Search PubMed⌕ Search

Biomedical subjects

P Clark

Publications and source records attributed to P Clark.

At least 109 records · Page 6Linked to original sources
PubMed1997

Tetrahydrobiopterin binding to macrophage inducible nitric oxide synthase: heme spin shift and dimer stabilization by the potent pterin antagonist 4-amino-tetrahydrobiopterin.

The characteristics of tetrahydrobiopterin (H4biopterin) binding to pteridine-free recombinant macrophage inducible nitric oxide synthase expressed in Escherichia coli were investigated with a special focus given to effects caused by 2,4-diamino-5,6,7, 8-tetrahydro-6-(l-erythro-1,2-dihydroxypropyl)pteridine (4-amino-H4biopterin), a novel pterin-based inhibitor of nitric oxide synthase. The 4-amino compound completely inhibited enzyme stimulation by 10 microM H4biopterin with a half-maximally active concentration of 7.2 +/- 0.39 microM, whereas H2biopterin and sepiapterin were much less potent. Binding studies using [3H]H4biopterin at 4 degrees C revealed biphasic association of the radioligand according to two first-order reactions with apparent rate constants of 2.2 and 0.05 min-1, each accounting for approximately 50% of total binding. Dissociation of [3H]H4biopterin occurred with rate constants of 0.005 and 0.0028 min-1 in the absence and presence of l-arginine, respectively. Specific binding of 10 nM [3H]H4biopterin was antagonized by unlabeled H4biopterin and its 4-amino analog with half-maximal effects at 84 +/- 6 and 34 +/- 3.2 nM, respectively. Binding of H4biopterin and 4-amino-H4biopterin was accompanied by a partial low spin to high spin conversion of the heme that was completed by l-arginine. Similarly, the active cofactor and the inhibitory 4-amino derivative both induced significant formation of stable protein dimers that survived during SDS electrophoresis, suggesting that the allosteric effects caused by H4biopterin do not explain sufficiently the essential role of the pteridine cofactor in NO biosynthesis.

Animals↗
PubMed1997

Preferential adhesion to and survival on patterned laminin organizes myogenesis in vitro.

We have examined a potential role for differential adhesiveness in muscle development using an in vitro model which employed the culture of myoblasts and myotubes, (conditionally immortal myogenic cells, H2k(b)-tsA58), on micropatterned surfaces. These surfaces are made up of multiple alternating tracks of hydrophobic organosilane-treated glass and untreated glass (track width ranging from 5 to 100 microm). We found that myoblasts were aligned on patterns in the presence of serum, by adhering to the tracks of untreated glass, which had preferentially adsorbed serum attachment factors. However, as serum attachment factors are not sufficient for maintenance of adhesion of mature myotubes, we determined whether precoating patterns with laminin, which maintains adhesion, could still provide a differential adhesive cue. Laminin preferentially adsorbs to the hydrophobic regions resulting in alternating tracks that have adsorbed laminin or serum attachment factors. Myoblasts were less well aligned on these patterns as they could adhere both to the untreated glass and to laminin on the previously hydrophobic tracks, but did show a preference for laminin. However, cell alignment increased upon differentiation into myotubes and continued to increase as the myotubes matured. We found that the alignment of myoblasts and myotubes on patterns increased as track width increased. In addition, adhesion to laminin was required for long term survival of the myotubes. Myotubes that had formed on nonlaminin surfaces began to detach after 2 days of differentiation. Although we found that myoblasts preferentially clustered on laminin tracks, this arrangement did not influence the diameter of the myotubes formed, upon differentiation. Instead, the number of myotubes per track increased with track width, while the myotube diameter remained constant. This uniformity of myotube diameter suggests that a mechanism exists which restricts the ability of myoblasts to undergo lateral fusion. Overall, these findings suggest that differential adhesiveness could be an important mechanism for formation and survival of myotubes, and by using these patterns we have demonstrated a mechanism controlling the formation of linear myotubes by restricting the geometry of cell-cell adhesion.

Animals↗
PubMed1997

Correlation between cervical cytologic results and Gram stain as diagnostic tests for bacterial vaginosis.

OBJECTIVE: Our purpose was to determine the reliability of the Papanicolaou smear in making the diagnosis of bacterial vaginosis with the vaginal Gram stain used as the diagnostic standard. STUDY DESIGN: We conducted a prospective, blinded, cross-sectional study of 210 consecutive patients referred to the Colposcopy Clinic for evaluation of abnormal cervical cytologic results. Each patient had a standard Papanicolaou smear and Gram stain of vaginal discharge. The sensitivity, specificity, positive predictive value, and negative predictive value of the Papanicolaou smear were determined with the Gram stain used as the standard for diagnosis of bacterial vaginosis. RESULTS: Of the 210 patients, 80 (38.1%) had Gram stains that were positive for bacterial vaginosis and 118 (56.2%) had negative Gram stains. Twelve (5.7%) had intermediate Gram stains that were also considered negative. Of the 80 patients with positive Gram stains, 44 had cervical smears consistent with bacterial vaginosis and 36 had negative smears. Of the 130 patients with negative Gram stains, two had a positive cervical smear. Therefore, compared to the Gram stain, cervical cytologic test results had a sensitivity of 55% and a specificity of 98%. The respective positive predictive and negative predictive values were 96% and 78%. CONCLUSION: Compared to Gram stain of vaginal secretions, the cervical Papanicolaou smear has fair sensitivity (55%) and excellent positive predictive value (96%) in diagnosing bacterial vaginosis.

Bacteria, Anaerobic↗
PubMed1997

Studies to detect carriers of haemophilia A in German shepherd dogs using diagnostic DNA polymorphisms in the human factor VIII gene.

The current study investigated whether the DNA polymorphisms in the human FVIII gene, that are used for the diagnosis of carriers of haemophilia A, were diagnostically useful in dogs. Genomic DNA from 20 German Shepherd dogs (13 females, three normal males and four haemophilic males) was tested using five restriction site polymorphisms [HindIII/F8 (exon 17-18), Taq I/ST14.1, BclI/ST14.1, BclI F8 (exon 17-18) and Bgl II/DX13]. The DNA probes (with the exception of DX13) all hybridized to the canine DNA at high stringency, indicating significant homology between the human and canine FVIII gene. A BclI polymorphism (13.5/13.5 + 12.8 kb) was detected with the ST14.1 probe.

Animals↗
PubMed1997

CO2 laser safety considerations in facial skin resurfacing.

Carbon dioxide lasers have been used increasingly in the field of aesthetic plastic surgery, specifically for facial resurfacing procedures. As many plastic surgeons are now venturing into the arena of laser surgery for the first time, it is paramount to understand basic laser safety principles to protect our patients, the operating room personnel, and the laser surgeon. This article reviews basic laser principles and practices and delineates the safety requirements needed to perform laser resurfacing using the CO2 laser system. We subjected several common objects present in the operative field during resurfacing procedures to multiple passes of both the Coherent 5000 C laser and the Laser Industries (Sharplan) model 150XJ laser Silktouch to assess flammability and margins of safety. We tested endotracheal tubes, wet and dry towels, wet and dry gauze sponges, cottonoids, eye protectors, and ophthalmic ointments. Neither flame nor burn was incited in the moistened preparations. The dry objects tested produced flame. The plastic corneal protectors began to melt by the third pass and produced significant heat. Lastly, both the Lacrilube and Bacitracin ophthalmic ointments began to vaporize after three laser passes. On the basis of our findings in this study, we recommend guidelines for prudent and safe CO2 laser usage in facial skin resurfacing.

Fires↗
PubMed1997

Repetitive cycles of cyclophosphamide, thiotepa, and carboplatin intensification with peripheral-blood progenitor cells and filgrastim in advanced breast cancer patients.

PURPOSE: As an alternative to single-cycle cyclophosphamide, thiotepa, and carboplatin (CTCb) intensification, we evaluated the feasibility of administering one-quarter dose CTCb for four cycles with peripheral-blood progenitor-cell (PBPC) and filgrastim (granulocyte colony-stimulating factor [G-CSF]) in advanced-stage breast cancer patients. PATIENTS AND METHODS: From June 1992 to August 1993, 20 stage IIIB (n = 7) and IV (n = 13) breast cancer patients received 78 cycles of induction with doxorubicin 90 mg/m2 by intravenous (IV) bolus with G-CSF 5 microg/kg/d by subcutaneous injection (SC) repeated every 14 to 21 days for four cycles. PBPC were collected by 2-hour single-blood volume leukapheresis on 2 consecutive days at the time of hematologic recovery from each cycle of doxorubicin. Eighteen patients received 61 cycles of intensification with cyclophosphamide 1,500 mg/m2, thiotepa 125 mg/m2, and carboplatin 200 mg/m2 by IV continuous infusion with G-CSF 10 microg/kg/d SC and PBPC support repeated every 21 to 42 days for four cycles. RESULTS: Twelve of 20 patients (60%) completed all four planned cycles of doxorubicin induction followed by four cycles of one-quarter dose CTCb intensification. Statistically significantly decreases in the yield of mononuclear cells (MNC) (median slope per day, -0.032; P = .03), granulocyte-macrophage colony-forming unit (CFU-GM) (median slope per day, -0.57; P = .0008), and burst-forming unit-erythroid (BFU-E) (median slope per day, -1.18; P = .006) were observed over the course of the eight leukaphereses. Of 18 patients who began CTCb, 12 (67%) completed four cycles. Six patients were removed from study during intensification: two for progressive disease (PD), one refused further treatment, and three for dose-limiting hematologic toxicity. A fourth patient fulfilled the criteria for dose-limiting hematologic toxicity after cycle 4. The toxicity of the multiple cycle CTCb intensification regimen consisted of grade IV leukopenia, grade IV thrombocytopenia, and febrile neutropenia in 100%, 100%, and 26% of cycles, respectively. The median duration of each CTCb cycle was 24 days (range, 18 to 63), and the median duration of an absolute neutrophil count (ANC) < or = 500/microL and platelet count < or = 20,000/microL during each cycle was 6 days (range, 2 to 15) and 4 days (range, 0 to 38), respectively. CONCLUSION: It is feasible to administer repetitive cycles of one-quarter dose CTCb intensification with PBPC and G-CSF. Additional studies are required to determine whether multiple cycles of CTCb intensification might offer a therapeutic advantage over a single high-dose cycle.

Adult↗
PubMed1996

Crystal structures of 8-Cl and 9-Cl TIBO complexed with wild-type HIV-1 RT and 8-Cl TIBO complexed with the Tyr181Cys HIV-1 RT drug-resistant mutant.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is an important target for chemotherapeutic agents used in the treatment of AIDS; the TIBO compounds are potent non-nucleoside inhibitors of HIV-1 RT (NNRTIs). Crystal structures of HIV-1 RT complexed with 8-Cl TIBO (R86183, IC50 = 4.6 nM) and 9-Cl TIBO (R82913, IC50 = 33 nM) have been determined at 3.0 A resolution. Mutant HIV-1 RT, containing Cys in place of Tyr at position 181 (Tyrl81Cys), is highly resistant to many NNRTIs and HIV-1 variants containing this mutation have been selected in both cell culture and clinical trials. We also report the crystal structure of Tyrl81Cys HIV-1 RT in complex with 8-Cl TIBO (IC50 = 130 nM) determined at 3.2 A resolution. Averaging of the electron density maps computed for different HIV-1 RT/NNRTI complexes and from diffraction datasets obtained using a synchrotron source from frozen (-165 degrees C) and cooled (-10 degrees C) crystals of the same complex was employed to improve the quality of electron density maps and to reduce model bias. The overall locations and conformations of the bound inhibitors in the complexes containing wild-type HIV-1 RT and the two TIBO inhibitors are very similar, as are the overall shapes and volumes of the non-nucleoside inhibitor-binding pocket (NNIBP). The major differences between the two wild-type HIV-1 RT/TIBO complexes occur in the vicinity of the TIBO chlorine substituents and involve the polypeptide segments around the beta5-beta6 connecting loop (residues 95 to 105) and the beta13-beta14 hairpin (residues 235 and 236). In all known structures of HIV-1 RT/NNRTI complexes, including these two, the position of the beta12-beta13 hairpin or the "primer grip" is significantly displaced relative to the position in the structure of HIV-1 RT complexed with a double-stranded DNA and in unliganded HIV-1 RT structures. Since the primer grip helps to position the template-primer, this displacement suggests that binding of NNRTIs would affect the relative positions of the primer terminus and the polymerase active site. This could explain biochemical data showing that NNRTI binding to HIV-1 RT reduces efficiency of the chemical step of DNA polymerization, but does not prevent binding of either dNTPs or DNA. When the structure of the Tyr181Cys mutant HIV-1 RT in complex with 8-Cl TIBO is compared with the corresponding structure containing wild-type HIV-1 RT, the overall conformations of Tyr181Cys and wild-type HIV-1 RT and of the 8-Cl TIBO inhibitors are very similar. Some positional changes in the polypeptide backbone of the beta6-beta10-beta9 sheet containing residue 181 are observed when the Tyr181Cys and wild-type complexes are compared, particularlty near residue Val179 of beta9. In the p51 subunit, the Cys181 side-chain is oriented in a similar direction to the Tyr181 side-chain in the wild-type complex. However, the electron density corresponding to the sulfur of the Cys181 side-chain in the p66 subunit is very weak, indicating that the thiol group is disordered, presumably because there is no significant interaction with either 8-Cl TIBO or nearby amino acid residues. In the mutant complex, there are slight rearrangements of the side-chains of other amino acid residues in the NNIBP and of the flexible dimethylallyl group of 8-Cl TIBO; these conformational changes could potentially compensate for the interactions that were lost when the relatively large tyrosine at position 181 was replaced by a less bulky cysteine residue. In the corresponding wild-type complex, Tyr181 iin the p66 subunit has significant interactions with the bound inhibitor and the position of the Tyr181 side-chain is well defined in both subunits. Apparently the Tyr181 --> Cys mutation eliminates favorable contacts of the aromatic ring of the tyrosine and the bou

Antiviral Agents↗
PubMed1996

EPR spectroscopic characterization of neuronal NO synthase.

Neuronal NO synthase (nNOS) consists of a reductase domain that binds FAD, FMN, NADPH, and calmodulin, and an oxygenase domain that binds heme, tetrahydrobiopterin, and the substrate L-arginine. One flavin in resting nNOS exits as an air-stable semiquinone radical. During NO synthesis, electron transfer occurs between the flavins and heme iron. We have characterized the nNOS heme iron and flavin semiquinone radical by electron paramagnetic resonance (EPR) spectroscopy. Under anaerobic conditions, the flavin radical spin relaxation was very slow (8 HZ at 22 K) and was enhanced 13-fold by dissolved dioxygen via spin-spin coupling. The flavin radical, probably the semiquinone FMNH., was shown by progressive microwave power saturation and EPR saturation recovery under anaerobic conditions to be spin-spin coupled with the heme iron located in the nNOS oxygenase domain. Analysis of an nNOS preparation that was devoid of heme but contained the flavin radical revealed that spin-spin coupling increased the rate of flavin radical relaxation by a factor of 15. The presence of bound substrate (L-arginine) or the substate analogue Nomega-nitro-L-arginine methyl ester (NAME) had no effect on the flavin spin relaxation kinetics. The observed g values of the nNOS heme were 7.68, and 1.81 and were unchanged by occupation of the substrate binding site by L-arginine or NAME. The substrate also had no effect on the heme zero-field splitting parameter, D=5.2cm-1. Together, the data indicate that the flavin and heme redox centers are positioned near each other in nNOS, consistent with their participating in an interdomain electron transfer. The flavin radical is affected by dissolved oxygen, suggesting that its binding site within the reductase domain partially exposed to solvent, but is unaffected when substrate binds to the oxygenase domain. Substrate binding also appears to take place outside the first coordination shell of the nNOS heme iron.

Animals↗
PubMed1996

Analysis of proprioception in the posterior cruciate ligament-deficient knee.

The purpose of this study was to demonstrate loss of proprioception with posterior cruciate ligament (PCL) deficiency. Eight patients with isolated PCL-deficient knees were examined to determine whether or not the proprioceptive function of the knee joint was affected or impaired by the previous tear of the PCL. The patients were examined for perception of passive movement of the knee joint using a motorized apparatus which extended or flexed the patient's knee at a rate of 0.5 deg/s in a randomized sequence. The patient's normal contralateral knee was used as a control. This study demonstrated a significant difference in proprioception in the patient's PCL-deficient knee. All eight PCL-deficient knees clearly demonstrated slower perception of passive movement when compared with the contralateral normal knee. Injuries to the PCL resulting in impaired proprioception may play a role in knee instability, further damage and ultimately the degenerative changes frequently seen in the long-term follow-up of PCL-deficient knees.

Adult↗
PubMed1996

Risk factors for neonatal sepsis.

OBJECTIVE: To determine the associations between maternal characteristics, intrapartum events, and neonatal sepsis by multivariate analysis. METHODS: We enrolled 823 women from a high-risk population and analyzed maternal and neonatal demographic and outcome variables with univariate analysis and multivariate logistic modeling. RESULTS: Two-hundred sixteen women (26%) were colonized with group B streptococci, 82 (10%) developed chorioamnionitis, and 141 (17%) delivered prematurely. Culture-proven neonatal sepsis or meningitis was found in 15 of 833 (1.8%) neonates, and 101 of the remaining 818 (12.3%) infants were suspected to have sepsis or pneumonia. Multivariate analysis of risk factors for proven neonatal sepsis demonstrated a statistically significant association with decreasing gestational age, duration of internal monitoring for more than 12 hours (odds ratio [OR] 7.2, 95% confidence interval [CI] 1.6-32.2), maternal group B streptococcal infection (OR 4.2, 95% CI 1.4-13.1), chorioamnionitis (OR 4.4, 95% CI 1.2-16.1), and endometritis (OR 6.4, 95% CI 1.2-34.2). CONCLUSION: Through the use of multivariate modeling, we determined that chorioamnionitis or endometritis, preterm delivery, group B streptococcal colonization, and a prolonged duration of internal monitoring are independent risk factors for neonatal sepsis. We postulate that the presence of a foreign body that traverses the birth canal may facilitate ascending peripartal infection.

Confidence Intervals↗
PubMed1996

Patterns of dissociation in clinical and nonclinical samples.

Research has consistently found elevated mean dissociation scores in particular diagnostic groups. In this study, we explored whether mean dissociation scores for different diagnostic groups resulted from uniform distributions of scores within the group or were a function of the proportion of highly dissociative patients that the diagnostic group contained. A total of 1566 subjects who were psychiatric patients, neurological patients, normal adolescents, or normal adult subjects completed the Dissociative Experience Scale (DES). An analysis of the percentage of subjects with high DES scores in each diagnostic group indicated that the diagnostic group's mean DES scores were a function of the proportion of subjects within the group who were high dissociators. The results contradict a continuum model of dissociation but are consistent with the existence of distinct dissociative types.

Adolescent↗
PubMed1996

Rib fractures in major trauma.

BACKGROUND: To determine the mortality, hospital and intensive care unit (ICU) stay of rib fractures in patients admitted to Victorian hospitals for more than 1 day. METHODS: All patients fitting the entry criteria for the Victorian Major Trauma Study with fractured ribs were identified between 1 March 1992 and 28 February 1993. Aetiology, age, sex, associated injury and outcome were analysed. RESULTS: Patients with rib fractures had a higher mortality and length of hospital stay, but this was not significantly different from other trauma. A significantly higher percentage of patients required ICU care for rib fractures (44%) compared with the total group with blunt injury (24%). The majority of rib fractures resulted from motor vehicle accidents 361/541 (67%). Injuries occurring on the street/highway resulting in rib fractures were more likely to be major; 62% had Injury Severity Score (ISS) > 15. Fractured ribs occurred more commonly with increased age. Mortality for patients with fractured ribs versus total trauma group was higher in elderly patients. Univariate analysis showed rib fractures were a positive predictor of death but when adjusted for ISS and age, rib fractures became a negative predictor. Rib fractures were not predictors for length of ICU or hospital stay. CONCLUSION: The sample of rib fractures collected in this study underestimates the overall incidence. For those patients admitted to hospital with identified rib fractures, there is a trend towards higher mortality and morbidity. However, this association is better predicted by ISS and age.

Adolescent↗
PubMed1995

Absent pituitary gland in two brothers with an oral-facial-digital syndrome resembling OFDS II and VI: a new type of OFDS?

The oral-facial-digital syndromes (OFDS) comprise a group of heterogeneous genetic disorders. Considerable clinical overlap exists within the nine described types [Toriello, Clin Dysmorph 2:95-105, 1993], and with other entities such as Pallister-Hall (PH) syndrome and hydrolethalus syndrome, leading to difficulties in the classification of OFDS. We report on two brothers with findings overlapping OFDS II, VI, and Pallister-Hall syndrome who had congenital absence of the pituitary gland. This may represent a new type of OFDS or, alternatively, an example of phenotypic variability within the OFDS. It also emphasizes that agenesis of the pituitary gland can occur in a variety of syndromes with midline defects.

Face↗