Renal architecture of the dogfish Scyliorhinus caniculus (Chondrichthyes,Elasmobranchii)

Summary The excretory portion of the opisthonephric kidney of Scyliorhinus caniculus displays a mesial zone that is supplied with venous blood by the renal portal system and with arterial blood from the efferent arterioles of the glomeruli, and a zone of lateral bundles that is irrigated with arterial blood via arterioles in parallel to the afferent arterioles of the glomeruli. Each single nephron performs two large convolutions in the mesial tissue and two hairpin loops in the bundle. The nephron is differentiated into renal corpuscle (located between the two zones), neck segment (in the bundle), proximal segment I (beginning in the bundle, major convolution between the zones), proximal segment II (exclusively in the mesial zone), intermediate segment (beginning in the mesial tissue and ending in the bundle), distal segment (exclusively in the bundle) and collecting tubule (beginning in the bundle, with a large convolution in the mesial tissue and ending in the bundle) that joins the collecting duct-ureter system. In the bundles proximal and distal nephron segments, the end of the renal tubule and a central bundle vessel are arranged together and form a complex countercurrent system that is enclosed in a sheath of connective tissue. The bundles provide the structural basis for the creation of an environment with low urea concentration around the final portion of the renal tubules, which is consistent with previous experimental evidence of a significantly lower urea content of the bundles as compared with the blood and the mesial tissue in another marine elasmobranch, Raja erinacea. This condition is thought to lead to passive reabsorption of urea from the fluid of the end of the renal tubule. Separation of individual nephrons in the bundle zone appears to be correlated with the peculiar secondary structure that results from the folding of the bundles and may be in addition a requirement in conjunction with intermittent function of the glomeruli. The zonation of the renal tissue with formation of bundles with counter-current systems is characteristically found in marine Elasmobranchs and is considered to be the morphological correlate to the physiological ability of the marine Elasmobranchii to use urea for osmoregulation.     

Biochemical research on oogenesis. RNA accumulation during oogenesis of the dogfish Scyliorhinus caniculus

Four biochemical mechanisms have been shown to operate in the oocytes of amphibians and teleosts: (1) amplification of the 28 S and 18 S genes, (2) noncoordinate accumulation of 5 S RNA and 28 S + 18 S RNA, (3) storage of 5 S and transfer RNA made in excess by small oocytes within nucleoprotein particles, (4) expression of different 5 S genes in oocytes and somatic cells. We have tried to extend these observations to another group of vertebrates, i.e., selacians (Chondrichthya). Our data suggest that ribosomal gene amplification is low or absent in the oocytes of the dogfish Scyliorhinus caniculus. However, previtellogenic oocytes of this species accumulate more 5 S RNA than needed for ribosome assembly. Transfer and 5 S RNA present in small oocytes are probably not free in the cell sap. A substantial fraction of these RNAs sediments at 10 S when homogenates of immature ovaries are centrifuged in sucrose density gradients. In contrast to what we observed in amphibians and teleosts, 5 S RNA from ovaries of S. caniculus is identical in sequence to 5 S RNA from liver. Among the four mechanisms mentioned above, the second and probably the third one are used by the oocytes of S. caniculus. Mechanism (4) is absent in this species. No definitive conclusion can be drawn concerning mechanism (1), i.e., ribosomal gene amplification.     

Involvement of deoxyribonucleic acid polymerase beta in nuclear deoxyribonucleic acid synthesis.

The effects on DNA synthesis in vitro in mouse L929-cell nuclei of differential extraction of DNA polymerases alpha and beta were studied. Removal of all measurable DNA polymerase alpha and 20% of DNA polymerase beta leads to a 40% fall in the replicative DNA synthesis. Removal of 70% of DNA polymerase beta inhibits replicative synthesis by 80%. In all cases the nuclear DNA synthesis is sensitive to N-ethylmaleimide and aCTP (arabinosylcytosine triphosphate), though less so than DNA polymerase alpha. Addition of deoxyribonuclease I to the nuclear incubation leads to synthesis of high-molecular-weight DNA in a repair reaction. This occurs equally in nuclei from non-growing or S-phase cells. The former nuclei lack DNA polymerase alpha and the reaction reflects the sensitivity of DNA polymerase beta to inhibiton by N-ethylmaleimide and aCTP.     

Extraction and biochemical characterization of a nuclear deoxyribonucleic acid polymerase activity in bull spermatozoa.

Bull spermatozoa heads were separated from cytoplasmic contaminants, especially mitochondria-rich middle pieces, by centrifugation through 2.4M-sucrose. DNA polymerase activity was demonstrated by incubating nuclear heads for 1 h at 37 degrees C or for 20 h at room temperature in a medium containing detergent and dithiothreitol or 2-mercaptoethanol. Optimal DNA polymerase activity was detected after extraction in a medium containing 50 mM-borate, pH9, 1 mg of soya-bean trypsin inhibitor/ml and supplemented with either 20 mM-dithiothreitol and 4% Tween 80 or 100mM-2-mercaptoethanol and 10% Tween 80. The DNA polymerase reaction was Mg2+-dependent; Mn2+ or Ca2+ could not replace Mg2+ and all four deoxynucleoside triphosphates were required for optimal activity. The polymerase activity was pH-dependent (optimum between 8.2 and 10.5) and was a function of buffer composition and also of pH values. Optimal activity was obtained with 50 mM-Na+ or 150mM-K+ and was partially lowered by N-ethylmaleimide; it was inhibited by spermidine and by salmon protamines, but was greatly stimulated by calf thymus histones. It was also resistant to actinomycin D, netropsin and ethidium bromide. The present results suggest that bull spermatozoa heads contain a beta-type DNA polymerase activity.     

Glomerular bypass shunts and distribution of glomeruli in the kidney of the lesser spotted dogfish,Scyliorhinus caniculus

Summary Scanning electron microscopy of corroded resin casts of the renal vasculature of Scyliorhinus caniculus has revealed a novel vascular pathway arising from the afferent arteriole and bypassing the glomerulus. This glomerulus bypass shunt occurred in 36% of the glomerular casts examined. The shunt ran to join a peritubular network of capillaries and thereby offers the potential to vary the degree of glomerular perfusion and control the proportion of active glomeruli. In 29% of glomeruli two efferent arterioles drained the capillary knot. Glomeruli were located close to the dorsal margin of the posterior mass of the kidney, and towards the lateral edge of the anterior lobes of the kidney of female dogfish. In male dogfish, glomeruli were evenly distributed through the posterior mass of kidney, while in female dogfish 89% of glomeruli occurred in the posterior mass and 11% of glomeruli were located within the small anterior lobes.     

Novikoff hepatoma deoxyribonucleic acid polymerase. Purification and properties of a homogeneous beta polymerase.

Deoxyribonucleic acid polymerase-beta (EC 2.7.7.7) FROM THE Novikoff hepatoma has been purified over 200 000-fold (based on the increase in specific activity), by ammonium sulfate fractionation and chromatography on DEAE-Sephadex, phosphocellulose, hydroxylapatite, and DNA-cellulose. The enzyme is remarkably stable through all stages of purification until DNA-cellulose chromatography when it must be kept in buffers containing 0.5 M NaCl and 1 mg/ml bovine serum albumin for stability. The enzyme appears to be homogeneous as evidenced by a single stainable band when subjected to electrophoresis in polyacrylamide gels of different porosity. The stainable band corresponds to the DNA polymerase as determined by slicing sister gels and assaying for enzyme activity. The specific activity of the homogeneous preparation is about 60 000 units/mg. The enzyme lacks detectable exonuclease or endonuclease activity. It has a molecular weight of 32 000 as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. In sucrose gradients, the molecular weight is estimated at 31 000. The isoelectric point of the hydroxylapatite fraction enzyme is 8.5. The Novikoff beta-polymerase requires all four deoxyribonucleoside triphosphates, primer-template, and a divalent cation for maximal activity. The apparent Km for total deoxyribonucleoside triphosphate is 7-8 muM and for DNA 125 mug/ml. Activated DNA, rendered 7% acid soluble by DNase I, is the preferred primer-template, although a number of synthetic polynucleotides can by efficiently utilized, particularly in the presence of Mm2+ optimum is 7 mM; the Mn2+ optimum is 1 mM. The pH optimum is 8.4 in Tris-HCl or 9.2 in glycine buffer. The beta-polymerase is sstimulated about twofold by NaCl or KCl at an optimum of 50-100 MM, and the enzyme maintains considerable activity at high ionic strengths. The DNA polymerase is inhibited by ethanol, acetone, and a variety of known polymerase inhibitors. Glycols stimulate the enzyme as does spermine or spermidine. Unlike most beta-polymerases, the Novikoff enzyme is moderately sensitive to N-ethylmaleimide.     

Participation of deoxyribonucleic acid polymerase alpha in amplification of ribosomal deoxyribonucleic acid in Xenopus laevis.

Aphidicolin, a known inhibitor of eucaryotic deoxyribonucleic acid (DNA) polymerase alpha, efficiently inhibited amplification of ribosomal DNA during oogenesis in Xenopus laevis. DNA polymerase alpha, but not DNA polymerase gamma, as isolated from ovaries, was sensitive to aphidicolin. DNA polymerase beta was not detectable in Xenopus ovary extracts. Therefore, DNA polymerase alpha plays a major role in ribosomal ribonucleic acid gene amplification.     

Changes in deoxyribonucleic acid polymerase activities in synthesis of deoxyribonucleic acid during sporulation of Bacillus subtilis.

The deoxyribonucleic acid (DNA) polymerase activities in Bacillus subtilis strains Marburg 168 (thy-trp2) and D22, a DNA polymerase I-deficient mutant, were measured at various stages of sporulation. The DNA polymerase I activity, which had decreased after the exponential growth, began to increase at the early stage of sporulation, reached a maximum and then again decreased. The activity of neither DNA polymerase II nor III was observed to change so drastically as that of DNA polymerase I during sporulation. The incorporation of [3H]deoxythymidine 5'-triphosphate ([3H]dTTP) into Brij 58-treated permeable cells increased during sporulation. The stimulation of [3H]dTTP incorporation into the cells by irradiation with ultraviolet light was also observed to coincide with DNA polymerase I activity. In strain D22 the activities of DNA polymerase II and III were almost constant with time. Neither change of [3H]dTTP incorporation into Brij 58-treated cells nor stimulation of incorporation by irradiation with ultraviolet light was observed.     

Ultrastructural and chemical study of the chromatin during spermiogenesis of the fish Scyliorhinus caniculus (L.) (author's transl)

Electron microscopic, cytochemical and biochemical techniques were applied to study structural aspects and changes in nuclear components during the spermiogenesis of Scyliorhinus caniculus. Five major stages of nuclear differentiation were recognized and characterized by variations in the organization and chemical properties of chromatin. Stage I is analogous to a somatic nucleus with heterogeneous chromatin. At the second stage, the nuclear content is dispersed but the chromatin fibers are of the same diameter as those of the stage I. The nuclear elongation begins at stage III, the DNP fibers running preferentially parallel to the long axis of the nucleus. During these early modifications of chromatin structure appear two new basic nuclear proteins (S 1 and S 2) which migrate faster than histones but typical histones remain assosciated with these nuclei. In later elongation stage (stage IV), the chromatin fibers organize in a helical form and fuse side by side giving lamellar systems which have a reticular structure. At the end of this stage, the nuclear material has become uniformly compact. These late variations in chromatin organization are parallel to the association of chromatin with new basic nuclear proteins (S 3, S 4, Z 1, Z 2 and Z 3). The cytochemical and electrophoretical properties of one of these proteins (S 4) which appears at the end of spermiogenesis are similar to those of a protamine. In stage V, the chromatin is homogeneous and the nucleus assumes a helical configuration beginning at the posterior end. The deoxyribonucleoproteins of the mature sperm show some novel chemical characters, including the appearance of a stable nuclear acidophilia with the ALFERT and GESCHWIND method and extraction with 0.25 N HCl of one of the basic protein fractions newly appeared in late spermiogenesis (Z 3), two other fractions (Z 1 and Z 2) being extracted with a more drastic procedure. The other fractions described before are no more detectable.     

Structural and enzymological characterization of the homogeneous deoxyribonucleic acid polymerase from Mycoplasma orale.

We have purified the DNA polymerase from Mycoplasma orale to homogeneity. The protein structure of the enzyme was declined by sodium dodecyl sulfate gel electrophoresis, which revealed a single band of 116 000 daltons that was coincident with the polymerase activity profile in the final step of DNA--cellulose chromatography, and by two-dimensional gel analysis, which demonstrated a single protein species at pI = 6.8 that was congruent with enzyme activity and contained the same 116 000 polypeptide. although severe enzyme aggregation occurs during nondenaturing gel electrophoresis, a monomer species can be resolved with a Mr of 140 000 by the Ferguson plot analysis. Gel filtration and velocity gradient centrifugation yield a Stokes radius of 4.8 nm and a sedimentation coefficient of 5.6 S, respectively, from which Mr values of 106 000--128 000 can be computed. The different size values suggest that the polymerase molecule is asymmetric. The purified enzyme has a specific activity of approximately 6 x 10(5) units/mg of protein and in completely devoid of exodeoxyribonuclease and endodeoxyribonuclease activities, at exclusion limits of 10(-4)--10(-6%) of the polymerase activity. The mechanism of polymerization is moderately processive, with an average of 14 +/- 4 nucleotides incorporated per binding event, and the "effective template length" on activated DNA is approximately 40 nucleotides.