the erythrocyte cell surface.     

Effects of a mixture of a saturated with an unsaturated fatty acid on secretion of the very low density lipoprotein by the liver.

Palmitic acid (16:0) and palmitoleic acid (16:1), as the complex with bovine serum albumin, were infused at rates of 62 and 124 μmoles/hr into an albumin-buffer medium perfusing livers isolated from normal fed male rats. In other experiments, equimolar mixtures (124 μmoles/hr, total) of 16:0 + 16:1, or myristate (14:0) + 16:1 were infused. The output of triglyceride when 16:1 was infused was greater than when equivalent amounts of 14:0 or 16:0 were infused; output with equimolar mixtures of 14:0 and 16:1, or 16:0 and 16:1 was intermediate between that of saturated and unsaturated fatty acids alone. Rate-zonal mobility of the VLDL in the ultracentrifuge was more rapid as the quantity of 16:1 available to the liver increased, but did not change with increasing amounts of 16:0. The rate-zonal mobility of the mixtures of 14:0 and 16:1, or of 16:0 and 16:1, was not different than that of 16:1 alone. The ratios of phospholipid and cholesterol relative to triglyceride in the VLDL decreased with increasing output of triglyceride and with unsaturation of the fatty acid. Ratios resulting from mixtures of the fatty acids appeared to be in an intermediate position. The composition and properties of the secreted VLDL clearly are dependent on the structure and quantity of FFA available to the liver; with mixtures of saturated and unsaturated fatty acids, the unsaturated fatty acid seems to exert a dominant effect.     

Renal cortical cells in primary monolayer culture. Enzymatic changes and morphological observations.

Stress fibers and bands of intermediate filaments (100 Å) were studied in cultured non-muscle cells using laser microbeam techniques. Wavelengths of 532, 537 and 280 nm were used, and no artificial chromophores were employed. Lesions were assayed using a combination of phase contrast, polarizing and transmission electron microscopy (TEM). (1) Stress fibers 1–2 μm in diameter were narrowed or completely servered by irradiation at 532, 537 and 280 nm. Stress fibers could be grouped into two classes: (a) those whose severed ends separated during the first few seconds following laser irradiation (46% of fibers irradiated); (b) those fibers which showed no movements (54%). Microtubules which paralleled stress fibers persisted in the presence of colcemid for up to 5 h, and alignment of the severed stress fiber ends was maintained even in their absence. Injured stress fibers appear to be repaired within 1 h of irradiation. (2) Bands of 100 Å filaments were induced in non-muscle cells in secondary cultures of neonatal rat heart by exposure to colcemid. Lesions which appeared as phase dense spots were induced in these bands by irradiation at 532, 537 and 280 nm. The positions of the lesions in the band relative to one another did not change over several hours despite movements of the entire band. These studies demonstrate that (a) stress fibers may be an excellent system in which to study subcellular repair; (b) induced bands of 100 Å filaments probably move passively in the cells containing them; (c) laser irradiation of cytoplasmic filaments in non-muscle cells does not require the introduction of an artificial chromophore.     

Migration inhibition of alloimmune mouse macrophages by soluble transplantation antigens

Specific allogeneic transplantation antigens were solubilized and shown to inhibit the migration of alloimmune macrophages. Alloimmune macrophages treated with trypsin prior to antigen exposure migrated in the presence of the specific soluble antigens. The arming of nonimmune macrophages and the rearming of trypsinized immune macrophages with hyperimmune serum was readily observable using the migration inhibition test, whereas immune serum was ineffective.     

Uridine-cytidine kinase. Kinetic studies and reaction mechanism.

The initial velocity pattern has been determined for uridine-cytidine kinase purified from the murine mast cell neoplasm P815. With either uridine or cytidine as phosphate acceptor, and ATP as phosphate donor, the pattern observed was one of intersecting lines, ruling out a ping-pong reaction mechanism, and suggesting that the reaction probably proceeds by the sequential addition of both substrates to the enzyme to form a ternary complex, followed by the sequential release of the two products. This pattern was obtained whether the reaction was run in 0.01 m potassium phosphate buffer, pH 7.5, or in 0.1 m Tris-HCl, pH 7.2. When analyzed by the Sequen computer program, the data indicated an apparent K_m of the enzyme for uridine of 1.5 × 10^?4m, an apparent K_m for cytidine of 4.5 × 10^?5m, and a K_m for ATP, with uridine or cytidine as phosphate acceptor, of 3.6 × 10^?3m or 2.1 × 10^?3m, respectively. The V was 1.83 μmol phosphorylated/min/mg enzyme protein for the uridine kinase reaction and 0.91 μmol for the cytidine kinase reaction.     

Testosterone and photoperiod interact to regulate locomotor activity in male hamsters

Testicular size, plasma testosterone levels, copulatory behavior, and daily locomotor activity are reduced in male hamsters after 10 weeks of exposure to short days. The role of testosterone in the short day-induced decline in locomotor activity was investigated, determining whether or not photoperiod could alter the effect of testosterone on activity. Castrated adult hamsters were allowed to acclimate to running wheels (wired to digital counters) and then were kept on either long (L:D 14:10) or short (L:D 6:18) days for 60 days. On Day 60, half of the animals on each light cycle were implanted with 12-mm-long testosterone-filled Silastic capsules; half received empty capsules. Digital counting of wheel-running activity continued for another 140 days. Blood samples taken on Day 200 confirmed L:D 14:10 and L:D 6:18 testosterone-treated hamsters had equivalent plasma testosterone levels. After an initial decline in activity, L:D 14:10 animals exhibited a progressive rise in mean running activity (from ~2000 to ~5000 wheel revolutions per day) through 100 days after the initiation of testosterone treatment. In contrast, activity levels in testosterone-treated L:D 6:18 animals remained uniform (~2000 wheel revolutions per day) during this time, indicating exposure to short days rendered the hamsters less sensitive to the stimulatory effect of testosterone on activity. Of further interest was a marked increase in activity after 160–200 short days in animals treated with either testosterone-filled or empty capsules. It appears the total amount of daily locomotor activity in the hamster is modulated by circulating testosterone levels in a manner which is dependent upon the environmental photoperiod.