Biomedical subjects
Publications and source records attributed to T Takano.
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DNA is probably the most discussed and least observed of all biological macromolecules. Although its role in biology is a central one, with many examples such as operators and restriction sites where specific base sequences have control functions or interact with specific enzymes, the structures that DNA can adopt have been based until now only on sequence-averaged fibre diffraction patterns. Recent improvements in triester synthesis methods have made possible the preparation of sufficient homogeneous DNA of predetermined sequence for crystallization and X-ray structure analysis. We report here the first single-crystal structure analysis of more than a complete turn of right-handed B-DNA, with the self-complementary dodecamer sequence d(CpGpCpGPApApTpTpCpGpCpG) or CGCGAATTCGCG.
In the refinement of the X-ray diffraction structures of molecules, it is conventional to introduce atomic 'temperature factors' of the Debye-Waller form to characterize the widths of the electron density peaks corresponding to the atoms. Although these factors are known to include a variety of contributions other than thermal fluctuations of the atomic positions, recent progress in the refinement of protein structures has led to inferences concerning atomic mobilities from the temperature factor data for several proteins. Atomic position fluctuations can be calculated independently by the molecular dynamics method, in which the classical equations of motion for the atoms of an equilibrated protein are solved on a computer. We now show that the X-ray diffraction and dynamical simulation methods yield similar pictures of the atomic mobility in tuna ferrocytochrome c.
The DNA tetramer d(CpGpCpG) or CGCG crystallizes from high-salt solution as a left-handed double helix, the Z' helix. Its structure differs from that of the other known left-handed helix, Z-DNA, by a Cl'-exo sugar pucker at deoxyguanosines rather than C3'-endo, and these represent two alternative solutions to the same steric constraint arising from the syn glycosyl bond orientation. The apparent molecular basis for the Z to Z' transition in going from intermediate to high salt is substitution of a bound anion for water at guanine amino groups, and consequent charge repulsion of anions and backbone phosphates.
A practical procedure is described for obtaining milligram quantities of a small (29 nucleotide) Eco RI restriction fragment of DNA containing the Escherichia coli lac operator. A yield of 10--15 mg of operator is obtained from 1 kg of wet cell paste. The resultant operator is shown to be homogeneous and competitively active in filter assays. Two separable but interconvertible forms of lac operator exist in solution, probably linear duplex and hairpin isomers. Only the presumed linear form is active in binding lac repressor by competition assay, but the two isomers are interconvertible by heating to 80 degrees C. The methods described here should be generally applicable for purifying other restriction fragments from plasmids.
Tuna ferrocytochrome c and ferricytochrome c have been refined independently at high resolution (1.5 A and 1.8 A) to crystallographic residual errors of 17.3% and 20.8%, respectively. Small but significant conformational differences are seen surrounding a buried water molecule that is hydrogen bonded to Asn-52, Tyr-67, and Thr-78. In the oxidized state, this water molecule is 1.0 A closer to the heme and the heme has moved 0.15 A out of its heme crevice; both changes lead to a more polar microenvironment for the heme. Chemical modification studies, patterns of evolutionary conservatism, structural differences in bacterial cytochromes, and x-ray studies all agree that the "active site" for cytochrome c is bounded by lysines 8, 13,27, 72, 79, 86, and 87 (thus containing the evolutionary conservative 72-87 loop) and has the buried water molecule just below its surface and the opening of the heme crevice slightly to one side.
The activity of ornithine decarboxylase (OD-Case:L-ornithine carboxy-lyase, EC 4.1.1.17) in rabbit costal chondrocytes in culture increased markedly after addition of parathyroid hormone (PTH), reaching a maximum 4 to 5 hr after PTH addition. The increase in ODCase activity was followed by increase in the intracellular concentrations of polyamines, especially putrescine, which increased in 6 hr to about 3-fold that of untreated cultures. The induction of ODCase by PTH was not observed in L, 3T3, HeLa, buffalo rat liver, or BHK cells. Retinyl acetate and retinoic acid both inhibited expression of the differentiated phenotype of chondrocytes by rabbit costal chondrocytes in culture within 3 days after their addition, as judged by morphological change and decrease in sulfate incorporation into glycosaminoglycans but did not inhibit cell proliferation. PTH could not induce an increase in ODCase in de-differentiated cells that had been pretreated with retinyl acetate or retinoic acid for 3 days. but 4 days after removal of the retinoids, these de-differentiated cells regained the ability to synthesize ODCase in response to PTH. These facts suggest that the induction of ODCase and the formation of putrescine by PTH are good markers of the differentiated phenotype of cultured chondrocytes.
Age related changes of the thyroid gland of CF1 mice ranging in age from 15 days to 24 months were studied morphologically and biochemically. The mean diameter of follicles becomes larger with advancing age due to fusion of adjacent follicles, and there is also an increasing variability of follicle diameter. The volumetric density of lysosomes in the cytoplasm of epithelial cells lining follicles increases with age. In the younger (seven days to 1.5-month-old) mice, most lysosomes are small and homogeneously dense; these are considered to be the primary lysosomes necessary for the hydrolysis of reabsorbed colloid droplets. The heterogeneously dense, large lysosomes containing lipid-like droplets increase in number with age, especially in mice over 15 months. The specific activities of acid phosphatase, beta-glucuronidase and beta-galactosidase show a relative decrease with age after six months; although there may be an absolute increase. Some of the lysosomes observed in old animals may be residual bodies.
The characteristics of the virion-associated RNA-dependent DNA polymerase (RDPase) of a baboon endogenous virus, M7, were studied extensively; the optimal conditions for the exogenous RDPase reaction were obtained with 0.4 mM-Mn2+, 110 mM-NaCl, 24 microgram/ml poly(rA). oligo(dT)12-18, at pH 7.6 in the presence of 0.01 % Brij-58. Under these conditions, the incorporation of 3H-TMP proceeded up to 90 min at a speed of 0.1 pmol TMP/microgram virus protein/min. Poly(rC). oligo (dG)12-18 and poly(rCm). oligo (dG)12-18 served as the template-primers in the exogenous reaction with 3 mM-Mg2+ and 0.4 mM-Mn2+, respectively. Polyuridylic acid, bleomycin, rifampicin, spermidine and inorganic phosphate significantly inhibited the RDPase activity of BaEV. The RDPase of BaEV requires a higher concentration of NaCl, a lower pH and milder conditions of detergent treatment than those of other mammalian retroviruses.
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The behavior of hyperplastic nodules following an in vivo short-term screening test for hepatocarcinogens was studied. Rats were injected ip with 200 mg/kg body weight of diethylnitrosamine (DEN), given basal diet containing 200 ppm of N-2-fluorenylacetamide (2-FAA) (group 1), 1000 ppm of the alpha-isomer of 1,2,3,4,5,6,-hexachlorocyclohexane (alpha-BHC) (group 2) or basal diet (group 3) from week 3 to week 8, and then given basal diet and tap water. They were subjected to partial hepatectomy at the end of week 3. Animals were killed at weeks 4, 6, 8, 10, 20, 30, 40, and 50. A significant disappearance of hyperplastic nodules following the cessation of carcinogen treatment was observed in group 1, but was not evident in groups 2 and 3. With gamma-glutamyltranspeptidase (GGTase) as a positive marker and adenosine triphosphatase (ATPase) as a negative marker, hyperplastic nodules were classified into 3 different phenotypic categories, i.e., (1) GGTase-positive and ATPase-negative, (2) GGTase-positive, and (3) ATPase-negative. The percentages of GGTase-positive and ATPase-negative hyperplastic nodules were almost 80 approximately 90% in group 1 and 70 approximately 80% in groups 2 and 3. Some of the hyperplastic nodules were necrotic from week 8 in groups 1 and 2, and from week 20 in group 3. Subsequently, the numbers of necrotic hyperplastic nodules increased with time. Hepatocellular carcinomas were found at weeks 30, 40, and 50 in group 1, and at weeks 40 and 50 in group 2. Significantly higher incidences of cancer were found in group 1 than in group 2. The hepatocellular carcinomas were also classified enzyme-histochemically into 3 different phenotypic categories as for hyperplastic nodules, but the percentage (20%) of GGTase-positive and ATPase-negative hepatocellular carcinomas was significantly lower than that (70 approximately 90%) of GGTase-positive and ATPase-negative hyperplastic nodules in each group.