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Biomedical subjects

D M Miller

Publications and source records attributed to D M Miller.

At least 127 records · Page 7Linked to original sources
PubMed1992

Structure and applications of intermolecular DNA triplexes.

Current DNA binding drugs are not sequence specific. Triplex-forming oligonucleotides will bind targeted duplex DNA sites in a sequence-specific manner. A new class of DNA binding molecules based on triple-helical DNA formation promises a sequence-specific method of targeting discrete regions of DNA. DNA modifying molecules linked to third strands have been shown to modify only regions of DNA to which they were targeted. Current research will increase the understanding of triplex DNA structure and will lead to improved DNA binding drugs.

Base Composition↗
PubMed1992

A pressure regulator for the cuff of a tracheal tube.

A regulator is described for the maintenance of a constant pressure difference above airway pressure during positive pressure ventilation in a cuffed tracheal tube. It comprises a tubular threshold valve which is powered by the anaesthetic gas supply source to a breathing system. The valve is interposed between the anaesthetic gas supply machine and the breathing system creating a pressure differential. The upstream pressure is transmitted to the pilot tube supplying the cuff of a tracheal tube via an upstream connection. The valve is suitable only for breathing systems that require a constant gas supply. The regulator was evaluated during anaesthesia, using a modified Mallinkrodt Hi-Lo jet ventilation tube to obtain simultaneous pressure measurements within the cuff and the lumen of the tracheal tube. A greater pressure was demonstrated in the cuff than in the airway and the two traces were approximately parallel throughout the respiratory cycle. The device should prevent excessive cuff inflation pressure and solves the problem of forgetting to let the cuff down before extubation.

Equipment Design↗
PubMed1992

Expression and regulation of the cystic fibrosis gene during rat liver regeneration.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) are responsible for cystic fibrosis. The CFTR gene has recently been identified and encodes a 6.5-kb mRNA transcript. Recent observations showing that CFTR expression increases during differentiation of epithelial cells suggested that CFTR may also be regulated in the liver in response to partial hepatectomy (PH). We studied the expression of CFTR in rat regenerating liver and investigated the mechanisms that regulate CFTR RNA levels during a 120-h period after PH. Northern and slot-blot analysis revealed a liver-specific biphasic increase of CFTR mRNA levels, which peaks at 2 and 24 h post-PH. In contrast to these findings, the mode of regulation of the homologous gene MDR-1 showed a clearly different pattern. Nuclear run-on analysis demonstrated increased levels of CFTR transcription corresponding to the time points where an increase in CFTR message was observed. Similarly, the beta-actin gene, which increases transiently during liver regeneration, showed increased nuclear run-on activity 4 h posthepatectomy, indicating that the nuclei were functional. No increase of MDR-1 gene transcription was detected, confirming the previous finding that the increase in MDR-1 mRNA level in regenerating liver results from a post-transcriptional event such as message stabilization. This study indicates that expression of the CFTR gene is regulated during the regenerative process of the liver. The data also suggest that the increase in CFTR and MDR expression levels result from two distinct regulatory mechanisms.

Animals↗
PubMed1992

The best asthma care. A case problem in continuous quality improvement.

In the future, health care delivery systems may need to respond to requests from payers to demonstrate the quality of care they provide for types of patients or diseases. Here, asthma is used as an example of what such a response might look like in the context of continuous quality improvement.

Asthma↗
PubMed1992

Characterization of the binding of transforming growth factor-beta 1, -beta 2, and -beta 3 to recombinant beta 1-latency-associated peptide.

Preprotransforming growth factor-beta 1 (TGF beta 1) is a 390-amino acid precursor polypeptide that undergoes a number of processing steps to yield mature TGF beta 1 (amino acid residues 279-390) and a pro portion (residues 30-278) termed beta 1-latency-associated peptide (beta 1LAP). The dimeric form of beta 1LAP has been shown to associate noncovalently with the mature growth factor, resulting in inactivation of biological activity. To further characterize this interaction, the mature TGF beta 1 was radioiodinated and used to determine dissociation constants. A cross-linking method using the bifunctional covalent cross-linker bis-(sulfosuccinimidyl)suberate was found to be the best approach for measuring the amount of bound growth factor. The efficiency of cross-linking was constant within each experiment and varied between 45-55%. Saturation plots and their associated Scatchard analyses indicate apparent Kd values between 1.1-1.8 nM. Competition of TGF beta 1 binding to beta 1LAP by TGF beta 2 and TGF beta 3 (two closely related growth factors) revealed that the latter also bind beta 1LAP tightly, with apparent Kd values of 1.9 and 0.4 nM, respectively.

Animals↗
PubMed1992

Ultrasonographic appearance of the ovaries of dogs during the follicular and luteal phases of the estrous cycle.

Ultrasonography of the ovaries of 10 bitches was performed daily, using a 7.5-Mhz transducer with a built-in stand-off pad, from the onset of proestrus until the onset of metestrus. Ovarian size, shape, location, echogenicity, follicular development, and apparent ovulation were monitored. Blood samples were collected twice daily for luteinizing hormone determination and daily for progesterone determination. Vaginal smears were made daily for cytologic evaluation. Ultrasonograms were evaluated independent of hormonal and cytologic data, and the day of ovulation was noted. Initially, the ovaries were uniform and had an echogenicity that was equal to or slightly greater than that of the renal cortex. Follicles appeared as focal hypoechoic to anechoic rounded structures. Ovaries were easier to identify as follicular development progressed. Ovarian size increased with time. Apparent ovulation was characterized by a decrease in number of follicles seen from 1 day to the next, but 1 or more follicles remained in at least 1 ovary of 7 of 10 bitches. The ovaries had an oval shape that became rounded after ovulation. At some time after ovulation, all bitches had cystic (anechoic) structures indistinguishable from follicles. These structures increased in echogenicity and decreased in size with time and may have been follicles that did not ovulate, corpora hemorrhagica, fluid-filled corpora lutea, or cystic luteinized follicles. Time of ovulation determined by ultrasonography paralleled that predicted on the basis of hormonal data in 9 of 10 bitches and with cytologic findings in all bitches.

Animals↗
PubMed1992

Regulation of c-myc transcription initiation and elongation.

The c-myc protooncogene plays an important role in the regulation of cellular proliferation and differentiation. Selective downregulation of c-myc expression induces differentiation of leukemic cells, whereas constitutive overexpression prevents differentiation of the same cell types. The c-myc gene is comprised of three exons, the first of which is largely untranslated. Both c-myc mRNA and the c-myc gene product have very short half-lives, resulting in a very sensitive regulation of this important gene. Both transcriptional and posttranscriptional regulation of the c-myc gene expression have been documented. Transcriptional regulation can occur at the level of transcriptional initiation or elongation. A number of c-myc promoter-binding proteins have been identified and characterized. These include positive and negative regulatory factors. It appears that the c-myc gene is very tightly regulated in response to positive and negative growth stimuli.

Animals↗
PubMed1992

Results of conservative management of cervical intraepithelial neoplasia.

Cryotherapy and laser surgery have been the most frequently used conservative methods to treat cervical intraepithelial neoplasia (CIN) in the past decade. This report documents our experience using these modalities to treat 2773 patients between the years 1984-1989. One thousand eight hundred eleven women received laser surgery and the remaining 962 were treated with cryotherapy. In the first 2 years of the study period, only 78 patients were treated with laser surgery. Conversely, only 69 of the 979 patients treated in 1988 and 1989 had cryotherapy. As greater experience was gained with laser surgery, the success rates rose from 58.3% in 1984 to 95.5% in 1988. The success rate was similar for all grades of CIN. Overall, 11.2% of all patients were lost to follow-up. Among patients treated with laser surgery, 4.8% had postoperative bleeding that required either packing or, in two instances, sutures for hemostasis. Success with these methods appeared to be related to the size of lesion and not to the degree of histologic abnormality. The shift toward increasing use of laser surgery in our clinic was due to its precision in destroying identified lesions in the transformation zone. Our results indicate that both cryotherapy and laser surgery are simple, effective methods for the treatment of CIN.

Adult↗
PubMed1991

Mercury-induced H2O2 production and lipid peroxidation in vitro in rat kidney mitochondria.

Mercuric ion (Hg(II)) causes oxidative tissue damage in kidney cortical cells. We studied the in vitro effects of Hg(II) on hydrogen peroxide (H2O2) production by rat kidney mitochondria, a principal intracellular target of Hg(II). In mitochondria supplemented with a respiratory chain substrate (succinate or malate/glutamate) and an electron transport inhibitor (antimycin A (AA) or rotenone), Hg(II) (30 nmol/mg protein) increased H2O2 formation approximately 4-fold at the ubiquinone-cytochrome b region (AA-inhibited) and 2-fold at the NADH dehydrogenase region (rotenone-inhibited). Concomitantly, Hg(II) increased iron-dependent lipid peroxidation 3.5-fold at the NADH dehydrogenase region, but only by 25% at the ubiquinone-cytochrome b region. The mitochondrial concentration of reduced glutathione (GSH) decreased both with incubation time and Hg(II) concentration. Hg(II), at a concentration of 12 nmol/mg protein, caused almost complete depletion of measurable GSH in substrate-supplemented mitochondria after a 30-min incubation. In electron transport-inhibited mitochondria, Hg(II) caused greater depletion of GSH in rotenone-inhibited than in AA-inhibited mitochondria, consistent with the effects of Hg(II) on lipid peroxidation. These results suggest that Hg(II) at low concentrations depletes mitochondrial GSH and enhances H2O2 formation in kidney mitochondria under conditions of impaired respiratory chain electron transport. The increased H2O2 formation by Hg(II) may lead to oxidative tissue damage, such as lipid peroxidation, observed in mercury-induced nephrotoxicity.

Animals↗
PubMed1991

Determination of the thickness of the unstirred layers in the moving-drop method for measuring aqueous-nonaqueous interfacial transport rates.

A technique for measuring the thickness of the unstirred layer in the aqueous phase surrounding a rising octanol drop is described. Measurements of the rate of movement of both acetic and valeric acids from the drop into buffered solutions were made using an apparatus previously described (Miller, D.M. (1986) Biochim. Biophys. Acta 856, 27-35). The rate of movement of valeric acid was shown to be dependent on the pH of the buffer while that of acetic acid was not. The thickness of the unstirred layer in the buffered solution (w.u.l.) was found to be 7.0 microns, and the thickness of the unstirred layer in the drop (o.u.l.) was estimated to have an upper limit of 4.6 microns. The rate constant for the movement of valeric acid from the octanol phase to the buffer, kow, has a value restricted to the range 21 to 46 microns s-1 while that for movement in the opposite direction, kwo, ranges from 600 to 1300 microns s-1. The permeability of the w.u.l. is 120 microns s-1, while that of the o.u.l. has a lower limit of 37 microns s-1. Thus diffusion through the w.u.l. was not rate-limiting during movement into the drop, but was during movement outward. The rate of uptake of acetic acid was found to be 39 microns s-1 and was independent of pH, from which it was concluded that the resistance of the w.u.l. was negligible for this compound.

Acetates↗
PubMed1991

Evidence that interfacial transport is rate-limiting during passive cell membrane permeation.

The octanol to water overall transfer rate constants (k'ow), the octanol/water partition coefficients (Kpc) and the diffusion coefficients in octanol (Do) and water (Dw) were measured for 36 compounds. A plot of Do/k'ow as a function of Do.Kpc/Dw was shown to fit a straight line for compounds having a high Kpc, and from the slope of this line, the thickness of the unstirred layer in the water phase was found to be 9.1 microns. Using this value and the data provided by compounds having a low Kpc, an estimate of 1.2 microns was obtained for the thickness of the unstirred layer in the octanol phase. This provided estimates for the true interfacial rate constants, kow (for movement of a compound from octanol into water) and kwo (for movement in the opposite direction), which were corrected for the effects of the unstirred layers. kow proved to be constant to within one order of magnitude for a series of compounds whose Kpc values ranged over three orders of magnitude, while kwo for these same compounds varied directly as Kpc. Assuming that octanol has solvent properties similar to those of the lipid bilayer present in natural membranes, the permeability of these membranes to a given compound can be shown to be equal to kwo/2. Comparing the permeabilities calculated in this way to measured permeabilities of natural membranes showed them to be of the same order or smaller. These data are consistent with a proposal that the rate-limiting step in passive membrane permeation is not the rate of diffusion within the membrane itself, but rather, transfer of the permeant across the interfaces separating the lipid phase of the cell membrane from the aqueous phases on either side of it.

1-Octanol↗
PubMed1991

Mithramycin blocks transcriptional initiation of the c-myc P1 and P2 promoters.

The c-myc protooncogene plays an important role in the regulation of cellular proliferation. Mithramycin, a DNA binding antibiotic which binds G-C-rich DNA, inhibits c-myc expression in both differentiating and nondifferentiating cells. The G-C-rich nature of the c-myc promoter suggests that mithramycin may act by directly inhibiting promoter function. The mithramycin binding sites in the c-myc promoter regions were determined by DNAse I footprinting. Particularly prominent mithramycin binding is noted in the regions just 5' of the P1 and P2 promoter TATA boxes. Gel retardation experiments performed in the presence of mithramycin demonstrate that drug binding can prevent the formation of discrete complexes between HeLa cell nuclear proteins and c-myc promoter DNA fragments. Mithramycin also directly blocks the binding of the transcription factor Sp1 to the P1 promoter region. In vitro run-off transcription demonstrates that mithramycin can completely inhibit the in vitro function of both the P1 and P2 promoters. These data suggest that mithramycin inhibits transcription of the c-myc protooncogene by blocking the binding of important regulatory factors, thus preventing formation of the c-myc transcription initiation complex.

Base Sequence↗
PubMed1991

Expression of cloned bovine adrenal rhodanese.

A cDNA for the enzyme rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) has been cloned from a bovine adrenal library. An initiator methionine codon precedes the amino-terminal amino acid found in the isolated protein. Rhodanese is synthesized in the cytoplasm and transferred to the mitochondrial matrix. Thus, any amino-terminal sequence required for organelle import is retained in the mature protein. Furthermore, the DNA sequence shows that there are three additional amino acids, Gly-Lys-Ala, at the carboxyl terminus that are not found by protein sequencing. Additionally, comparison of the published amino acid sequence with that encoded by the open reading frame revealed three differences in the amino acid sequence. Comparison of the bovine and chicken liver sequences shows an overall level of 70% sequence homology, but there is complete identity of all residues that have been implicated in the function of the enzyme. When two mammalian cells, cos-7 and 293 cells, were transiently transfected with a plasmid containing the rhodanese coding region, rhodanese activity in lysates increased approximately 20-fold. Fluorograms of denaturing polyacrylamide gels detected a large increase in a polypeptide that co-migrated with the native protein and reacted with anti-rhodanese antibodies. Nondenaturing gels showed two active species that co-migrated with the two major electrophoretic forms purified by current procedures. Escherichia coli, transformed with a plasmid containing the rhodanese coding region, showed a 15-fold increase in rhodanese activity over baseline values. When the E. coli recombinant protein was analyzed on a nondenaturing gel, only one species was observed that co-electrophoresed with the more electropositive variant seen in purified bovine liver rhodanese. This single variant could be converted by carboxypeptidase B digestion to a form of the enzyme that co-migrated with the more electronegative species isolated from bovine liver. Thus, two major, enzymatically active electrophoretic variants, commonly observed in mammalian cells, can be accounted for by carboxyl-terminal processing without recourse to other post-translational modifications.

Adrenal Glands↗
PubMed1991

Reactivity of Hg(II) with superoxide: evidence for the catalytic dismutation of superoxide by Hg(II).

Mercuric ion, a well-known nephrotoxin, promotes oxidative tissue damage to kidney cells. One principal toxic action of Hg(II) is the disruption of mitochondrial functions, although the exact significance of this effect with regard to Hg(II) toxicity is poorly understood. In studies of the effects of Hg(II) on superoxide (O2-) and hydrogen peroxide (H2O2) production by rat kidney mitochondria, Hg(II) (1-6 microM), in the presence of antimycin A, caused a concentration-dependent increase (up to fivefold) in mitochondrial H2O2 production but an apparent decrease in mitochondrial O2- production. Hg(II) also inhibited O(2-)-dependent cytochrome c reduction (IC50 approximately 2-3 microM) when O2- was produced from xanthine oxidase. In contrast, Hg(I) did not react with O2- in either system, suggesting little involvement of Hg(I) in the apparent dismutation of O2- by Hg(II). Hg(II) also inhibited the reactions of KO2 (i.e., O2-) with hemin or horseradish peroxidase dissolved in dimethyl sulfoxide (DMSO). Finally, a combination of Hg(II) and KO2 in DMSO resulted in a stable UV absorbance spectrum [currently assigned Hg(II)-peroxide] distinct from either Hg(II) or KO2. These results suggest that Hg(II), despite possessing little redox activity, enhances the rate of O2- dismutation, leading to increased production of H2O2 by renal mitochondria. This property of Hg(II) may contribute to the oxidative tissue-damaging properties of mercury compounds.

Animals↗
PubMed1991

Cloning and characterization of a human c-myc promoter-binding protein.

A human cDNA clone encoding a c-myc promoter-binding protein was detected by screening a HeLa cell lambda phage expression cDNA library. The library was screened by using an XhoI-NaeI human c-myc P2 promoter fragment as a probe. The recombinant phage encoded a fusion protein, myc-binding protein 1 (MBP-1), which had an apparent molecular size of 40 kDa. A corresponding protein with a molecular size of 35 kDa was present in a HeLa cell extract. Sequence analysis of the cloned gene reveals an open reading frame of 1,038 bp with a 3' untranslated region of 378 bp. The predicted protein sequence contains a proline-rich region in the amino terminus but does not demonstrate a known DNA-binding domain. DNase I footprint analysis demonstrates that MBP-1 binds to the sequence just 5' of the TATA box sequence of the human c-myc P2 promoter. MBP-1 cDNA hybridizes to a 1.4-kb mRNA from HeLa and HL-60 cells, indicating that the cDNA insert (1,416 bp) is a full-length clone. Coexpression of the MBP-1 protein repress transcription from the human c-myc promoter, suggesting that MBP-1 may act as a negative regulatory factor for the human c-myc gene.

Amino Acid Sequence↗
PubMed1991

Mithramycin inhibits SP1 binding and selectively inhibits transcriptional activity of the dihydrofolate reductase gene in vitro and in vivo.

The promoter of the human dihydrofolate reductase (DHFR) gene contains two consensus binding sites for the DNA binding protein Sp1. DNAse protection and gel mobility shift assays demonstrate binding of recombinant Sp1 to both decanucleotide Sp1 binding sequences which are located 49 and 14 base pairs upstream of the transcription start site. The more distal of the two binding sites exhibits a somewhat higher affinity for Sp1. The G-C specific DNA binding drug, mithramycin, binds to both consensus sequences and prevents subsequent Sp1 binding. Promoter-dependent in vitro transcription of a DHFR template is selectively inhibited by mithramycin when compared to the human H2b histone gene. A similar effect is also noted in vivo. Mithramycin treatment of MCF-7 human breast carcinoma cells containing an amplified DHFR gene induces selective inhibition of DHFR transcription initiation, resulting in a decline in DHFR mRNA level and enzyme activity. This selective inhibition of DHFR expression suggests that it is possible to modulate the overexpression of the DHFR gene in methotrexate resistant cells.

Base Sequence↗
PubMed1991

Role of immune cells in the Pavlovian conditioning of specific resistance to cancer.

It has been demonstrated that significant protection against YC8 lymphoma can be induced in mice preimmunized with normal DBA/2 spleen cells. The DBA/2 spleen cells used as alloantigens share minor histocompatibility determinants with the YC8 tumor. We have used this model to investigate the nature of the immunity conferred by treatment with the alloantigen and infer that the conditioned resistance observed was maintained by the same effector mechanism. The results demonstrated that repeated immunization of tumor bearing mice with the alloantigen had some beneficial effect as shown by the slower rate of growth of the tumor, and an increase in median survival time over controls. The observations showed however that once tumor was present in vivo, the use of potent tumor specific vaccine can help in increasing survival but can no longer produce high incidence of regressions and cures. Conditioning can potentiate the effects of this treatment by increasing survival and cure.

Animals↗