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Biomedical subjects

J H Miller

Publications and source records attributed to J H Miller.

At least 19 recordsLinked to original sources
PubMed1992

A repair system for 8-oxo-7,8-dihydrodeoxyguanine.

Active oxygen species can damage DNA and may play a role in aging and carcinogenesis. We have tested MutY glycosylase for activity on undamaged mispairs as well as mispairs formed with the oxidatively damaged substrates, 8-oxo-7,8-dihydrodeoxyguanine (GO) or 8-oxo-7,8-dihydrodeoxyadenine (AO). MutY acts as a glycosylase on four of the heteroduplexes tested, A/G, A/GO, A/C, and A/AO, removing the undamaged adenine from each substrate. Genetic data suggest that the primary substrate for MutY glycosylase in vivo is the A/GO mispair. We present biochemical evidence demonstrating that MutY glycosylase is an important part of a repair system that includes the MutM and MutT proteins. The GO repair system is dedicated to the repair of the oxidatively damaged guanine and the mutations it can induce.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1992

Polarity of transport of 2-deoxy-D-glucose and D-glucose by cultured renal epithelia (LLC-PK1).

At least two types of glucose transporter exist in cultured renal epithelial cells, a Na(+)-glucose cotransporter (SGLT), capable of interacting with D-glucose but not 2-deoxy-D-glucose (2dglc) and a facilitated transporter (GLUT) capable of interacting with both D-glucose and 2dglc. In order to examine the polarity of transport in cultured renal epithelia, 2dglc and D-glucose uptakes were measured in confluent cultures of LLC-PK1 cells grown on collagen-coated filters that permitted access of medium to both sides of the monolayer. The rates of basolateral uptake of both 1 mM glucose (Km 3.6 mM) and 1 mM 2dglc (Km 1.5 mM) were greater than apical uptake rates and the (apical-to-basolateral)/(basolateral-to-apical) flux ratio was high for glucose (9.4) and low for 2dglc (0.8), thus, confirming the lack of interaction of 2dglc with the apical SGLT. Specific glucose transport inhibitor studies using phlorizin, phloretin and cytochalasin B confirmed the polarised distribution of SGLT and GLUT in LLC-PK1 cells. Basolateral sugar uptake could be altered by addition of insulin (1 mU/ml) which increased 2dglc uptake by 72% and glucose uptake by 50% and by addition of 20 mM glucose to the medium during cell culture which decreased 2dglc uptake capacity at confluence by 30%. During growth to confluence, 2dglc uptake increased to a maximum, then decreased at the time of confluence, coincident with a rise in uptake capacity for alpha-methyl-D-glucoside, a hexose that interacts only with the apical SGLT. It was concluded that the non-metabolisable sugar 2dglc was a useful, specific probe for GLUT in LLC-PK1 cells and that GLUT was localised at the basolateral membrane after confluence.

Animals↗
PubMed1992

Evidence that MutY and MutM combine to prevent mutations by an oxidatively damaged form of guanine in DNA.

It has been previously shown both in vivo and in vitro that DNA synthesis past an oxidatively damaged form of guanine, 7,8-dihydro-8-oxoguanine (8-oxoG), can result in the misincorporation of adenine (A) opposite the 8-oxodG. In this study we show that MutY glycosylase is active on a site-specific, oxidatively damaged A/8-oxoG mispair and that it removes the undamaged adenine from this mispair. Strains that lack active MutY protein have elevated rates of G.C----T.A transversions. We find that the mutator phenotype of a mutY strain can be fully complemented by overexpressing MutM protein (Fpg protein) from a plasmid clone. The MutM protein removes 8-oxoG lesions from DNA. In addition, we have isolated a strain with a chromosomal mutation that suppresses the mutY phenotype and found that this suppressor also overexpresses MutM. Finally, a mutY mutM double mutant has a 25- to 75-fold higher mutation rate than either mutator alone. The data strongly suggest that MutY is part of an intricate repair system directed against 8-oxoG lesions in nucleic acids and that the primary function of MutY in vivo is the removal of adenines that are misincorporated opposite 8-oxoG lesions during DNA synthesis.

Bacterial Proteins↗
PubMed1992

Amino acid substitution analysis of E. coli thymidylate synthase: the study of a highly conserved region at the N-terminus.

Amino acid substitution analysis within a highly conserved region of Escherichia coli thymidylate synthase (TS), using suppression of amber mutations by tRNA suppressors, has yielded a bank of 124 new mutationally altered TS proteins. These mutant proteins have been used to study the structure-function relationship of the Escherichia coli TS protein at the N-terminus corresponding to residues 20 through 35. This region contains a block of amino acids whose sequence has been well conserved among other known TS proteins from various organisms. Positions 20 through 25 contain a surface loop structure and positions 26 through 35 encompass a beta-strand. We find that residues surrounding a beta-bulge structure within the beta-strand are particularly sensitive to amino acid substitution, suggesting that this structure is maintained by a highly ordered packing arrangement. Three residues in the surface loop that are present at the base of the substrate binding pocket are also sensitive to amino acid substitution. The remainder of the conserved sites, including those at the dimer interface, are tolerant to most, if not all, of the substitutions tested.

Amino Acid Sequence↗
PubMed1992

DNA inversions between short inverted repeats in Escherichia coli.

Using site-specific mutagenesis in vitro, we have constructed Escherichia coli strains that allow the detection of the inversion of an 800-bp segment in the lac region. The invertible segment is bounded by inverted repeats of either 12 or 23 bp. Inversions occurring at these inverted repeats will restore the Lac+ phenotype. Inversions can be detected at both short homologies at frequencies ranging from 0.5 x 10(-8) to 1 x 10(-7). These events, which have been verified by DNA sequence analysis, are reduced up to 1000-fold in strains deficient for either RecA, RecB or RecC. They are not reduced in strains deficient in the RecF, J pathway. These results show that the RecB,C,D system can mediate rearrangements at short sequence repeats, and probably plays a major role in cellular rearrangements.

Base Sequence↗
PubMed1992

Thrombocytosis and hyposplenism in an infant with fetal hydantoin syndrome.

An infant boy with fetal hydantoin syndrome was evaluated for the finding of thrombocytosis. Delayed splenic maturation was identified by a decreased splenic uptake of 99mTc sulfur colloid and by an increased number of pitted erythrocytes at 6 months of age. We speculate that the patient's thrombocytosis may be related to delayed maturation of splenic function and conclude that hyposplenism and secondary thrombocytosis may be associated with fetal hydantoin syndrome.

Abnormalities, Drug-Induced↗
PubMed1992

Cloning and sequencing of Escherichia coli mutR shows its identity to topB, encoding topoisomerase III.

We have cloned and sequenced the mutR gene from Escherichia coli, which results in an increased frequency of spontaneous deletions, by using a strain carrying a Tn10 derivative inserted into mutR. The analysis of 1,286 bp of mutR sequence shows that this gene is identical to the topB gene, which encodes topoisomerase III. The increased deletion formation is the first reported phenotype for cells lacking topoisomerase III, and this suggests that topoisomerase III is involved in reactions that normally reduce the levels of spontaneous deletions.

Base Sequence↗
PubMed1992

Interaction of calcium with plasma membrane of epithelial (MDCK) cells during junction formation.

We have previously shown that upon transferring confluent monolayers of Madin-Darby canine kidney (MDCK) cells from low- to normal-Ca2+ medium, cytosolic Ca2+ increases and tight junctions (TJs) assemble and seal, but the increase in cytosolic Ca2+ does not seem to be necessary for junction formation. In the present work we establish that these are in fact two independent phenomena. We first measured unidirectional Ca2+ fluxes across the plasma membrane of MDCK cells to find suitable inhibitors and tested their effects on the ability of Ca2+ to seal the TJ. Likewise, we studied a variety of multivalent cations. We observed that 1) Ca2+ triggering of junction formation does not depend on its entering the cell, 2) cations like La3+ may impair the influx of Ca2+ without affecting the sealing of TJs, and 3) only Cd2+ is able to block both Ca2+ penetration and junction formation; however, 4) Cd2+ itself cannot trigger junction formation. We interpret that Ca2+ triggers junction formation by acting mainly on an extracellular membrane site and that this site has a higher Ca2+ selectivity than the mechanisms for Ca2+ translocation across the membrane.

Animals↗
PubMed1992

Sodium-sensitive, probenecid-insensitive p-aminohippuric acid uptake in cultured renal proximal tubule cells of the rabbit.

In the intact kidney, renal proximal tubule cells accumulate p-aminohippurate (PAH) via a basolateral, probenecid- and sodium-sensitive transport system. Primary cultures of rabbit proximal tubule cells retain sodium-glucose co-transport in culture, but little is known about PAH transport in this system. Purified proximal tubule cells from a rabbit were grown in culture and assessed for PAH and alpha-methyl-D-glucoside uptake capacities as well as proximal tubule marker enzyme activities. Control PAH uptake on collagen-coated filters (20 +/- 3 pmol/mg protein.min; n = 8) was not significantly different from uptake in the presence of 1 mM probenecid (19 +/- 4 pmol/mg protein.min; n = 8). Uptake from the basal side of the cell was 3.9 +/- 0.7 times greater than that from the apical side. In multi-well plate studies, the uptake was significantly reduced by removing sodium from the medium and stimulated by coating the wells with collagen. Glutarate (10 mM) had no effect on the uptake of PAH. Other differentiated proximal tubule characteristics were retained in culture, including the ability to form domes and to transport glucose by a phlorizin-sensitive system. Phlorizin-sensitive 1 mM alpha-methyl-D-glucoside uptake was 134 +/- 42 pmol/mg protein.min (n = 7; P less than 0.02). The proximal tubule marker enzymes alkaline phosphatase and gamma-glutamyltranspeptidase, increased in activity in the cultures after confluence. It was concluded that whereas some differentiated properties were retained during primary culture of rabbit proximal tubule cells, the PAH transport system was selectively lost or modified from that present in the intact kidney.

Alkaline Phosphatase↗
PubMed1992

Binding and functional selectivity of himbacine for cloned and neuronal muscarinic receptors.

The binding potencies for the putative M2-selective antagonist himbacine were determined in radioligand binding and in functional response assays in neuronal tissue and Chinese hamster ovary cells containing transfected muscarinic receptors. Himbacine was shown to bind to all five cloned muscarinic receptor subtypes in the order of potencies: hM2 = hM4 > hM3 > hM1 > hM5 (Kd values were 4, 7, 59, 83 and 296 nM, respectively). Himbacine was shown to bind to M2 receptors in rat heart and brain stem with Kd values of 6.9 and 4.6 nM, respectively. In rat brain tissues with complex mixtures of muscarinic receptors, and using the radioligand [3H] +/- -5,11-dihydro-11-([(2-(2-[(dipropylamino)methyl]-1- peperidinyl)ethyl)amino]carbonyl)-6H-pyrido(2,3-b)(1,4)benzodiazep ine-6-one to demarcate M2 and M4 receptors, himbacine was shown to bind to 80% of cortical or striatal receptors with Kd values of 4.5 and 3.8 nM, respectively, consistent with the involvement of M2 and/or M4 receptors in both these brain regions. Himbacine was a potent blocker of oxotremorine-M-mediated cyclic AMP inhibition in rat striatum (4.4 nM) and in N1E-115 neuroblastoma cells (10.6 nM), responses mediated by M4 receptors. Himbacine also reversed oxotremorine-M-mediated inhibition of evoked acetylcholine release from hippocampal tissue with a Kd value of 8.6 nM, a value consistent with the involvement of M2 or M4 receptors. At the cortical postsynaptic muscarinic receptors involved with phosphoinositide turnover (putative M1 and M3 receptors), himbacine was 21-fold less potent. Himbacine appears to be a potent muscarinic antagonist that displays selectivity for M2 or M4 receptors, as compared to M1 or M3 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1992

Color Doppler ultrasound of the genitourinary tract.

This paper discusses the role of color Doppler ultrasonography (CDU) as it applies to the genitourinary tract. CDU is beneficial in evaluating both the native and allograft kidney in the diagnosis of renal arterial and venous stenosis, arteriovenous fistulas, pseudoaneurysms, arterial and venous thrombosis, and in the characterization and staging of renal masses. CDU has redefined the role of ultrasonography in the evaluation of the scrotum as it relates to testicular torsion, inflammatory disorders and varicoceles. Early results of CDU in prostate carcinoma and benign prostate hypertrophy have shown limited success. Identifying and assessing the neurovascular bundle may have an impact on the staging of prostate carcinoma. It is hoped that the future addition of ultrasound contrast agents will further establish the role of color Doppler in the diagnosis of a variety of genitourinary diseases and disorders.

Female Urogenital Diseases↗
PubMed1992

Distinguishing soft-tissue hemangiomas from vascular malformations using technetium-labeled red blood cell scintigraphy.

Soft-tissue vascular lesions in children can be classified as either hemangiomas or vascular malformations. The distinction between the two has important prognostic and therapeutic implications. Over the past 8 years, we have evaluated 64 vascular lesions with the technetium-labeled red blood cell (Tc-RBC) scan. Twenty-eight lesions imaged as hemangiomas with intense focal uniform uptake. This diagnosis was confirmed in 27 lesions, or 96 percent. Thirty-six lesions imaged as vascular malformations with abnormal vessels or diffusely increased activity. This diagnosis was confirmed in 35 lesions, or 97 percent. Overall, the Tc-RBC scan was 97 percent accurate in distinguishing hemangiomas from vascular malformations. It is particularly useful when the clinical diagnosis of the lesion may not be evident. Not only can biopsy be avoided, but parents can be reassured at an earlier age and given accurate information regarding prognosis.

Arteriovenous Malformations↗
PubMed1992

Using PCR to extend the limit of oligonucleotide synthesis.

A method has been developed to allow one to extend the practical limit of oligonucleotide synthesis by coupling the synthesis reaction to a subsequent PCR. Given that DNA synthesizers are capable of producing reasonable yields of oligonucleotides that are 125-150 bases in length, this method could be used to recover the minute amount of full-length product present in mixtures extended well beyond the established limits. This technology could be applied to gene synthesis and mutagenesis.

Base Sequence↗
PubMed1991

Potencies and stereoselectivities of enantiomers of huperzine A for inhibition of rat cortical acetylcholinesterase.

The stereoselectivities and mechanisms of the inhibition of rat cortical acetylcholinesterase by the enantiomers of huperzine A were determined. (-)-Huperzine A was the more potent enantiomer with a Ki value of 8 nM. (+)-Huperzine A inhibited the enzyme 38-fold less potently with a Ki value of 300 nM. Racemic huperzine A was about two-fold less potent than the more active isomer, (-)-huperzine A. The mechanism of inhibition of acetylcholinesterase for all three drugs was of the mixed linear competitive type.

Alkaloids↗
PubMed1991

MutM, a protein that prevents G.C----T.A transversions, is formamidopyrimidine-DNA glycosylase.

We have cloned chromosomal DNA bordering an insert that inactivates mutM. Sequencing of this clone has revealed that the insertion element is located between the promoter and structural gene for formamidopyrimidine-DNA glycosylase (Fapy-DNA glycosylase). An overproducing clone of Fapy-DNA glycosylase complements the original mutM strain that had been isolated after EMS mutagenesis. Thus, we conclude that MutM is actually Fapy-DNA glycosylase. mutM has previously been characterized as a mutator strain that leads specifically to G.C----T.A transversions. This in vivo characterization correlates well with the mutagenic potential of one of the lesions Fapy-DNA glycosylase removes, 8-oxo-7,8-dihydro-2'-deoxyguanine (8-OxodG).

8-Hydroxy-2'-Deoxyguanosine↗