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Biomedical subjects

J M Clark

Publications and source records attributed to J M Clark.

At least 217 records · Page 12Linked to original sources
PubMed1982

Inhibition of DNA replication and repair by anthralin or danthron in cultured human cells.

The comparative effects of the tumor promoter anthralin and its analog, danthron, on semiconservative DNA replication and DNA repair synthesis were studied in cultured human cells. Bromodeoxyuridine was used as density label together with 3H-thymidine to distinguish replication from repair synthesis in isopycnic CsCl gradients. Anthralin at 1.1 microgram inhibited replication in T98G cells by 50%. In cells treated with 0.4 or 1.3 microM anthralin and additive effect was observed on the inhibition of replication by ultraviolet light (254 nm). In cells irradiated with 20 J/m2, 2.3 microM anthralin was required to inhibit repair synthesis by 50%. Thus there was no selective inhibitory effect of anthralin on repair synthesis. Danthron exhibited no detectable effect on either semiconservative replication or repair synthesis at concentrations below about 5.0 microM. Neither compound stimulated repair synthesis in the absence of ultraviolet irradiation. Thus, anthralin and danthron do not appear to react with DNA to form adducts that are subject to excision repair. Although both compounds appear to intercalate into supercoiled DNA in vitro to a limited extent, the degree of unwinding introduced by the respective drugs does not correlate with their relative effects on DNA synthesis in vivo. Therefore the inhibitory effect of anthralin on DNA replication and repair synthesis in T98G cells does not appear to result from the direct interaction of the drug with DNA.

Anthracenes↗
PubMed1982

Successful therapy of cerebral air embolism with hyperbaric oxygen at 2.8 ATA.

A 60-year-old male patient suddenly developed blindness, agitation, and disorientation 36 h after coronary bypass surgery. Onset of symptoms followed efforts to clear an air-filled radial artery cannula. Seven hours after onset of symptoms, initial compression to 2.8 ATA (60 fsw), 100% oxygen (U.S. Navy Table 6), steroids, intravenous fluids, and antiplatelet drugs were used for therapy. The patient's agitation and disorientation dictated that we avoid initial compression to 6 ATA (165 fsw), contrary to conventional practice in therapy of air embolism, and instead immediately give oxygen at 2.8 ATA. After a second treatment with USN Table 6, given 6 h after the first, the patient's vision and mental state returned to normal. He subsequently had an uneventful recovery from surgery and cerebral air embolism.

Atmospheric Pressure↗
PubMed1981

High yield culture of human fibroblasts on microcarriers: a first step in production of fibroblast-derived interferon (human beta interferon).

Production of large quantities of diploid human fibroblasts in small culture volumes can be achieved using microcarrier culture techniques. Modification of the microcarrier culture procedure to include a virtually static attachment period and a reduced culture volume during the first 6 hr of culture was shown to double the attachment efficiency of the cells, improve the culture growth rate and increase final cell yields. Using the modified culture procedure yields greater than 2 x 10(6) cells/ml could be regularly obtained when 7--8 viable cells/microcarrier were used to inoculate a culture containing 3 mg of Cytodex 1 microcarriers/ml. A protocol for the mass production of human beta interferon from microcarrier cultures is described. After priming the culture with beta interferon the superinduction step involves use of a poly (I) . poly(C)/DEAE-Dextran complex. Modifications to the microcarrier culture procedure and to the interferon production sequence enable the production of 3 x 10(8) IU of interferon from a 5 liter microcarrier culture of human fibroblasts. This quantity of interferon corresponds to a yield of approximately 30 IU/10(3) cells or 2 x 10(4) IU/mg of microcarrier.

Cells, Cultured↗
PubMed1981

Characterization of polysaccharide accumulation in a cell division defective mutant of Escherichia coli 15T-.

Escherichia coli 15T-R1, a temperature-dependent cell division mutant, grows into filaments of various lengths (200 to 500 microgram) at 24 degrees C, but divides essentially normally at 37 degrees C. When grown to late-exponential phase at the restrictive temperature, the elongated cells showed discrete areas of increased density at polar regions and other sites in the cytoplasm, when viewed by phase contrast microscopy. Electron microscopy of preparations specifically stained for polysaccharide revealed clusters of granules with a similar distribution pattern to that of the dense areas seen by phase contrast microscopy. The granules were susceptible to alpha-amylase digestion, and chemical analysis of the extracted and purified polysaccharide showed that it consisted of polyglucose, including glycogen. At 24 degrees C the R1 cells contained about twice as much polyglucose and four times as much glucogen as at 37 degrees C.

Cell Division↗
PubMed1981

Optimizing culture conditions for the production of animal cells in microcarrier culture.

Cytodex 1 microcarriers have been used for the successful culture of more than 80 different types of animal cells--including primary cells, normal diploid cell strains and established or transformed cell lines. culture volumes have ranged from a few milliliters for diagnostic studies to over several hundred liters for vaccine production. Experience with this wide variety of cell types and culture volumes has enabled the identification of several parameters critical for obtaining maximum cell yields from microcarrier cultures. The most vital stage for the successful microcarrier culture of many cell types was the initial stage of the culture cycle. To achieve high cell yields, it was necessary to use culture procedures which maximized plating efficiency and final cell yield could be further increased by ensuring that the inoculation cell density exceeded a critical viable cell/microcarrier ratio. Modifications of the standard microcarrier culture procedures included reducing initial culture volume, reducing the initial stirring speed and/or supplementing the medium during the early stages of the culture cycle. Control of pH, nutrient supply, and gas tension were all critical throughout the culture cycle. Results with low-serum and serum-free media indicate that the requirement for fetal calf serum in the microcarrier culture of Vero and MRC-5 cells can be reduced or even eliminated. Large scale microcarrier culture equipment should take into account the modified culture procedures which are often required to achieve the full potential of this culture method. The design of a new flexible culture system suitable for pilot and production scale cultures is presented. This system accomodates a wide variety of culture and production procedures and through a recirculation system permits: (a) "in-line" monitoring and control of culture parameters; (b) provides an efficient gas exchange capacity which obviates the need for fermenter headspace and sparging; and (c) allows for maximal utilization of medium components and rapid harvesting of medium or cell products.

Animals↗
PubMed1981

Excision of pyrimidine dimers from nuclear deoxyribonucleic acid in ultraviolet-irradiated Dictyostelium discoideum.

A sensitive endonuclease assay was used to study the fate of pyrimidine dimers introduced by ultraviolet irradiation into the nuclear deoxyribonucleic acid of the cellular slime mold Dictyostelium discoideum. Analysis of the frequency of T4 endonuclease V-induced single-strand breaks by alkaline sucrose gradient sedimentation showed that strain NC4 (rad+) removed greater than 98% of the dimers induced by irradiation at 40 J/m2 (254 nm) within 215 min after irradiation. HPS104 (radC44), a mutant sensitive to ultraviolet irradiation, removed 91% under these conditions, although at a significantly slower rate than NC4: only 8% were removed during the 10- to 15-min period immediately after irradiation, whereas NC4 excised 64% during this interval. HPS104 thus appears to be deficient in the activity(ies) responsible for rapidly incising ultraviolet-irradiated nuclear deoxyribonucleic acid at the sites of pyrimidine dimers.

DNA Repair↗
PubMed1981

Effects of acute and chronic hypercapnia on oxygen tolerance in rats.

Groups of 16-52 normal or CO2-adapted rats were exposed top 100% O2 or to O2 with 60 Torr PICO2 (O2-CO2) at pressures of 1.0, 1.5, 2.0, 3.0, and 4.0 ATA. Exposure durations for 50% mortality (LD50) in normal rats at 4.0, 3.0, 2.0, 1.5, and 1.0 ATA O2 were 6.3, 9.3, 17.2, 27.4, and 76.1 h, respectively. Corresponding LD50 values for normal rats exposed to O2-CO2 were 2.0, 2.9, 16.3, 24.8, and 74.8 h. Survival times of CO2-adapted rats exposed to O2 were nearly identical to those of normal rats. LD50 values for CO2-adapted rat exposed to O2-CO2 were 4.1, 7.5, 17.9, 23.6, and 65.4 h, respectively. These data confirm acceleration of O2 intoxication by acute hypercapnia at 4.0 and 3.0 ATA, but they show less prominent effects at 2.0, 1.5, and 1.0 ATA. Hypercapnia adaptation clearly has a protective effect in rats exposed to O2-CO2 at 4.0 and 3.0 ATA. At 2.0, 1.5, and 1.0 ATA, where acute hypercapnia has less effect, the effects of CO2 adaptation are also less prominent. The observed changes in oxygen tolerance can be explained by cerebral vasodilation with increased brain oxygenation in acute hypercapnia and by significant amelioration of this response during chronic hypercapnia.

Acid-Base Equilibrium↗
PubMed1981

Hyperbaric oxygen therapy with subtotal extirpation surgery in the management of radionecrosis of the mandible.

Radiation-induced obliterative endarteritis causes ischemia and hypoxia in tissue wounds, making them prone to dehiscence, infection and delayed healing. The successful use of hyperbaric oxygen as an adjunct to intensive antibiotic and surgical therapy in the treatment of radionecrosis of the mandible is well established. This report details our experience in four cases of radionecrosis of the mandible of long duration, prior to acceptance of the patients into our hyperbaric oxygen protocol.

Aged↗
PubMed1981

A comparison of various laboratory scale culture configurations for microcarrier culture of animal cells.

When developing procedures for microcarrier culture it is important to use equipment which results in even suspension of microcarriers with minimal shear forces and which permits maximum growth of the culture. A variety of different laboratory scale culture systems were investigated including traditional magnetic spinner flasks, modified spinner flasks, cultures stirred with bulb-shaped rods and a fluid lift culture system. All systems were compared with respect to growth of either MRC-5 or Vero cells on Cytodex microcarriers. Although most of the earlier work with microcarriers has been performed with traditional magnetic spinner flasks, recently modified equipment resulted in better plating efficiencies and greater cell yields. Cultures stirred with a bulb-shaped rod and contained in flasks with contoured bases were particularly useful for cells having a low plating efficiency and provided thorough stirring of the medium with minimum shear forces. A fluid lift system based on upward perfusion of medium through a culture contained in a chromatographic column is described. This technique could provide a useful alternative for suspending microcarriers and provides for accurate in-line control of gas tensions.

Animals↗
PubMed1981

Serum supplements and serum-free media: applicability for microcarrier culture of animal cells.

Several different combinations of serum supplements and also serum-free media were examined for their ability to support the growth of MRC-5 and Vero cells on Cytodex microcarriers. Greater economy of serum was achieved for routine microcarrier culture by selecting the type of serum supplement, on the basis of whether the supplement was to support attachment and growth of cells at low densities or growth of cells at later stages in the culture cycle. Further economy was achieved by altering the serum concentration according to the requirements of each stage of the culture cycle. More consistent results were obtained when batches of serum were selected on the basis of quality control tests with microcarrier culture as well as with monolayer culture. Low serum and serum-free media were able to support the growth of cells in microcarrier cultures. A mixture of DME/Medium 199 (50/50) containing foetal calf serum (0.5%, v/v), BSA and EGF supported growth of MRC-5 and Vero cells to nearly the same extent as DME/Medium 199 containing 10% (v/v) foetal calf serum. Serum-free media was used to achieve cell yields at least one half of those attained with serum-supplemented media. Maximum growth was obtained when DME/Medium 199 was supplemented with fibronectin, BSA, insulin, transferrin, putrescine, fetuin and EFG. Fetuin was omitted and replaced by dexamethasone and trace metals with only a slight reduction in cell yield. Fibronectin was essential in all serum-free media.

Animals↗
PubMed1981

Alternative surfaces for microcarrier culture of animal cells.

As part of an effort to broaden the applicability and efficiency of microcarrier cell culture various alternative new microcarriers were synthesized. The microcarriers were compared as substrates for the growth of several types of cells and with respect to binding of proteins from the culture medium. Cross-linked dextran has been found to be the most suitable material for a microcarrier matrix and was used as the matrix for the new microcarriers. One type of microcarrier was synthesized so that the charges necessary for cell attachment were present only in the surface layer of the microcarrier in the form of N,N,N-trimethyl-2-hydroxyaminopropyl groups. The resulting microcarriers had a very low capacity to bind proteins from the culture medium (e.g. albumin and IgG) and such proteins could be removed from the cultures more efficiently than when using previous microcarriers. A new principle was used for the development of the other type of microcarrier. A surface layer of cross-linked denatured collagen provided the surface for growth of cells. These microcarriers provided a "natural" substrate for cell growth and allowed improved attachment and spreading of cells with epithelial morphology. Harvesting cells from these microcarriers with proteolytic enzymes was more efficient than with previous microcarriers. The two alternative microcarriers should result in improved process efficiency and have a potential value in the preparation of live vaccines.

Animals↗
PubMed1980

Translation initiation site of the coat protein messenger ribonucleic acid of the cowpea strain of tobacco mosaic virus.

The 5'-terminus of the coat protein messenger RNA of the cowpea strain of tobacco mosaic virus contains a m7G(5')ppp(5')Gp... cap group. Removal of this cap group followed by introduction of a 5'-terminal [32P]phosphomonoester onto the decapped RNA and use of this 32P-labeled RNA in nucleotide sequence studies establishes that the 5'-terminal nucleotide sequence of this RNA is m7G(5')ppp(5')GUAUUUGAUGAUGGCAUACUCGAUUCCGACUCCCUAGCU.... The codons following the two consecutive AUG sequences match the N-terminal amino acids of the coat protein of the cowpea strain of tobacco mosaic virus. Translation initiation complexes between wheat germ 80S ribosomes and this messenger RNA protect regions adjacent to the two consecutive AUG sequences of this messenger RNA from attack by ribonucleases. Therefore, one or both of these AUG sequences serve as sites for the initiation of translation of the coat protein messenger RNA of the cowpea strain of tobacco mosaic virus.

Base Sequence↗
PubMed1980

Protection of satellite tobacco necrosis virus ribonucleic acid by wheat germ 40S and 80S ribosomes.

Wheat germ ribosomes combine with the AUG codon at positions 30-32 from the 5' terminus of in vitro radioiodinated satellite tobacco necrosis virus (STNV) ribonucleic acid (RNA) to form initiation complexes that protect specific regions of the RNA from attack by ribonucleases. Wheat gern 80S ribosomes convey partial protection to a region 20-52 nucleotides from the 5' terminus of STNV RNA and convey protection against high levels of ribonucleases to a region 20-44 nucleotides from the 5' terminus of STNV RNA. Wheat germ 40S ribosomes convey partial protection to a region 3-52 nucleotides from the 5' terminus of STNV RNA and convey protection against high levels of ribonucleases to a region 10-47 nucleotides from the 5' terminum of STNV RNA. Characterization of these levels of protection against attack by specific ribonucleases establishes that wheat germ 40S and 80S ribosomes form initiation complexes with a linear conformation of STNV RNA lacking the 5'-terminal stem and loop anticipated by Leung and co-workers [Leung, D. W., Browning, K. S., Heckman, J. E., RajBhandary, U. L., & Clark, J. M., Jr. (1979) Biochemistry 18, 1361-1366] for the 5' terminus of STNV RNA.

Base Sequence↗
PubMed1980

Elastase-like enzymes in human neutrophils localized by ultrastructural cytochemistry.

The thiol ester N-t-Boc-L-alanine-p-nitrothiophenyl ester (Boc-Ala-SNp) was synthesized and applied as an ultrastructural cytochemical substrate for intracellular elastase-like enzymes. Mature human neutrophils incubated with Boc-Ala-SNp and gold ions generate an electron-dense reaction product, gold p-nitrothiophenolate, which is found in the nuclear membrane, Golgi complex, endoplasmic reticulum, mitochondria, and granules of these cells. Enzyme activity against Boc-Ala-SNp is also observed in developing monkey bone marrow neutrophils and in other blood cells. The intracellular neutrophil enzyme activity is elastase-like because it is characterized by a slightly alkaline pH optimum and is inactivated by exposure of the cells to general and specific active site inhibitors of neutrophil elastase. This substrate appears to have important potential for use in ultrastructural studies of intracellular elastase-like enzymes.

Animals↗