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Biomedical subjects

M R Rice

Publications and source records attributed to M R Rice.

9 recordsLinked to original sources
PubMed2000

Recognition of native DNA methylation by the PvuII restriction endonuclease.

Recognizing the methylation status of specific DNA sequences is central to the function of many systems in eukaryotes and prokaryotes. Restriction-modification systems have to distinguish between 'self' and 'non-self' DNA and depend on the inability of restriction endonucleases to cleave their DNA substrates when the DNA is appropriately methylated. These endonucleases thus provide a model system for studying the recognition of DNA methylation by proteins. We have characterized the interaction of R.PVU:II with DNA containing the physiologically relevant N4-methylcytosine modification. R.PVU:II binds (N4m)C-modified DNA and cleaves it very slowly. Methylated strands in hemimethylated duplexes were cleaved at a higher rate than in fully methylated duplexes, in parallel with a higher binding affinity for hemimethylated DNA. The co-crystal structures of R.PVU:II-DNA, together with a mutagenesis study, have implicated specific amino acids in recognition of the methylatable base; one of these is His84. We report that replacing His84 with Ala reduced the rate of cleavage of unmodified DNA but, in contrast, slightly increased the cleavage of (N4m)C-modified DNA.

Base Sequence↗
PubMed2000

Snowmobile injuries and deaths in children: a review of national injury data and state legislation.

BACKGROUND: Snowmobiling is a popular family sport, with annual expenditures over $9 billion. The size and speed of snowmobiles make them potentially dangerous to children. Pediatric snowmobile-related trauma has not been studied in the United States. METHODS: We analyzed 291 pediatric snowmobile- related injuries and 75 deaths reported to the Consumer Product Safety Commission from 1990 to 1998. We reviewed snowmobile legislation in the states that reported at least 1 death to the Consumer Product Safety Commission during this time period. RESULTS: The most common sites of injury were the extremities (48.8%) and the head, neck, and face (28.2%). Head and neck injuries were the predominant cause of death (66.7%). The most common diagnosis was contusion/abrasion (30.9%), followed by laceration (22%), fracture (20.3%), and strain/sprain (14.4%). Nonfatal injuries most often involved ejection from the snowmobile (26.1%), but striking a stationary object was the most common mechanism in fatal crashes. The review of state legislation revealed that few age restrictions or helmet laws exist. Children as young as 8 years old may legally operate a snowmobile in some states. Often, restrictions do not apply to snowmobile use on private property, where 43% of pediatric snowmobile-related injuries occurred. CONCLUSIONS: Head, neck, and face injuries are common nonfatal injuries and are the most common cause of death. State legislation often lacks age restrictions on private property, and laws requiring helmet use are rare. Legislators have not addressed the dangers of pediatric snowmobile-related injuries. Helmet laws and age restrictions similar to those enacted for motorcycle riders are necessary and appropriate.

Adolescent↗
PubMed1999

Substrate recognition by the Pvu II endonuclease: binding and cleavage of CAG5mCTG sites.

The Pvu II restriction endonuclease (R. Pvu II) cleaves CAG downward arrowCTG sequences as indicated, leaving blunt ends. Its cognate methyltransferase (M. Pvu II) generates N4-methylcytosine, yielding CAGN4mCTG, though the mechanism by which this prevents cleavage by R. Pvu II is unknown. The heterologous 5-methylcytosinemethylation CAG5mCTG has also been reported to prevent cleavage by R. Pvu II and this has been used in some cloning methods. Since this heterologousmethylation occurs at the native methylated base, it can provide insights into the detection of DNAmethylation by R. Pvu II. We found that the cloned gene for R. Pvu II could not stably transform cells protected only by M. Alu I (AG5mCT) and then determined that R. Pvu II cleaves CAG5mCTG in vitro, even when both strands are methylated. DNase I footprint analysis and competition experiments reveal that R. Pvu II binds to CAG5mCTG specifically, though with reduced affinity relative to the unmethylated sequence. These results provide biochemical support for the publishedstructures of R. Pvu II complexed with DNA containing CAGCTG and CAG5-iodoCTG and support a model for how methylation interferes with DNA cleavage by this enzyme.

5-Methylcytosine↗
PubMed1999

Reducing hemoglobin oxygen affinity does not increase hydroxyl radicals after acute subdural hematoma in the rat.

Extensive evidence is available to show the importance of ischemia after severe human head injury. We have previously shown that pharmacologically increasing the release of oxygen in brain tissue where the local oxygen pressure is low reduces infarct size in animal models. To study the possible negative effects of this strategy, we tested the effect of an allosteric modifier of hemoglobin (RSR13) on free radical production in the rat acute subdural hematoma (ASDH) model, both under normoxic as well as under hyperoxic, normobaric conditions. When compared to baseline, induction of ASDH resulted in a significant increase (p < 0.05) in 2,3-DHBA (2,3 dihydroxybenzoic acid, produced from salicylate after attack by hydroxyl radicals) at 30 and 60 min postinduction, both for the control group (39% and 91%) as well as the RSR13-treated group (41% and 62%). The 2,5-DHBA also increased significantly (p < 0.05) in the drug-treated animals at the 30- and 60-min time points when compared to baseline (49% and 77%). At all time points, except the 30-min, the increase in 2,3-DHBA was less marked in the RSR13 animals than in the control group. Similarly, the 2,5-DHBA increase after ASDH was lower at all time points except for the 30-min time point in the RSR13-treated group. These results indicate that enhanced tissue oxygen release by the allosteric modifier of hemoglobin RSR13 does not increase hydroxyl radical production after ASDH. Clinical trials are needed to test this compound in humans after severe head injury.

Aniline Compounds↗
PubMed1998

Increased free radical production due to subdural hematoma in the rat: effect of increased inspired oxygen fraction.

Acute subdural hematoma (ASDH) complicates about 15%-20% of severe head injury patients and is one of the major causes for bad outcome, yet the pathomechanisms involved are not well understood. This study has employed a recently developed technique to determine whether ASDH induces free radicals in the underlying brain. We also studied the effect of increased inspired oxygen fraction (FiO2) on free radical production, both in the normal rat brain and after ASDH induction. Twelve male Sprague Dawley rats were studied over 5 h (2 h of FiO2 = 30%, 3 h of FiO2 = 100%). Hydroxyl radical production was measured with microdialysis using the salicylate trapping technique by quantitating the 2,3 dihydroxy benzoic acid (2,3 DHBA) and 2,5 dihydroxy benzoic acid (2,5 DHBA), degradation products, in either noninjured brain (n = 6) or after ASDH (n = 6). Both 2,3 DHBA and 2,5 DHBA increased significantly by 39% and 108%, respectively, after the induction of the SDH (p < 0.05). By increasing the FiO2 to 100%, 2 h after ASDH induction, the 2,3 DHBA and 2,5 DHBA further increased only slightly (ns). After increasing the FiO2 to 100% in the noninjured group, the mean level of 2,3 DHBA increased by 56% (p = 0.06, ns). The level of 2,5 DHBA in the dialysate increased significantly by 56% (p < 0.05), when the FiO2 was increased to 100% ASDH results in a significant increase in free radical production. At the same time, prolonged increase in FiO2 does not lead to further increase in free radical production in the injured brain.

Acute Disease↗
PubMed1997

Use of an inducible regulatory protein to identify members of a regulon: application to the regulon controlled by the leucine-responsive regulatory protein (Lrp) in Escherichia coli.

Procedures were developed to facilitate the identification of genes that belong to a given regulon and characterization of their responses to the regulator. The regulon controlled by the Escherichia coli leucine-responsive regulatory protein (Lrp) was studied by isolating random transcriptional fusions to lacZ, using lambda placMu53 and a strain in which lrp is under isopropylthio-beta-D-galactopyranoside (IPTG)-inducible control. Fusions exhibiting IPTG-responsive beta-galactosidase activity were cloned by integrating the suicide vector pIVET1 via homologous recombination at lacZ, followed by self-ligating digested chromosomal DNA. We verified the patterns of lacZ expression after using the plasmid clones to generate merodiploid strains with interrupted and uninterrupted copies of the same sequence. If the merodiploid expression pattern was unchanged from that shown by the original fusion strain, then the cloned fusion was responsible for the regulatory pattern of interest; a difference in the expression pattern could indicate that the original strain carried multiple fusions or that there were autogenous effects of having interrupted the fused gene. Using these procedures, we generated a fusion library of approximately 5 x 10(6) strains; approximately 3,000 of these strains were screened, yielding 84 Lrp-responsive fusions, and 10 of the 84 were phenotypically stable and were characterized. The responses of different fusions in a given operon to in vivo Lrp titrations revealed variations in expression with the position of insertion. Among the newly identified members of the regulon is an open reading frame (orf3) between rpiA and serA. Also, expression of a fusion just downstream of dinF was found to be Lrp dependent only in stationary phase.

Bacterial Proteins↗
PubMed1997

Effects of the allosteric modification of hemoglobin on brain oxygen and infarct size in a feline model of stroke.

BACKGROUND AND PURPOSE: Cerebral ischemia and stroke are leading causes of morbidity and mortality. An approach to protecting the brain during ischemia is to try to increase the delivery of oxygen via the residual blood flow through and around ischemic tissue. To test this hypothesis, we used a novel oxygen delivery agent, RSR-13 (2-[4-[[(3,5-dimethylanilino)-carbonyl]-methyl]phenoxy]-2-methylpr opionic acid). Intravenous administration of RSR-13 increases oxygen delivery through allosteric modification of the hemoglobin molecule, resulting in a shift in the hemoglobin/oxygen dissociation curve in favour of oxygen delivery. METHODS: We studied RSR-13 in a feline model of permanent middle cerebral artery occlusion to assess its effects on cerebral oxygenation and infarct size. A randomized, blinded study of RSR-13 (n = 6) versus 0.45% saline (n = 12) was conducted, after an RSR-13 dose-escalation study (n = 4). Drug was administered as a preocclusion bolus followed by a continuous infusion for the duration of the experiment (5 hours). Brain oxygen was measured continuously with the use of a Clark oxygen electrode. Infarct size was measured at 5 hours after occlusion with computer-assisted volumetric analysis. RESULTS: The drug treatment group had consistently higher mean brain oxygen tension than controls (33 +/- 5 and 27 +/- 6 mm Hg, respectively) and significantly smaller infarcts (21 +/- 9% versus 33 +/- 9%, respectively, P < .008). We observed an inverse relationship between the dose response of RSR-13 (the shift in the hemoglobin/oxygen dissociation curve) and infarct size. CONCLUSIONS: These results are evidence that allosteric hemoglobin modification is protective to the brain after acute focal ischemia, providing a new opportunity for neuroprotection and raising the possibility of enhancing the protective effect of thrombolysis and ion channel blockade.

Aniline Compounds↗
PubMedDate not supplied

Effect of 4'-doxorubicin analogs on heterotransplantation of human tumors in congenitally athymic mice.

The antitumor activity of three new doxorubicin (DX) derivatives with less cardiotoxicity than the parent compound was tested against several human tumors representative of some of the major classes of human cancer. The tested DX derivatives, modified on the 4' position of the amino sugar, were 4'-epiDX, 4'-deoxyDX, and 4'-O-methylDX. Fourteen human tumors (three breast tumors, three lung tumors, three melanomas, two ovarian tumors, one prostate tumor, one sarcoma, and one larynx tumor) serially transplanted in athymic mice were used to screen the antineoplastic activity of the 4'-DX derivatives. BALB/c nude mice were treated iv with equitoxic doses of each as a single agent (less than or equal to LD10) on a weekly basis for 3-4 weeks, starting when the tumor became relatively large. 4'-EpiDX, which has a higher threshold limit of cardiac toxicity in man, was found active against breast, lung (epidermoid and oat cell carcinoma), prostate, and ovarian tumors. This drug showed particularly good activity against melanomas. 4'-DeoxyDX was active against breast and prostate tumors, while 4'-0-methylDX was active against breast and ovarian tumors and possibly sarcoma.

Animals↗