Search PubMed⌕ Search

Biomedical subjects

D M Miller

Publications and source records attributed to D M Miller.

At least 163 records · Page 9Linked to original sources
PubMed1990

Buffer solutes as stabilizers of 35S-amino acids: a study of volatility, radiochemical purity and biological activity.

It is well established that volatile radioactive compounds are associated with preparations of 35S-amino acids, but misconceptions still exist. The addition of stabilizers to these preparations has created a false sense of security regarding the evolution of these volatiles from cell culture medium. Experiments presented here show that the commercially used buffers L-lysine, tricine and 3,4-pyridine-dicarboxylic acid all reduced, but did not eliminate, the evolution of 35S volatiles from tissue culture medium. In each case, the rate of release was approximately 12 nCi/mCi/day (or 0.001%), compared to approximately 44 nCi/mCi/day in the buffer's absence. None of six alternative buffers tested are more effective than the commercially used buffers. We found that the release of labeled volatiles is dependent on the nature of the culture medium, but is no greater from crude 35S-amino acid preparations than from purified 35S-methionine. These commercially used buffers are also effective in maintaining radiochemical purity and are compatible with biological activity; however, because these buffers do not entirely eliminate the release of radioactivity into the gas phase, routine safe-handling procedures should be practiced when working with these products.

Amino Acids↗
PubMed1989

Studies of ascorbate-dependent, iron-catalyzed lipid peroxidation.

We have previously observed that both Fe(II) and Fe(III) are required for lipid peroxidation to occur, with maximal rates of lipid peroxidation observed when the ratio of Fe(II) to Fe(III) is approximately one (J. R. Bucher et al. (1983) Biochem. Biophys. Res. Commun. 111, 777-784; G. Minotti and S. D. Aust (1987) J. Biol. Chem. 262, 1098-1104). Consistent with the requirement for both Fe(II) and Fe(III), ascorbate, by reducing Fe(III) to Fe(II), stimulated iron-catalyzed lipid peroxidation but when the ascorbate concentration was sufficient to reduce all of the Fe(III) to Fe(II), ascorbate inhibited lipid peroxidation. The rates of lipid peroxidation were unaffected by the addition of catalase, superoxide dismutase, or hydroxyl radical scavengers. Exogenously added H2O2 also either stimulated or inhibited ascorbate-dependent, iron-catalyzed lipid peroxidation apparently by altering the ratio of Fe(II) to Fe(III). Thus, it appears that the prooxidant effect of ascorbate is related to the ability of ascorbate to promote the formation of a proposed Fe(II):Fe(III) complex and not due to oxygen radical production. The antioxidant effect of ascorbate on iron-catalyzed lipid peroxidation may be due to complete reduction of iron.

Animals↗
PubMed1989

Inhibition of superoxide and ferritin-dependent lipid peroxidation by ceruloplasmin.

Ceruloplasmin (CP) was found to inhibit xanthine oxidase and ferritin-dependent peroxidation of phospholipid liposomes, as evidenced by decreased malondialdehyde formation. Ceruloplasmin was also shown to inhibit superoxide-mediated mobilization of iron from ferritin, in a concentration-dependent manner, as measured spectrophotometrically using the iron(II) chelator bathophenanthroline sulfonate. Ceruloplasmin failed to function as a peroxyl radical-scavenging antioxidant as evidenced by its inability to inhibit free radical-initiated peroxidation of linoleic acid, suggesting that CP inhibited lipid peroxidation by affecting the availability of ferritin-derived iron. In addition, CP scavenged xanthine oxidase-derived superoxide as measured spectrophotometrically via its effect on cytochrome c reduction. However, the extent of the superoxide scavenging of CP did not quantitatively account for its effects on iron release, suggesting that CP inhibits superoxide-dependent mobilization of ferritin iron independently of its ability to scavenge superoxide. The effects of CP and apoferritin on iron-catalyzed lipid peroxidation in systems containing exogenously added ferrous iron was also investigated. In the absence of apoferritin, CP exhibited a concentration-dependent prooxidant effect. However, CP-dependent, iron-catalyzed lipid peroxidation was inhibited by the addition of apoferritin. Apoferritin did not function as a peroxyl radical-scavenging antioxidant but was shown to incorporate iron in the presence of CP. These data suggest that CP inhibits superoxide and ferritin-dependent lipid peroxidation largely via its ability to reincorporate reductively mobilized iron back into ferritin.

Animals↗
PubMed1989

Myosin heavy chain gene amplification as a suppressor mutation in Caenorhabditis elegans.

In the nematode, Caenorhabditis elegans, the body wall muscles contain paramyosin and two different types of myosin heavy chain, MHC A and MHC B. In mutants that do not express MHC B or that express defective paramyosin, muscle structure is disrupted and movement is impaired. Second site mutations in the sup-3 locus partially reverse these defects and are correlated with a 2- to 3-fold increase in the accumulation of the MHC A isoform. The sup-3 mutations occur at a high frequency (10(-4] after ethyl methanesulfonate (EMS) mutagenesis. This is comparable to the average EMS-induced mutation rate per gene in C. elegans. In this paper we show that the sup-3 mutation is an amplification of the structural gene for the MHC A protein, myo-3. We employed genomic Southern hybridization with MHC gene-specific probes in order to measure the copy number of the myo-3 gene relative to that of the MHC B gene, unc-54. We have identified the putative amplification junctions for these sup-3 alleles using a set of cosmid clones which encompass myo-3 region. Although it has been suggested that gene amplification plays an important role in evolution, there are few known cases of gene amplification in the germ line cells of multicellular organisms. The results shown here provide a clear example of a heritable gene amplification event that occurs at a high frequency in the germ line. Similar events may thus represent the initial event in the evolution of new function and in the formation of multigene families.

Animals↗
PubMed1989

Compensatory ovarian hypertrophy occurs by a mechanism distinct from compensatory growth in the regenerating liver.

The mechanism by which compensatory ovarian growth occurs is complex and not completely understood. To compare the molecular events in compensatory ovarian growth with those known to occur in other compensatory growth processes such as the regenerating liver, the temporal pattern of proto-oncogene expression and dexoyribonucleic acid synthesis was investigated in rat ovarian tissue after unilateral castration. One hundred fifty female rats were subjected to either a left hemioophorectomy or a sham oophorectomy. Twenty-four rats from each group were put to death at 3 and 14 days after the initial procedure and the ovaries were weighed. There was a mean compensatory weight increase in the right ovaries of the hemioophorectomy group of 7.9% at 3 days and 22.5% at 14 days. The temporal pattern of proto-oncogene expression was determined by removing the right ovary from six rats in each group at 4, 8, 12, 24, 36, and 48 hours after the initial procedure. The ovaries were paired into three samples in each group for each time point and the ribonucleic acid was extracted. Dot blot hybridization was performed on each ribonucleic acid sample with radiolabeled complementary deoxyribonucleic acid probes for the proto-oncogenes c-myc, c-HA-ras, and c-fos. There was no significant increase in proto-oncogene expression in the right ovaries of the hemioophorectomy group when compared with the right ovaries of the sham oophorectomy group. The temporal pattern of dexoyribonucleic acid synthesis was determined by removing the right ovary from three rats in each group at 8, 12, 24, 36, and 48 hours after the initial procedure. Each rat had been injected intraperitoneally with [3H]thymidine 2 hours before the right oophorectomy. The specific activity of dexoyribonucleic acid extracted from each ovarian sample did not demonstrate a significant increase in ovarian dexoyribonucleic acid synthesis after hemioophorectomy or any significant difference in dexoyribonucleic acid synthesis between the hemioophorectomy and the sham oophorectomy groups. This report concludes that compensatory ovarian growth occurs by a mechanism distinct from compensatory growth in the regenerating liver.

Animals↗
PubMed1989

Alloxan- and glutathione-dependent ferritin iron release and lipid peroxidation.

The diabetogenic action of alloxan is believed to involve oxygen free radicals and iron. Incubation of glutathione (GSH) and alloxan with rat liver ferritin resulted in release of ferrous iron as assayed by spectrophotometric detection of ferrous-bathophenanthroline complex formation. Neither GSH nor alloxan alone mediated iron release from ferritin. Superoxide dismutase (SOD) and catalase did not affect initial rates of iron release whereas ceruloplasmin was an effective inhibitor of iron release. The reaction of GSH with alloxan resulted in the formation of the alloxan radical which was detected by ESR spectroscopy and by following the increase in absorbance at 310nm. In both instances, the addition of ferritin resulted in diminished alloxan radical detection. Incubation of GSH, alloxan, and ferritin with phospholipid liposomes also resulted in lipid peroxidation. Lipid peroxidation did not occur in the absence of ferritin. The rates of lipid peroxidation were not affected by the addition of SOD or catalase, but were inhibited by ceruloplasmin. These results suggest that the alloxan radical releases iron from ferritin and indicates that ferritin iron may be involved in alloxan-promoted lipid peroxidation.

Alloxan↗
PubMed1989

Enhancement of monocyte-mediated tumoricidal activity by multiple interferon-alpha species.

Twenty-one interferon (IFN)-alpha species were evaluated for their ability to enhance monocyte-mediated lysis of the human melanoma cell line, A375. A wide variation in the potency of the different species in inducing monocyte tumoricidal action was observed. In addition, many IFN-alpha species were found to induce as much or more tumoricidal activity than recombinant IFN-gamma. The degree of monocyte activation induced by the various species generally correlated with their antiviral activity. Those which were better at inducing monocyte tumoricidal action also gave the highest antiviral specific activities. Studies were conducted to determine if the relative potency of the IFN-alpha species could be changed by altering certain parameters of the cytotoxicity assay. All IFN-alpha species tested required only 30 min in culture with the monocytes to induce activation. There were no changes in the relative potency of the species when cytotoxicity was measured at different times, nor when the effector:target ratio was altered. Competitive binding studies revealed that those IFN-alpha species which induced little activity in the bioassays were also generally poor in their ability to bind the IFN-alpha receptor on human monocytes, while the IFN-alpha species which induced relatively more activity in the bioassays were better able to bind to the IFN-alpha receptor. These data indicate that there are dramatic differences in activities among the IFN-alpha species which may, in part, be explained by different binding affinities. In addition, the differences observed among the IFN-alpha species demonstrate the need for further functional and structural characterization of the individual IFN-alpha species which could lead to a more effective clinical application of IFN-alpha.

Antiviral Agents↗
PubMed1989

Dissociation of cellular proliferation and c-myc expression by buttercup extract.

Buttercup extract (BE), an extract of the buttercup plant (Zanthoriza simplicissima), inhibits RNA and DNA synthesis by HL-60 promyelocytic leukemia cells. Exposure of these cells to 3% BE for 48 hours results in dramatic inhibition of RNA synthesis without loss of cell viability. The effect of BE is partially reversible over 12-24 hours with the level of RNA synthesis returning nearly to control levels during this time period. DNA synthesis is also reversibly inhibited by exposure to BE. Despite the inhibition of RNA synthesis in HL-60 cells, there is no decrease in the level of c-myc mRNA, even at high BE concentrations. The level of gene-specific mRNA for the c-Ha-ras, c-fms, and c-mos genes in these cells also remained constant during exposure to BE. Ribosomal RNA is not degraded during 24 hours of BE treatment in vitro, suggesting that BE does not maintain the relative mRNA level for these genes by selective degradation of other RNA species. The inhibition of RNA and DNA synthesis by BE without a corresponding alteration in the level of expression of the c-myc gene suggests that this agent dissociates c-myc expression and cellular proliferation in these cells.

Cell Division↗
PubMed1989

Mithramycin blocks protein binding and function of the SV40 early promoter.

Specific interactions between DNA and transcription factors are necessary for transcription initiation. These interactions provide a potential target for the selective inhibition of eukaryotic gene expression. Mithramycin is a DNA binding antibiotic which, in the presence of Mg2+, binds G-C containing sequences in the minor groove. The SV40 early promoter contains six G-C decanucleotide sequences, which are binding sites for the transcriptional activating factor, Sp1. Each of the six Sp1 binding sites of this promoter is protected from DNAse 1 digestion by mithramycin binding. Mithramycin binding to the G-C rich sequences in the SV40 early promoter prevents subsequent protein binding to these sequences. The gel retardation of the SV40 early promoter fragment incubated with a HeLa cell extract is completely abrogated by pretreatment of the DNA fragment with mithramycin. The functional significance of mithramycin binding is reflected in the ability of mithramycin to block promoter function. Mithramycin inhibits promoter dependent transcription in an in vitro runoff transcription system in a concentration dependent manner. This suggests that mithramycin prevents transcriptional activation of the SV40 early promoter by blocking binding of transcriptional activating proteins to G-C rich promoter regions.

Base Sequence↗
PubMed1989

Stimulation of protooncogene expression by partial hepatectomy is not tissue-specific.

We have characterized the early protooncogene response and the later cell proliferative response in the kidneys and livers of normal rats cross-circulated with partially hepatectomized animals. Increase c-myc and C-Ha-ras expression was observed in the kidneys of totally hepatectomized rats, as well as those of their cross-circulated partners. This indicates that the initial response to hepatectomy is not organ-specific, although the later DNA synthetic response of the kidney is only approximately one-tenth that of regenerating liver. Expression of c-myc and c-Ha-ras is dramatically increased in the livers of both hepatectomized and nonhepatectomized, parabiotic (cross-circulated) rats within 1 hr of partial hepatectomy, confirming the presence of a circulating factor which stimulates protooncogene expression early in regeneration. DNA synthesis was also stimulated in the livers of the cross-circulated animals between 20 and 26 hr following hepatectomy, but only to a level one-eighth that of the livers of hepatectomized animals. Normal rats cross-circulated with totally hepatectomized animals also demonstrated an early increase in hepatic c-myc and c-Ha-ras expression, indicating that regeneration must be stimulated by an extrahepatic signal. Our data suggest that the early increase in protooncogene expression is a non-organ-specific response to partial hepatectomy which does not insure subsequent cellular proliferation. The organ specificity of liver regeneration must involve an event separate from the early stimulation of protooncogene expression.

Animals↗
PubMed1989

Mouse fibroblasts transformed with the human c-myc gene express a high level of mRNA but a low level of c-myc protein and are non-tumorigenic in nude mice.

Overexpression of a transfected c-myc gene under the control of a strong promoter causes the phenotypic changes of malignant transformation. We have investigated the effect of exogenous c-myc gene expression in NIH3T3 cells transfected with an intact human c-myc gene which is expressed under the control of its own promoter. The presence of the exogenous c-myc gene in the transfected cells was documented by Southern blot analysis. The transfected clones contained 20-40 copies of the human c-myc gene and demonstrated a proportional elevation in the level of c-myc mRNA which had a normal half life of 20-30 min. Despite the high level of c-myc mRNA, the transfected cells contained a relatively low level of c-myc protein and expression appears to be regulated at a post-transcriptional level. The transfected cells exhibited a decreased serum requirement for growth and formed colonies in soft agar, but were non-tumorigenic in Balb/c athymic nude mice. The morphologic evidence of transformation in the absence of tumorigenesis suggests that transfection of murine fibroblasts with multiple copies of the c-myc gene expressed under the control of its own promoter may cause partial transformation.

Animals↗
PubMed1988

Muscular dystrophy in a litter of golden retriever dogs.

Clinicopathologic findings from two golden retriever dogs with an inherited, progressive, degenerative muscle disease that were studied until 27 and 40 months of age are described. Initial clinical signs included stilted gait and simultaneous advancement of their pelvic limbs. Further gait restriction and muscle hypertrophy eventually occurred. Serum creatine kinase was dramatically elevated (greater than 10,000 U/L). There were persistent "spontaneous" high-frequency discharges (pseudomyotonia) on electromyographic evaluation. Features of both muscle fiber degeneration (hyaline fibers, myophagocytosis) and regeneration (small basophilic fibers) were seen on light microscopy. Similar ultrastructural changes (fiber hypercontraction, increased myoblasts) were present. On morphometric histochemical evaluation, mean fiber diameter of both type 1 and 2 fibers was increased compared with controls in two of three muscles examined. There was no apparent fiber type predominance. Scattered ragged red fibers were seen, but this appeared to be a nonspecific finding of either muscle fiber regeneration or degeneration. These findings and potential contributing pathophysiologic mechanisms are discussed in relation to Duchenne muscular dystrophy.

Animals↗
PubMed1988

Selective inhibition of c-myc expression by the ribonucleic acid synthesis inhibitor mithramycin.

Expression of the c-myc proto-oncogene has been shown to correlate with the rate of cellular proliferation and malignant transformation in a number of cell types. JEG-3 choriocarcinoma cells demonstrate c-myc transcript levels that are greater than those of nonmalignant trophoblastic tissue at any stage of gestation. Southern blot analysis documents c-myc gene amplification in JEG-3 cells, with a gene copy number of approximately 20. The methylation pattern and genomic structure of the amplified c-myc oncogene in JEG-3 choriocarcinoma cells are identical to those of normal placenta. Treatment of JEG-3 cells with mithramycin, a ribonucleic acid synthesis inhibitor, results in a dramatic decrease in c-myc expression relative to that of the c-Ha-Ras gene. The apparent selectivity of mithramycin for c-myc expression represents the only example, to date, of the selective pharmacologic modulation of oncogene expression.

Cell Line↗
PubMed1988

Enclosed afferent reservoir breathing systems. Description and clinical evaluation.

A new group of breathing systems, namely the Enclosed Afferent Reservoir (EAR) systems, is described. They allow for the selective elimination of alveolar gas in association with both spontaneous and controlled ventilation. A comparison with the Bain system in controlled ventilation demonstrates greater efficiency in eliminating carbon dioxide. A fresh gas flow (VF) of 70 ml kg-1 min-1 using an EAR system gave mild hypocarbia which equated to a VF of 100 ml kg-1 min-1 using the Bain system. Smaller minute volumes of ventilation are required for optimal performance than with the Bain system. The minimum recommended minute volume of ventilation (VI) should equal VF plus anatomical deadspace ventilation (VDanat). The pattern of ventilation appears to have little influence upon the efficiency of carbon dioxide elimination when using an EAR system, whereas the Bain system does appear to be affected.

Anesthesia, General↗