Mitochondrial DNA sequence evolution in sharks: rates, patterns, and phylogenetic inferences

Abundant representation of sharks in the fossil record makes this group a superb system in which to investigate rates and patterns of molecular evolution and to explore the strengths and weaknesses of phylogenetic inferences from molecular data. In this report, the molecular evolution of the cytochrome b gene in sharks is described and the information related to results from phylogenetic analysis of the data evaluated in the light of a phylogeny derived independently of the molecular data. Across divergent lineages of sharks there is evidence for significant substitution rate variation, departure from compositional equilibrium, and substantial homoplasy; nevertheless, the signal of evolutionary history is evident in patterns of shared transversions and amino acid replacements.      

Metabolic rate and directional nucleotide substitution in animal mitochondrial DNA

There is marked heterogeneity of nucleotide composition in mitochondrial DNA across divergent animals. Differences in nucleotide composition presumably reflect differences in directional nucleotide substitution for A+T or G+C nucleotides. In mitochondrial DNA, there is A+T directional nucleotide substitution in most (if not all) animals surveyed, and the magnitude of directional A+T nucleotide substitution differs greatly within and among groups. Differences in directional nucleotide substitution among lineages of mammals can be explained by changes in metabolic physiology. This relationship is thought to be mediated by the effect of oxygen radicals because these toxic compounds are by-products of aerobic metabolism and are known mutagens. Association between metabolism and nucleotide composition provides additional evidence in favor of the hypothesis that rates and patterns of nucleotide substitution in mitochondrial DNA can be influenced by factors that impinge on rates of endogenous DNA damage.      

Elemental distribution in striated muscle and the effects of hypertonicity: Electron probe analysis of cryo sections

A method of rapid freezing in supercooled Freon 22 (monochlorodifluoromethane) followed by cryoultramicrotomy is described and shown to yield ultrathin sections in which both the cellular ultrastructure and the distribution of diffusible ions across the cell membrane are preserved and intracellular compartmentalization of diffusabler ions can be quantitated. Quantitative electron probe analysis (Shuman, H., A.V. Somlyo, and A.P. Somlyo. 1976. Ultramicros. 1:317-339.) of freeze-dried ultrathin cryto sections was found to provide a valid measure of the composition of cells and cellular organelles and was used to determine the ionic composition of the in situ terminal cisternae of the sarcoplasmic reticulum (SR), the distribution of CI in skeletal muscle, and the effects of hypertonic solutions on the subcellular composition if striated muscle. There was no evidence of sequestered CI in the terminal cisternae of resting muscles, although calcium (66mmol/kg dry wt +/- 4.6 SE) was detected. The values of [C1](i) determined with small (50-100 nm) diameter probes over cytoplasm excluding organelles over nuclei or terminal cisternae were not significantly different. Mitochondria partially excluded C1, with a cytoplasmic/ mitochondrial Ci ratio of 2.4 +/- 0.88 SD. The elemental concentrations (mmol/kg dry wt +/- SD) of muscle fibers measured with 0.5-9-μm diameter electron probes in normal frog striated muscle were: P, 302 +/- 4.3; S, 189 +/- 2.9;C1, 24 +/- 1.1;K, 404 +/- 4.3, and Mg, 39 +/- 2.1. It is concluded that: (a) in normal muscle the "excess CI" measured with previous bulk chemical analyses and flux studies is not compartmentalized in the SR or in other cellular organelles, and (b) the cytoplasmic C1 in low [K](0) solutions exceeds that predicted by a passive electrochemical distribution. Hypertonic 2.2 X NaCl, 2.5 X sucrose, or 2.2 X Na isethionate produced: (a) swollen vacuoles, frequently paired, adjacent to the Z lines and containing significantly higher than cytoplasmic concentrations of Na and Cl or S (isethionate), but no detectable Ca, and (b) granules of Ca, Mg, and P = approximately (6 Ca + 1 Mg)/6P in the longitudinal SR. It is concluded that hypertonicity produces compartmentalized domains of extracellular solutes within the muscle fibers and translocates Ca into the longitudinal tubules.     

Periodic operation of immobilized live cell bioreactor for the production of candicidin

A lumped model for cell growth and secondary metabolite production in an immobilized live cell bioreactor has been developed. This model is applied here to simulate the performance of an immobilized bioreactor under steady-state conditions and under conditions of periodically varying concentration of a growth-limiting substrate. The results of the simulation study were experimentally verified in the case of the production of the antibiotic candicidin by Streptomyces griseus in an immobilized bioreactor with forced periodic operation. The results of the studies suggest that periodically operated immobilized live cell bioreactors can provide a potent alternative for the production of non-growth-associated biochemicals, as compared to free cell fermentations, pulsed fermentations with process cycle regeneration, and nonregenerated bioreactors. This work has demonstrated that by frequent pulsing of the growth limiting nutrient, stable extended production can be obtained at high specific cellular productivities.     

Immobilization of Brevibacterium flavum cells on collagen for the production of glutamic acid in a recycle reactor

In this study, live cells of Brevibacterium flavum were immobilized for the production of glutamic acid. The reason for such a choice was that glutamic acid fermentation is an extensively studied fermentation and one which requires the viability of entire cellular faculties for the acid production. Brevibacterium flavum was chosen because it is an industrially used bacterium, and is very potent via a vis glutamic acid production. Studies were performed to find aeration and agitation conditions for optimal growth and glutamic acid productivity. Experiments were also done to find the optimum harvesting time. The cell activity peaks during the run of fermentation, and the time at which the peak occurs, was found. Conventional methods for immobilizing the cells on collagen were found to be lacking. The pH and drying were the two main reasons for loss of viability of the cells; the latter being more important. A modified immobilization procedure has been devised, which can immobilize live cells at any given pH and ionic strength, in contrast to the conventional method which requires the pH to be above 11 or below 3. This new method involves dialysis of collagen in suitable dialysis bags against water at pH7 (or buffer at any desired pH). The dialysed collagen blended at 20,000 rpm, resulted in a very smooth dispersion, unnoticeably different from collagen dispersion prepared at pH 11. The dispersed collagen was then cast and dried at an elevated temperature, and high air flow rate over the cast membrane, decreasing the time of drying from 6–8 hr ( in the conventional method) to 1.5–2 hr. The membrane has been tested for glutamic acid producing capabilities in a column reactor with the membrane spirally wound. The reactor has been operated under continuous conditions for 5–10 days with stable activities.     

Phenotypic conversion of cultured mouse embryo cells by aza pyrimidine nucleosides.

Cells of the $\text{C3H}\text{10}\text{T}\text{1}\text{2}\text{CL8}$ line, which are nonmyoblastic in nature, form functional myotubes when treated with low concentrations of 5-azacytidine. Further characterization of the myotubes revealed that they arise from the fusion of mononucleated precursors and not as a result of endoreplication. They accumulate histochemically detectable myosin ATPase activity as well as acetylcholine receptors capable of binding radioactively labeled α-bungarotoxin. The deoxy analog, 5-aza-2′-deoxycytidine, induced myogenic conversion at one-tenth of the maximally effective concentration of 5-azacytidine. The ability of both analogs to induce myotube formation and to cause cytotoxicity was strongly influenced by cotreatment with certain pyrimidine nucleosides. These effects were consistent with a requirement for metabolism of both aza compounds to phosphorylated derivatives and with a mechanism of action based on their incorporation into DNA. Concentrations of the analogs causing myogenic conversion did not substantially alter rates of DNA, RNA, or protein synthesis as measured by precursor incorporation into intact cells. The induction of myotubes by 5-azacytidine in cells synchronized by two different methods required that treatment with the analog was carried out at a critical phase early in S phase. Thus the mechanism of drug action appears to be linked to specific DNA synthesis.