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Biomedical subjects

J R Cook

Publications and source records attributed to J R Cook.

At least 37 records · Page 2Linked to original sources

Fourteen internal transcribed spacers in the circular ribosomal DNA of Euglena gracilis.

Cytoplasmic ribosomes from Euglena gracilis contain 16 rRNA components. These include the typical 5 S, 5.8 S and 19 S rRNAs that are found in other eukaryotes as well as 13 discrete small RNAs that interact to form the equivalent of eukaryotic 25-28 S rRNA (accompanying paper). We have utilized DNA sequencing techniques to establish that genes for all of these RNAs, with the exception of 5 S rRNA, are encoded by the 11,500 base-pair circular rDNA of E. gracilis. We have determined the relative positions of the coding regions for the 19 S rRNA and the 14 components (including 5.8 S rRNA) of the large subunit rRNA, thereby establishing that the genes for each of these rRNAs are separated by internal transcribed spacers. We conclude that sequences corresponding to these spacers are removed post-transcriptionally from a high molecular weight pre-rRNA, resulting in a multiply fragmented large subunit rRNA. Internal transcribed spacers, in positions analogous to some of these additional Euglena rDNA spacers, have been found in the rDNA of other organisms and organelles. This finding supports the view that at least some internal transcribed spacers may have been present at an early stage in the evolution of rRNA genes.

Animals

Acquired myasthenia gravis in a cat with thymoma.

A 4-year-old castrated Abyssinian cat was evaluated for profound neuromuscular weakness. Results of electromyography and repetitive nerve stimulation tests were normal. Thoracic radiography revealed a cranial mediastinal mass, which was excised and identified as a thymoma. Serum acetylcholine receptor antibodies were detected at high concentration, supporting a diagnosis of acquired myasthenia gravis. Clinical signs of disease responded to treatment with pyridostigmine and corticosteroids.

Animals

Alpha-fetoprotein mRNA: variation of half-life in rat hepatoma cell lines and destabilization by sodium butyrate.

The half-life of alpha-fetoprotein (AFP) mRNA was determined in two rat hepatoma cell lines. In the 7777 cell line, AFP mRNA half-life is 30-35 h, consistent with earlier observations (Innis and Miller, 1979). However, the half-life in McA-RH8994 cells is only 14-16 h. Dexamethasone does not affect AFP mRNA half-life in either cell line, indicating that glucocorticoid-mediated alterations in AFP mRNA levels in these cells is at the transcriptional level. The naturally-occurring fatty acid sodium butyrate did, however, cause significantly more rapid degradation of AFP mRNA in McA-RH8994 cells, decreasing the half-life from 14-16 h to only 5-6 h. The ability of sodium butyrate to block glucocorticoid action on AFP gene expression in this cell line is therefore due to both transcriptional and post-transcriptional effects.

Animals

The evaluation of chicken spermatozoa using fluorescent staining in a 96-well format.

An efficient method for the evaluation of spermatozoa using the fluorescent stains carboxyfluorescein diacetate and propidium iodide and an automated fluorescence concentration analyzer was adapted for chicken semen. Arbitrary fluorescence units representing either live or dead spermatozoa were strongly correlated with percentage of added dead spermatozoa and with the direct fluorescent microscope counts of live, dead, and damaged cells.

Animals

Amplitude and latency characteristics of spinal cord motor-evoked potentials in dogs.

The motor-evoked potential can be reliably recorded in anesthetized dogs by use of percutaneous placement of active recording electrodes near the dorsal lamina of the vertebral column. Two types of responses were observed in this study; short (less than 5.5 ms at T9-10)- and long (greater than 5.8 ms at T9-10)-latency waves. Short-latency waves are larger in amplitude and appear with higher stimulus intensities than do long-latency waves. Short-latency waves are conducted at greater than 80 m/s and may not reflect pyramidal tract activation. The safety of using higher intensity stimuli to generate short-latency waves has not been determined.

Animals

Microporosity of the substratum regulates differentiation of MDCK cells in vitro.

We have analyzed the ability of the physical substratum to modulate both the ultrastructural and protein synthetic characteristics of the Madin-Darby canine kidney (MDCK) renal cell line. When MDCK cells were seeded on Millipore Millicell CM microporous membrane cell culture inserts they demonstrated a more columnar organization with an increase in cell density sixfold greater than the same cells seeded on conventional plastic substrata. After 1 wk postseeding on the microporous membrane a partial basal lamina was noted, with a contiguous basement membrane being apparent after 2 wk. One-dimensional sodium dodecyl sulfate gel electrophoresis was used to analyze detergent-solubilized proteins from MDCK cells maintained on plastic substrata vs. microporous membranes. When proteins were pulse-labeled with [35S]methionine, a 55 kDa protein was evident in the cytosolic extract of cells grown on collagen, laminin, and nontreated plastic substrata; but this labeled protein was not evident in similar extracts from cells grown on collagen and laminin-coated microporous membranes. To test if the polarized, basement-membrane secreting phenotype of the MDCK cells could be generated on a microporous membrane without pretreatment with any extracellular matrix (ECM) components, cells were seeded on the Millipore Millicell HA (cellulosic) microporous membrane. This type of substrata does not need a coating of ECM components for cell attachment. A partial basement membrane was formed below cells where the basal surface of the cell was planar, but not in areas where the cell formed large cytoplasmic extensions into the filter. This led us to the conclusion that the microporous nature of the substrata can dictate both ultrastructural and protein synthetic activities of MDCK cells. Furthermore, we suggest that both the planar nature of the basal surface and the microporosity of the substrate are corequisites for the deposition of the basement membrane.

Animals

Enhancement of transformed cell growth in agar by serine protease inhibitors.

We investigated the effects of three serine protease inhibitors (leupeptin, soybean trypsin inhibitor, and aprotinin) on the serum-free growth of two transformed cell lines in soft agar. Aprotinin markedly enhanced the growth of rat embryo fibroblasts that had been transformed by polyoma middle T antigen (PyMLV-REF52), while having only a slight effect on the colonial growth of SV40 transformed Balb/c 3T3 cells (SV3T3-Aga). Leupeptin and soybean trypsin inhibitor, on the other hand, significantly enhanced the growth of SV3T3-Aga cells while having little effect on PyMLV-REF52 growth. We observed no stimulatory effect of any of the protease inhibitors on serum-free monolayer growth. Under conditions of excess aprotinin, PyMLV-REF52 cells were found to be unresponsive to epidermal growth factor (EGF) at a concentration that would normally stimulate agar colony growth. However, aprotinin was not capable of supporting colony formation with transforming growth factor-beta. These results indicate that aprotinin acts primarily as a protease inhibitor in spite of its structural homology to EGF and that EGF may promote the soft agar growth of these cell lines either by inhibiting proteolysis directly or by enhancing the synthesis of a serine protease inhibitor.

Antigens, Polyomavirus Transforming

Methylation of the alpha-fetoprotein gene in isogenic rat hepatoma and liver cell lines.

We have found that DNA methylation is inversely correlated with alpha-fetoprotein (AFP) gene expression in a series of isogenic rat hepatoma cell lines. The 5' end of the gene is extensively demethylated in AFP-producing cells and is highly methylated in cell lines which do not produce AFP. Glucocorticoid affects markedly the synthesis of AFP in the hepatoma cells. However, methylation patterns of cell lines which were treated with dexamethasone were not different from those of control cells, indicating that glucocorticoid action on AFP gene expression does not alter DNA methylation in this region of the gene.

Animals

Mechanism of the dexamethasone effect on alpha-fetoprotein gene expression in McA-RH8994 rat hepatoma cells.

We have investigated the mechanism by which dexamethasone (DEX) increases alpha-fetoprotein (AFP) secretion by the McA-RH8994 rat hepatoma cell line. In contrast to the 7777 cell line, DEX was found to increase the amount of hybridizable cytoplasmic AFP mRNA in McA-RH8994 cells. This effect was dose-dependent, reversible after removal of DEX, and was partially blocked by progesterone in excess, indicating a receptor-mediated response. In vitro nuclear transcription experiments showed that DEX increased AFP gene transcription in McA-RH8994 cells. Cycloheximide blocked the effect of DEX on AFP mRNA levels in both the McA-RH8994 and 7777 cell lines, showing that ongoing protein synthesis is required for glucocorticoids either to enhance or to suppress AFP gene expression in these cell lines.

Animals

Genetic regulation of sn-glycerol-3-phosphate dehydrogenase in brown adipose tissue.

The levels of sn-glycerol-3-phosphate dehydrogenase (GPDH) were determined in the brown adipose tissue (BAT) of different inbred strains of mice. The BAT of the BALB/cJ strain contains twice as much enzyme activity per milligram protein as do other strains. The appearance of this difference is developmentally dependent, since it is not detected in BAT until 25-30 days postpartum. Genetic analysis of this strain difference has shown that the mechanism of inheritance involves at least two genes, one of which is linked to the Gdc-1 structural locus on chromosome 15. Determinations of GPDH synthesis by immunoprecipitation of GPDH protein labeled in vivo with [3H]leucine, and of GPDH mRNA by Northern blot analysis, establish that in BALB/cJ mice higher rates of enzyme synthesis are determined by elevated levels of GPDH mRNA. It was also found that cold stress increases GPDH mRNA levels in all the strains examined.

Adipose Tissue, Brown

Physical properties of a plasmid-like DNA from Euglena gracilis.

A small circular extrachromosomal DNA of the flagellate protozoan Euglena gracilis has been characterized as having a contour length of 11.3 kb, with a consistent restriction map. The buoyant density (rho = 1.717) and melting temperature (tm = 89 degrees C) both indicate a base content of 59% G + C. The DNA is found in both wild-type cells and those lacking plastids. The copy number is estimated to be about 1000.

DNA Restriction Enzymes

Effect of sodium butyrate on alpha-fetoprotein gene expression in rat hepatoma cells in vitro.

Sodium butyrate has been reported to induce cellular differentiation and reduce the tumorigenicity of certain tumor cells. We have examined the effects of butyrate on alpha-fetoprotein (AFP) gene expression in 7777 and McA-RH8994 rat hepatoma cells and have found that nontoxic concentrations of the drug decrease AFP mRNA levels in both cell lines. However, McA-RH8994 requires a 10-fold lower concentration (0.5 mM) of butyrate to affect a 50% reduction in AFP mRNA levels within 48 h. At 2 mM, sodium butyrate reduces AFP mRNA levels in McA-RH8994 cells by at least 90% after 48 h, while having little effect on the expression of either the 7S RNA or Harvey-ras genes. Time-course studies show that the effect of butyrate on McA-RH8994 AFP mRNA levels is immediate and is accompanied by an accumulation of cells in the G1/G0 phase of the cell cycle. Sodium butyrate was found to reduce AFP mRNA levels in both dexamethasone-treated 7777 and McA-RH8994 cells; dexamethasone decreases AFP mRNA levels in the former cell line and increases AFP mRNA levels in the latter. Therefore, it is unlikely that butyrate acts simply by reducing the dexamethasone receptor concentration in 7777 cells.

Animals

Culture pH, CO2 tension, and cell division in Euglena gracilis Z.

Growth characteristics of Euglena gracilis Z as functions of culture pH, CO2 tension, temperature, and lighting regime were investigated. The results are consistent with the possibility that cell division is preceded by a lowered intracellular pH. Also consistent with this possibility is the finding that division rhythmicity can be induced by periodic changes in CO2 tension. It is suggested that the rhythmicity is induced by changes in intracellular pH produced by carbonic acid.

Carbon Dioxide

Simple procedure for determining octanol--aqueous partition, distribution, and ionization coefficients by reversed-phase high-pressure liquid chromatography.

The described simple, accurate, and precise reversed-phase high-pressure liquid chromatographic procedure is in excellent agreement with 1-octanol shake-flask partition or distribution coefficients over a 3.5 log range. A chemically bonded octadecylsilane support is persilated and coated with 1-octanol. With 1-octanol-saturated buffers as mobile phases, a stable baseline (compared to 1-octanol adsorbed on silica) is obtained rapidly, and the log relative retention times are highly correlated with unit slope to log distribution or partition coefficients obtained from the classical shake-flask procedures. Only relatively basic, unhindered pyridines deviate, probably because of binding with residual silinol sites. In addition, if the apparent pKa or pKab of an ionizable compound lies within the pH operating range of the column support, the apparent pKa or pKab usually can be determined simultaneously with log P by measuring the log distribution coefficient at several pH values. The procedure gives rapid results, requires little material, and can tolerate impurities.

Chemistry, Pharmaceutical