Coexistence of Tetranychus urticae (Acarina: Tetranychidae) with different capacities for diapause: comparative life-history traits

Summary Individuals of the two-spotted spider mite, Tetranychus urticae Koch (Acarina: Tetranychidae), with and without a capacity for diapause, coexist in central Japan. Diapause appears to be adaptive because females in diapause suffered less mortality than non-diapausing individuals when frozen at –24°C for more than 4 h. However non-diapausing females showed good survival up to 4 h of freezing. Active non-diapausing mites survived on rose leaves in Kyoto (35°N) throughout the winters of 1988–1989 and 1989–1990. Cultures of mites with low (LD) and high (HD) diapause capacities at 18°C and 9L-15D photoperiod were successfully selected from the rose population and from a population on chrysanthemum in Nara (34.4°N). Their life-history traits at 15, 20, 25 and 30°C were characterized. HD and LD mites from both populations were of similar ages at first reproduction at 15–30° C. However, at temperatures 20° C, HD individuals produced more eggs than LD individuals, resulting in higher fecundity and intrinsic rate of natural increase. These traits allow HD individuals, which stop breeding between October and April, to increase faster in summer than LD individuals. This provides a mechanism, together with climatic fluctuations, in maintaining the coexistence of diapausing and non-diapausing T. urticae in Kyoto where winter conditions are rarely lethal to the non-diapausing individuals.     

Three-dimensional SEM study of arteriovenous anastomoses in the dog's tongue using corrosive resin casts

Arteriovenous anastomoses (AVAs) participate in the regulation of blood flow. Although it has been speculated that AVAs in the dog's tongue play a role in the regulation of the body temperature, no published work is available on the structural characteristics of AVAs in the dog's tongue. The purpose of the present investigation was therefore to determine the frequency of AVAs and their structural characteristics by the fabrication of vascular corrosive resin casts and examination under a scanning electron microscope. This method permitted not only the visualization of the three-dimensional characteristics of AVAs, but also a clear differentiation between arterioles and venules. The total number of AVAs in the mucosal lamina propria of the dorsum of the left tongue half was 2,292. Several essential types such as S-shaped, hook-shaped, straight, bibranching and Y-shaped AVAs were observed, of which the S-shaped and similar types constituted the overwhelming majority; Y-shaped types have never been reported heretofore. This study also revealed that the locations where AVAs were most often distributed were, in descending order of frequency, the tip, the corpus and the root area of the tongue. This high frequency and strategic location of AVAs in the tongue strongly indicate that AVAs of the dog's tongue participate in the thermal regulation.     

Observation of inverted cubic phase in hydrated dioleoylphosphatidylethanolamine membranes

We report the observation of an inverted cubic phase in aqueous dispersions of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) by small-angle X-ray diffraction. DOPE is a paradigm in the study of nonlamellar phases in biological systems: it exhibits a well-known phase transition from the lamellar (L alpha) to the inverted hexagonal phase (HII) as the temperature is raised. The transition is observed to occur rapidly when a DOPE dispersion is heated from 2 degrees C, where the L alpha phase is stable, to 15 degrees C, where the HII phase is stable. We report on the induction of a crystallographically well-defined cubic lattice that is slowly formed when the lipid dispersion is rapidly cycled between -5 and 15 degrees C hundreds of times. Once formed, the cubic lattice is stable at 4 degrees C for several weeks and exhibits the same remarkable metastability that characterizes other cubic phases in lipid-water systems. X-ray diffraction indicates that the cubic lattice is most consistent with either the Pn3m or Pn3 space group. Tests of lipid purity after induction of the cubic indicate the lipid is at least 98% pure. The cubic lattice can be destroyed and the system reset by cycling the specimen several times between -30 and 2 degrees C. The kinetics of the formation of the cubic are dependent on the thermal history of the sample, overall water concentration, and the extreme temperatures of the cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

An auxiliary protein for DNA polymerase-delta from fetal calf thymus

An auxiliary protein which affects the ability of calf thymus DNA polymerase-delta to utilize template/primers containing long stretches of single-stranded template has been purified to homogeneity from the same tissue. The auxiliary protein coelutes with DNA polymerase-delta on DEAE-cellulose and phenyl-agarose chromatography but is separated from the polymerase on phosphocellulose chromatography. The physical and functional properties of the auxiliary protein strongly resemble those of the beta subunit of Escherichia coli DNA polymerase III holoenzyme. A molecular weight of 75,000 has been calculated from a sedimentation coefficient of 5.0 s and a Stokes radius of 36.5 A. A single band of 37,000 daltons is seen on sodium dodecyl sulfate gel electrophoresis, suggesting that the protein exists as a dimer of identical subunits. The purified protein has no detectable DNA polymerase, primase, ATPase, or nuclease activity. The ability of DNA polymerase-delta to replicate gapped duplex DNA is relatively unaffected by the presence of the auxiliary protein, however, it is required to replicate templates with low primer/template ratios, e.g. poly(dA)/oligo(dT) (20:1), primed M13 DNA, and denatured calf thymus DNA. The auxiliary protein is specific for DNA polymerase-delta; it has no effect on the activity of calf thymus DNA polymerase-alpha or the Klenow fragment of E. coli DNA polymerase I with primed homopolymer templates. Although the auxiliary protein does not bind to either single-stranded or double-stranded DNA, it does increase the binding of DNA polymerase-delta to poly(dA)/oligo(dT), suggesting that the auxiliary protein interacts with the polymerase in the presence of template/primer, stabilizing the polymerase-template/primer complex.     

Synthesis of the transferrin receptor by cultures of embryonic chicken spinal neurons

We have purified a glycoprotein from chicken sciatic nerves, sciatin, which has pronounced trophic effects on avian skeletal muscle cells in culture. Recent studies have shown that sciatin is identical to the iron-transport protein, transferrin, in terms of its physicochemical structure, immunological reactivity, and biological activity. To determine whether transferrin is synthesized and released by neuronal tissue, we incubated cultures of dissociated chicken spinal neurons in a medium free of L-leucine containing either L-3H-amino acids or L-[14C]leucine and immunoprecipitated transferrin with highly specific antibodies. The radiolabeled protein precipitated by rabbit heteroclonal, goat heteroclonal, or mouse monoclonal antitransferrin antibodies increased in specific activity in a linear manner for at least 30 min. Synthesis of this protein was abolished by the presence of puromycin (20 micrograms/ml) or cycloheximide (10(-5) M). The disappearance of the radiolabeled protein from cells was linear with a half-life (t 1/2) of 8-10 h. When immunoprecipitates were separated by SDS gel electrophoresis, a prominent band corresponding to transferrin (Mr 84,000) was visualized by staining with Coomassie Blue. However, when such gels were fluorographed, no radioactivity was apparent in the transferrin region of the gel although a prominent radioactive band was visualized at an Mr of 56,000. The protein of Mr 56,000 was not simply a degradation product of transferrin because this particular protein band was not generated by incubating radiolabeled transferrin with unlabeled neuronal homogenates. The protein of Mr 56,000 was purified from embryonic chicken brain and spinal cord by immunoabsorption chromatography on mouse monoclonal antitransferrin IgG conjugated to Sepharose 4B followed by affinity chromatography on immobilized transferrin. The purified protein bound radioiodinated transferrin and was precipitated by rabbit anti-chicken transferrin-receptor antibodies. Furthermore, this receptor protein was found to be localized on the plasma membrane of dorsal root ganglion neurons by immunocytochemistry using the peroxidase-antiperoxidase technique, and by blocking experiments, which showed that antitransferrin receptor IgG could inhibit the binding of fluorescein-conjugated transferrin at 4 degrees C to cultured neurons in vitro. From these data, we conclude that transferrin is not synthesized by cultures of chicken spinal cord neurons, but that the receptor for transferrin is synthesized by these cultures and is precipitated by antitransferrin antibodies as an antigen-receptor complex.     

Effect of base-pair stability of nearest-neighbor nucleotides on the fidelity of deoxyribonucleic acid synthesis

The influence of the stability of base pairs formed by nearest-neighbor nucleotides on misincorporation frequency has been studied with the large fragment of DNA polymerase I, the alternating DNA copolymers, poly(dI-dC) and poly-(dG-dC), as template-primers, and dGTP, dITP, and dCTP as substrates. We have utilized the difference in thermodynamic stability between the doubly H-bonded I X C base pair and triply H-bonded G X C base pair to examine the effects of base-pair stability of both the "preceding" and the "following" nucleotides on the frequency of insertion of a mismatched nucleotide, as well as on its stable incorporation into polynucleotide. The present studies demonstrate that the stability of the base pairs formed by nearest-neighbor nucleotides affects the frequency of incorporation of noncomplementary nucleotides. Misincorporation frequency is increased when the nearest-neighbor nucleotides form more stable base pairs with the corresponding nucleotides in the template and is decreased when they form less stable base pairs. The stability of the base pair formed by a nucleotide either preceding (5' to) or following (3' to) a misincorporated nucleotide influences misincorporation frequency, but by different mechanisms. The stability of base pairs formed by preceding nucleotides affects the rate of insertion of mismatched nucleotide but does not protect the mismatched nucleotide from removal by the 3' to 5' exonuclease activity. In contrast, the stability of a base pair formed by a following nucleotide determines whether a misincorporated nucleotide is extended or excised by affecting the ability of the enzyme to edit errors of incorporation.     

Phagocytosis of different matrix components by different cell types at bone-forming sites in cultured mouse calvariae

Summary Sites of bone formation on fragments of parietal bone of fetal-mice cultured for 10 days were examined by electron microscopy after addition of either ruthenium red or ferrocyanide to the postfixation fluid. Osteoclasts, osteoblast-like cells, and macrophages were the principal active cells at these formation sites. The mononuclear cells (osteoblast-like cells and macrophages) in the osteoid tissue showed evidence of having incorporated elements of calcified tissue. Osteoblast-like cells had phagocytized collagen fibrils and calcified bone matrix. This occurred more frequently in the calcifying area. Mononuclear macrophages showed not only phagocytosis and digestion of cellular debris and bone spicules in the osteoid, but also active incorporation of calcified bone matrix that had been detached from its surroundings by its pseudopod-like projections from long cytoplasmic processes. Collagen fibrils were seldom observed within the macrophages. These observations suggest that in our culture system osteoblast-like cells and macrophages at bone formation sites have a phagocytic role in bone remodeling.This study was supported in part by a grant from the Ministry of Education. Science and Culture of Japan (No. 59771321)     

Kinetic studies of adenosine kinase from L1210 cells: a model enzyme with a two-site ping-pong mechanism

Purified adenosine kinase from L1210 cells displayed substrate inhibition by high concentrations of adenosine (Ado), ATP, and MgCl2. When incubated with ATP and MgCl2, the enzyme was phosphorylated, and the phosphorylated kinase transferred phosphate to adenosine in the absence of ATP and MgCl2. Substrate binding, isotope exchange, and kinetic studies suggested that the enzyme catalyzes the reaction by means of a two-site ping-pong mechanism with the phosphorylated enzyme as an obligatory intermediate. Among many possible pathways within this mechanism probably a random-bi ordered-bi route is the preferred sequence in which the two substrates, adenosine and MgATP, bind in a random order to form the ternary complex MgATP . E . Ado followed by the sequential dissociation of MgADP and AMP. Dissociation constants of various enzyme-substrate and enzyme-product complexes and the first-order rate constant of the rate-limiting step were estimated.