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)     

The bialaphos biosynthetic genes ofStreptomyces hygroscopicus: Molecular cloning and characterization of the gene cluster

Summary We have isolated and studied the organization ofStreptomyces hygroscopicus genes responsible for the biosynthesis of the antibiotic herbicide bialaphos. Bialaphos production genes were cloned from genomic DNA using a plasmid vector (pIJ702). Three plasmids were isolated which restored productivity toS. hygroscopicus mutants blocked at different steps of the biosynthetic pathway. Subcloning experiments using other nonproducing mutants showed that four additional bialaphos production genes were also contained on these plasmids. A gene conferring resistance to bialaphos, which was independently cloned using the plasmid vector pIJ61, and an antibiotic-sensitive host (S. lividans), was also linked to the production genes. Cosmids were isolated which defined the location of these genes in a 16 kb cluster.     

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.     

Possible involvement of aminopeptidase, an ecto-enzyme, in the inactivation of bradykinin by intact neutrophils

The subcellular localization of the bradykinin-inactivating activity was studied using guinea-pig neutrophils and the following results were obtained. The bradykinin-inactivating activities were found to be present in the cytosol and membrane fractions but not in the granular and nuclear fractions. The bradykinin-inactivating activity of the cytosol fraction was inhibited by N-carbobenzoxy-Gly-Pro, an inhibitor of prolyl endopeptidase, whereas that of the membrane fraction was inhibited by bestatin, an inhibitor of aminopeptidase. Prolyl endopeptidase and aminopeptidase activities were located predominantly in the cytosol and membrane fractions, respectively, and their activities were inhibited by their respective inhibitors. Prolyl endopeptidase and aminopeptidase activities measured with synthetic substrates were competitively inhibited by bradykinin, suggesting that bradykinin is a possible substrate for prolyl endopeptidase and aminopeptidase. Intact neutrophils inactivated bradykinin rapidly. However, when neutrophils were modified chemically by diazotized sulfanilic acid, a poorly permeant reagent which inactivates ecto-enzymes selectively, both the bradykinin-inactivating activity and aminopeptidase activity of neutrophils decreased significantly without any inhibition of cytosol prolyl endopeptidase. The possibility that aminopeptidase, an ecto-enzyme, would be responsible for the inactivation of bradykinin by intact neutrophils was deduced from the results above, although both cytosol prolyl endopeptidase and membrane aminopeptidase could inactivate bradykinin.     

HPLC-studies on nonmercapt-mercapt conversion of human serum albumin

Human mercaptalbumin (HMA) and nonmercaptalbumin (HNA) could be separated by high-performance liquid chromatography (HPLC) at neutral pH. Using HPLC, the present authors found the nonmercapt-mercapt conversion (HNA----HMA) during hemodialysis and the mercapt-nonmercapt conversion (HMA----HNA) after hemodialysis in chronic renal failure, indicating HMA as the covalent carrier protein for sulfur-containing amino acids.     

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)