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Biomedical subjects

Robert Shapiro

Publications and source records attributed to Robert Shapiro.

At least 19 recordsLinked to original sources
PubMed2007

4-hydroxyequilenin-adenine lesions in DNA duplexes: stereochemistry, damage site, and structure.

The equine estrogens, equilin and equilenin, are major components of the drug Premarin, the most widely used formula for hormone replacement therapy. The derivative 4-hydroxyequilenin (4-OHEN), a major phase I metabolite of equilin and equilenin, autoxidizes to potent cytotoxic quinoids that can react in vitro and in vivo with cytosine and adenine in DNA. Unique cyclic adducts containing the same bicyclo[3.3.1]nonane-type connection ring are produced. Each base adduct has four stereoisomers. In order to elucidate the structural effects of A versus C modification, we have carried out molecular dynamics simulations of the stereoisomeric 4-OHEN-A adducts in DNA 11-mer duplexes and compared results with an earlier study of the C adducts (Ding, S., Shapiro, R., Geacintov, N.E., and Broyde, S. (2005) Equilenin-Derived DNA Adducts to Cytosine in DNA Duplexes: Structures and Thermodynamics, Biochemistry 44, 14565-14576). Similar stereochemical principles govern the orientations in DNA duplexes of the 4-OHEN-A adducts as for the analogous C adducts, with opposite orientations of the equilenin rings in stereoisomeric pairs of adducts characterized by near-mirror image circular dichroism (CD) spectra. However, the larger purine adducts have unique structural properties in the duplexes that distinguish their characteristics from those of the pyrimidine adducts. Significant differences are observed in terms of hydrogen bonding, stacking, bending, groove dimensions, solvent exposure, and hydrophobic interactions; also, each of the four stereoisomeric 4-OHEN-A adducts exhibit distinct structural features. Each base adduct and stereoisomer distorts the structure of the DNA duplex differently. These characteristics may manifest themselves in terms of differential nucleotide excision repair susceptibilities and mutagenic activities of the 4-OHEN-A and C adducts.

Adenine↗
PubMed2006

The effect of a single treatment of the Protonics system on lower extremity kinematics during gait and the lateral step up exercise.

INTRODUCTION: Patellofemoral pain (PFP) is often attributed to abnormal patellar tracking. The Protonics knee orthosis was developed to reduce femoral internal rotation by altering pelvic alignment via hamstring activation. The purpose of this research was to determine if a single treatment with the orthosis improved lower extremity alignment during gait and the lateral step up exercise. We hypothesized that anterior pelvic tilt, hip internal rotation and adduction, and external rotation of the tibia with respect to the femur would decrease after use of the brace. METHODS: Nineteen females (23.4+/-3.1 year, 1.66+/-0.05 m, 65.3+/-20.4 kg) with chronic PFP participated. Three-dimensional kinematic data were collected for each subject at 60 Hz during pre-treatment (PRE), after a placebo condition with the orthosis set at zero resistance (PLAC), and post-treatment (POST). Treatment consisted of having the subject perform the rehabilitation exercises recommended by the Protonics manufacturer. A repeated measures ANOVA was performed on each dependent variable (alpha=0.05). RESULTS: This investigation did not verify the changes in alignment proposed by the manufacturer as a result of acute application of the Protonics system. However, after the use of the brace, pelvic rotation and hip hike were decreased during the lateral step up exercise. CONCLUSION: Based on the results of this study, it was concluded that a single application of the Protonics system did not alter anterior pelvic tilt, hip internal rotation and adduction, or tibial external rotation during the lateral step up and gait.

Adult↗
PubMed2006

Mutagenic nucleotide incorporation and hindered translocation by a food carcinogen C8-dG adduct in Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4): modeling and dynamics studies.

Bulky carcinogen-DNA adducts commonly cause replicative polymerases to stall, leading to a switch to bypass polymerases. We have investigated nucleotide incorporation opposite the major adduct of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the DinB family polymerase, Dpo4, using molecular modeling and molecular dynamics (MD) simulations. PhIP, the most prevalent heterocyclic aromatic amine formed by cooking of proteinaceous food, is mutagenic in mammalian cells and is implicated in mammary and colon tumors. Our results show that the dG-C8-PhIP adduct can be accommodated in the spacious major groove Dpo4 open pocket, with Dpo4 capable of incorporating dCTP, dTTP or dATP opposite the adduct reasonably well. However, the PhIP ring system on the minor groove side would seriously disturb the active site, regardless of the presence and identity of dNTP. Furthermore, the simulations indicate that dATP and dTTP are better incorporated in the damaged system than in their respective mismatched but unmodified controls, suggesting that the PhIP adduct enhances incorporation of these mismatches. Finally, bulky C8-dG adducts, situated in the major groove, are likely to impede translocation in this polymerase (Rechkoblit et al. (2006), PLoS Biol., 4, e11). However, N2-dG adducts, which can reside on the minor groove side, appear to cause less hindrance when in this position.

Binding Sites↗
PubMed2006

Flexible 5-guanidino-4-nitroimidazole DNA lesions: structures and thermodynamics.

5-Guanidino-4-nitroimidazole (NI), derived from guanine oxidation by reactive oxygen and nitrogen species, contains an unusual flexible ring-opened structure, with nitro and guanidino groups which possess multiple hydrogen bonding capabilities. In vitro primer extension experiments with bacterial and mammalian polymerases show that NI incorporates C as well as A and G opposite the lesion, depending on the polymerase. To elucidate structural and thermodynamic properties of the mutagenic NI lesion, we have investigated the structure of the modified base itself and the NI-containing nucleoside with high-level quantum mechanical calculations and have employed molecular modeling and molecular dynamics simulations in solution for the lesion in B-DNA duplexes, with four partner bases opposite the NI. Our results show that NI adopts a planar structure at the damaged base level. However, in the nucleoside and in DNA duplexes, steric hindrance between the guanidino group and its linked sugar causes NI to be nonplanar. The NI lesion can adopt both syn and anti conformations on the DNA duplex level, with the guanidino group positioned in the DNA major and minor grooves, respectively; the specific preference depends on the partner base. On the basis of hydrogen bonding and stacking interactions, groove dimensions, and bending, we find that the least distorted NI-modified duplex contains partner C, consistent with observed incorporation of C opposite NI. However, hydrogen bonding interactions between NI and partner G or A are also found, which would be compatible with the observed mismatches.

DNA↗
PubMed2006

A comparison of individual joint contributions to multijoint position reproduction acuity in overhead-throwing athletes.

BACKGROUND: Measures of joint position sense often test single-axis motions isolating a single joint. Such methods hamper our ability to apply findings to functional multijoint activities. Using a functional, active, multijoint test, we measured upper-extremity position reproduction in overhead-throwers. We compared acuity among four joints and examined individual joint contributions or patterns of contribution among joints to overall task acuity. METHODS: We used an electromagnetic tracking device to measure reproduction of two functional upper-extremity positions. We calculated absolute, constant and variable error at four upper-extremity joints around multiple axes of rotation: three axes at the scapulothoracic and glenohumeral joints and two axes at the elbow and wrist. To represent individual joint acuity, we calculated 3-dimensional variable error. Additional 3-dimensional variable error scores using deviation in hand position (with reference to the thorax) represented overall task acuity. We used principle component analyses to identify contributions of individual joints or patterns of contribution among joints to overall task acuity for absolute, constant and variable error. FINDINGS: Scapulothoracic and glenohumeral joints displayed better acuity compared with elbow and wrist joints. When examined separately, absolute, constant and variable error scores did not indicate any individual joint contributed more to task acuity. When we examined the principle components of all error score measures together, a distinct proximal-to-distal pattern of joint contribution to overall task acuity emerged. INTERPRETATION: Proximal joints display better reposition acuity compared with distal joints, however proximal joints contribute more to acuity of the overall task. Our results indicate that upper-extremity joints do not function independently in repositioning tasks and measures of absolute, constant and variable errors combined over multiple joints may better represent upper-extremity function.

Adult↗
PubMed2006

Small molecule interactions were central to the origin of life.

Many scientists believe life began with the spontaneous formation of a replicator. This idea has been supported by "prebiotic" syntheses carried out by chemists using modern apparatus and purified reagents. The probability that such reactions would take place spontaneously on the early Earth is minute. These points are illustrated here by considering the often cited oligomerization of activated RNA components by clay minerals. A more likely alternative for the origin of life is one in which a collection of small organic molecules multiply their numbers through catalyzed reaction cycles, driven by a flow of available free energy. Although a number of possible systems of this type have been discussed, no experimental demonstration has been made. The inclusion of a "driver" reaction, directly coupled to the energy source, may lead to a solution.

Adenine↗
PubMed2005

Equilenin-derived DNA adducts to cytosine in DNA duplexes: structures and thermodynamics.

The drug Premarin is the most widely used formula for hormone replacement therapy. However, long-term exposure to estrogens from the Premarin drug increases the risk of breast cancer. Equilin and equilenin, major components of Premarin, are predominantly metabolized to 4-hydroxyequilenin (4-OHEN). The quinoids produced by 4-OHEN oxidation react with dG, dA, and dC to form unusual stable cyclic bulky adducts, with four stereoisomers identified for each base adduct. The 4-OHEN-dC adducts are most predominant. They are mutagenic in vitro and have been found in human tumor tissue. We have carried out molecular modeling and molecular dynamics simulations to investigate structures and thermodynamics of the four 4-OHEN-dC stereoisomeric adducts in DNA duplexes. Our results show that the structure of each stereoisomer adduct in duplex DNA is specifically governed by its unique stereochemistry. The bulky adducts, with an obstructed Watson-Crick edge and an equilenin ring system near perpendicular to the damaged cytosine, are located in the B-DNA major or minor groove, with the modified cytosine in the syn or anti conformation, respectively. The DNA duplex structures are distorted, in terms of Watson-Crick pairing at and near the lesion, stacking interactions, and groove dimensions. Stereochemistry determines the orientation of the equilenin rings with respect to the 5'- to 3'-direction of the modified strand, as well as the positioning of the equilenin moiety's methyl and hydroxyl groups for each stereoisomer. The unusual structures and the stereochemical effects underlie their biological processing as miscoding DNA lesions whose mutagenic properties may contribute to breast cancer.

Animals↗
PubMed2005

Structural and thermodynamic features of spiroiminodihydantoin damaged DNA duplexes.

Oxidation of guanine or 8-oxo-7,8-dihydroguanine can produce spiroiminodihydantoin (Sp) R and S stereoisomers. Both in vitro and in vivo experiments have shown that the Sp stereoisomers are highly mutagenic, causing G --> C and G --> T transversion mutations. Therefore, they are of interest as potential endogenous cancer causing lesions. However, their structural properties in DNA duplexes remain to be elucidated. We have employed computational methods to study the Sp lesions in 11-mer DNA duplexes with A, C, G, and T partners. Molecular dynamics simulations have been carried out to obtain ensembles of structures, and the trajectories were employed to analyze the structures and compute free energies. The structural and thermodynamic analyses reveal that the Sp stereoisomers energetically favor positioning in the B-DNA major groove, with minor groove conformers also low energy in some cases, depending on the partner base. The R and S stereoisomers adopt opposite orientations with respect to the 5' to 3' direction of the modified strand. Both syn and anti glycosidic bond conformations are energetically feasible, with partner base and stereochemistry determining the preference. The lesions adversely impact base stacking and Watson-Crick hydrogen bonding interactions in the duplex, and cause groove widening. The chemical nature of the partner base determines specific hydrogen bonding and stacking properties of the damaged duplexes. The structural characteristics may relate to observed mutagenic properties of the Sp stereoisomers, including possible stereoisomer-dependent differences.

Computational Biology↗
PubMed2005

Kinematic and EMG characteristics of simple shoulder movements with proprioception and visual feedback.

The objective of this study was to determine if simple, shoulder movements use the dual control hypothesis strategy, previously demonstrated with elbow movements, and to see if this strategy also applies in the absence of visual feedback. Twenty subjects were seated with their right arm abducted to 90 degrees and externally rotated in the scapular plane. Subjects internally rotated to a target position using a custom shoulder wheel at three different speeds with and without visual feedback. Kinematics were collected with a motion analysis system and electromyographic (EMG) recordings of the pectoralis major (PECT), infraspinatus (INFRA), anterior and posterior (ADELT, PDELT) deltoid muscles were used to evaluate muscle activity patterns during movements. Kinematics changed as movement speed increased with less accuracy (p<0.01). Greater EMG activity was observed in the PECT, PDELT, and INFRA with shorter durations for the ADELT, PDELT and INFRA. Movements with only kinesthetic feedback were less accurate (p<0.01) and performed faster (p<0.01) than movements with visual feedback. EMG activity suggests no major difference in CNS control strategies in movements with and without visual feedback. Greater resolution with visual feedback enables the implementation of a dual control strategy, allowing greater movement velocity while maintaining accuracy.

Adult↗
PubMed2005

Spiroiminodihydantoin lesions derived from guanine oxidation: structures, energetics, and functional implications.

Reactive oxygen species present in the cell generate DNA damage. One of the major oxidation products of guanine in DNA, 8-oxo-7,8-dihydroguanine, formed by loss of two electrons, is among the most extensively studied base lesions. The further removal of two electrons from this product can yield spiroiminodihydantoin (Sp) R and S stereoisomers. Both in vitro and in vivo experiments have shown that the Sp stereoisomers are highly mutagenic, causing G --> T and G --> C transversions. Hence, they are of interest as examples of endogenous DNA damage that may initiate cancer. To interpret the mutagenic properties of the Sp lesions, an understanding of their structural properties is needed. To elucidate these structural effects, we have carried out computational investigations at the level of the Sp-modified base and nucleoside. At the base level, quantum mechanical geometry optimization studies have revealed exact mirror image symmetry of the R and S stereoisomers, with a near-perpendicular geometry of the two rings. At the nucleoside level, an extensive survey of the potential energy surface by molecular mechanics calculations using AMBER has provided three-dimensional potential energy maps. These maps reveal that the range and flexibility of the glycosidic torsion angles are significantly more restricted in both stereoisomeric adducts than in unmodified 2'-deoxyguanosine. The structural and energetic results suggest that the unusual geometric, steric, and hydrogen bonding properties of these lesions underlie their mutagenicity. In addition, stereoisomer-specific differences indicate the possibility that their processing by cellular replication and repair enzymes may be differentially affected by their absolute configuration.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2005

Molecular recognition of human eosinophil-derived neurotoxin (RNase 2) by placental ribonuclease inhibitor.

Placental ribonuclease inhibitor (RI) binds diverse mammalian RNases with dissociation constants that are in the femtomolar range. Previous studies on the complexes of RI with RNase A and angiogenin revealed that RI utilises largely distinctive interactions to achieve high affinity for these two ligands. Here we report a 2.0 angstroms resolution crystal structure of RI in complex with a third ligand, eosinophil-derived neurotoxin (EDN), and a mutational analysis based on this structure. The RI-EDN interface is more extensive than those of the other two complexes and contains a considerably larger set of interactions. Few of the contacts present in the RI-angiogenin complex are replicated; the correspondence to the RI-RNase A complex is somewhat greater, but still modest. The energetic contributions of various interface regions differ strikingly from those in the earlier complexes. These findings provide insight into the structural basis for the unusual combination of high avidity and relaxed stringency that RI displays.

Amino Acid Sequence↗
PubMed2005

Utility of follow-up skeletal surveys in suspected child physical abuse evaluations.

OBJECTIVE: To evaluate the utility of a follow-up skeletal survey in suspected child physical abuse evaluations. METHODS: In this prospective study, follow-up skeletal surveys were recommended for 74 children who, after an initial skeletal survey and evaluation by the Child Abuse Team, were suspected victims of physical abuse. The number and location of the fractures were recorded for the initial skeletal survey and for the follow-up skeletal survey in each case. RESULTS: Forty-eight of the 74 (65%) children returned for a follow-up skeletal survey. The follow-up skeletal survey yielded additional information in 22 of 48 patients (46%). In three patients (6%) the additional information changed the outcome of cases; child abuse was ruled out in one of these patients and abuse was confirmed in two cases. In three other patients, the follow-up skeletal survey refuted tentative skeletal findings, but did not change the outcome because of other physical findings. CONCLUSION: A follow-up skeletal survey identified additional fractures or clarified tentative findings in children who were suspected victims of physical child abuse. The follow-up skeletal survey should be completed on all patients who have an initial skeletal survey performed for suspected physical child abuse and for whom child abuse is still a concern.

Child↗
PubMed2005

The emerging role of virtual colonoscopy.

A review of the current status of virtual colonoscopy is germane and topical. Clinicians need to be knowledgeable about this rapidly evolving and clinically relevant technology to understand the test benefits and limitations and to refer patients for this test appropriately. A critical review of the exponentially expanding literature on this subject is important. This article describes the imaging meth-ods, adjunctive techniques, and radiologic interpretation of CT colonography, and comprehensively and critically reviews the clinical data to help the clinician evaluate the current and potential applications of this technology.

Colonic Neoplasms↗
PubMed2005

Molecular dynamics of a food carcinogen-DNA adduct in a replicative DNA polymerase suggest hindered nucleotide incorporation and extension.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant of the carcinogenic heterocyclic aromatic amines in the human diet, and the major mutagenic effect of dietary PhIP is G-->T transversions. The major PhIP-derived DNA adduct is to C8 of guanine. We have investigated this adduct in a PhIP-induced mutational hotspot 5'-GGGA-3' of the Apc tumor suppressor gene, frequently mutated in mammalian colon tumors. We have carried out a molecular dynamics study to elucidate on a structural level nucleotide incorporation and extension opposite this major adduct during replication. The PhIP adduct was modeled into the ternary complex closed conformation of DNA polymerase RB69, at incorporation and extension positions, with normal cytosine or mismatched partner adenine. RB69 polymerase is a member of the B family as are most replicative eukaryotic DNA polymerases such as DNA polymerase alpha. These systems were subjected to molecular dynamics simulations with AMBER. Our results show that the adduct can reside on the major groove side of the modified DNA template opposite an incoming dCTP or dATP. In the case of the normal partner, disturbance to the active site is observed at the incorporation step, but there is less perturbance in the extension simulation. In the case of the mismatched partner, a less disturbed active site is observed during the incorporation step, but extension appears to be more difficult. Disturbances include adverse impacts on Watson-Crick hydrogen bonding in the nascent base pair, on the distance between the alpha-phosphate of the incoming dNTP and the primer terminus 3'-OH, and on critical protein interactions with the dNTP. However, in all of these cases, a near reaction ready distance (within 3.5 angstroms) between the 3'-terminal oxygen of the primer and the Palpha of the incoming nucleotide triphosphate is sampled occasionally (0.4-23.5% of the time). Thus, error-free bypass or the induction of a G-->T transversion mutation could occur at times and contribute to an extent to the mutagenic effect of PhIP. Polymerase stalling would be the more common outcome and in vivo could lead to switch to an error-prone bypass polymerase.

Amino Acids↗
PubMed2004

Age-related prevalence of cardiac valvular abnormalities warranting infectious endocarditis prophylaxis.

The goal of our study was to determine the prevalence of older patients with cardiac valvular abnormalities warranting endocarditis prophylaxis. We performed a retrospective analysis of 1,000 randomly selected echocardiograms (inpatients and outpatients) from our tertiary care institution. We found that the prevalence of valvular abnormalities increased significantly with age, and that 50% of patients > or =60 years of age warranted endocarditis prophylaxis using current guidelines. With the aging population of the United States and the negative consequences of widespread antibiotic prophylaxis, further investigation is needed to identify patients who are truly at risk for infectious endocarditis.

Adolescent↗
PubMed2004

Crystallographic studies on structural features that determine the enzymatic specificity and potency of human angiogenin: Thr44, Thr80, and residues 38-41.

Human angiogenin (Ang) is a potent inducer of blood vessel formation and is a member of the pancreatic ribonuclease superfamily. Its enzymatic activity is unusually weak and biased toward cleavage after cytidine nucleotides. As part of an ongoing investigation into the structural basis of Ang's characteristic activity, we have determined the crystal structures of three Ang variants having novel activity. (i) The structure of T44D-Ang indicates that Asp44 can participate directly in pyrimidine binding and that the intrinsic hydrogen-bonding capability of this residue largely governs the pyrimidine specificity of this variant. Unexpectedly, the mutation also causes the most extensive disruption of the C-terminus seen in any Ang variant thus far. This allows the side chain of Arg101 to penetrate the B(1) site, raising the possibility that it participates in substrate binding as occurs in ribonuclease 4. (ii) The structure of T80A-Ang supports the view that Thr80 plays little role in maintaining the obstructive conformation of the C-terminus and that its participation in a hydrogen bond with Thr44 selectively weakens the interaction between Thr44 and N3 of cytosine. (iii) ARH-II is an angiogenin/RNase A chimera in which residues 38-41 of Ang are replaced with the corresponding residues (38-42) of RNase A. Its structure suggests that the guest segment influences catalysis by subtle means, possibly by reducing the pK(a) of the catalytic lysine. The loss of angiogenic activity is not attributable to disruption of known cell-binding or nuclear translocation sites but may be a consequence of the chimera's enhanced ribonucleolytic activity.

Alanine↗
PubMed2004

The effect of a single treatment of the Protonics system on biceps femoris and gluteus medius activation during gait and the lateral step up exercise.

INTRODUCTION: Patellofemoral pain, a frequent complaint among women, is attributed in part to excessive femoral internal rotation, leading to patellofemoral malalignment. The Protonics resistive dynamic knee orthosis was designed to facilitate hamstring activation, leading to a less anteriorly tilted pelvis and less femoral internal rotation. This decrease in femoral internal rotation is thought to improve patellofemoral joint alignment, thereby reducing knee pain. In this position, the gluteus medius (GM) would be more effective against the force of gravity. Therefore, this study's purpose was to determine the effects of a single application of the Protonics orthosis on knee pain and biceps femoris (BF) and GM activation. METHODS: 21 females (23.4+/-3.1 years, 1.66+/-0.05 m, 65.3+/-20.4 kg) with a history of chronic PFP participated in the study. Data were collected during level walking and a lateral-step up exercise in three conditions: pre-treatment (PRE), a placebo condition collected after the orthosis was set at zero resistance (PLAC), and post-treatment (POST). PLAC and POST were performed after the orthosis had been removed from the subject's leg. Percent of gait cycle activated, integrated EMG (IEMG), and level of pain (VAS scale) were measured in each condition. An ANOVA was used to determine significance between conditions (alpha=0.05). RESULTS: Use of the Protonics orthosis did not result in any change in the level of knee pain, IEMG or duration of activation for the BF or GM. SUMMARY: Our results do not support an increase in hamstring activation or decrease in GM activation following a single treatment with the Protonics orthosis.

Adult↗