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PubMed1994

Autophosphorylation modulates the kinase activity and oncogenic potential of the Met receptor tyrosine kinase.

The met oncogene is activated by a genomic rearrangement that generates a hybrid protein containing tpr sequences at its amino terminus fused directly to the met (hepatocyte growth factor/scatter factor) receptor tyrosine kinase domain. The resultant p65Tpr-Met hybrid protein possesses tyrosine kinase activity and is constitutively phosphorylated on tyrosine in vivo. A substitution of the conserved lysine in the catalytic domain abolished kinase activity and the transforming potential of Tpr-Met demonstrating the requirement for kinase activity for transformation. To study the role of autophosphorylation at particular tyrosine residues on the transforming activity of Tpr-Met, the major autophosphorylation sites were identified by two dimensional phosphopeptide mapping. Two tyrosine residues in the catalytic domain, tyrosine 365 and tyrosine 366, were found to be the major autophosphorylation sites both in vitro and in vivo. Mutation of these sites singly or together modulates the biochemical and biological properties of Tpr-Met. Compared with the wild-type Tpr-Met, mutants have a reduced in vitro kinase activity when measured by levels of autophosphorylation and by their ability to phosphorylate an exogenous substrate and have little or no transforming activity. These results indicate that autophosphorylation at critical tyrosine residues is required for activation of the kinase activity of Tpr-Met and for cell transformation by this oncogene.

3T3 Cells↗

PubMedDate not supplied

Hemodynamic effects of acute tetrandrine and terlipressin administration on portal hypertensive rats.

The purpose of this study was to investigate the therapeutic effects of tetrandrine and terlipressin, alone or in combination, on anesthetized portal hypertensive rats. Portal hypertension was induced by partial portal vein ligation in Sprague-Dawley rats. Each portal hypertensive rat received one of three regimens: vehicle plus terlipressin, tetrandrine (.50 mg.kg-1.min-1) [corrected] plus terlipressin, or tetrandrine (.75 mg.kg-1.min-1) [corrected] plus terlipressin. Terlipressin dosage was 0.025 mg.kg-1.min-1 [corrected] infused for 3 min. Infusion of vehicle followed by terlipressin induced significant reduction of portal venous pressure (PVP, -24.3 +/- 1.5%) and prominent elevation of mean arterial pressure (MAP, 73.3 +/- 4.6%) as well as total peripheral resistance (TPR, 133 +/- 8%) from baseline, and there was a cardiodepressant response (cardiac index, CI, -30.6 +/- 3.8%; heart rate, HR, -10.4 +/- 2.1%). Infusion of tetrandrine (.50 mg.kg-1.min-1) [corrected] induced significant reduction of PVP (-7.2 +/- 0.9%), MAP (-12.5 +/- 1.6%), and TPR (-14.3 +/- 4.2%) from baseline. Infusions of tetrandrine followed by terlipressin induced significant reduction of PVP (-14.2 +/- 2.4%) but an increase in MAP (22.6 +/- 2.7%) and TPR (71.1 +/- 10.5%). Infusion of tetrandrine (.75 mg.kg-1.min-1) [corrected] induced significant reduction of PVP (-9.9 +/- 0.8%). MAP (-19.2 +/- 2.1%), and TPR (-24.1 +/- 2.1%) from baseline. Infusions of tetrandrine followed by terlipressin induced significant reduction of PVP (-14.0 +/- 1.1%) but an increase in MAP (14.0 +/- 2.7%) and TPR (75.2 +/- 5.2%). Compared with vehicle followed by terlipressin, tetrandrine significantly attenuated systemic pressor (MAP and TPR elevation), cardiodepressant (CI reduction) as well as portal hypotensive (PVP reduction) effects of terlipressin. Our results suggest that tetrandrine and terlipressin, alone or in combination, induced portal hypotensive effects in portal hypertensive rats. Combined tetrandrine and terlipressin administration ameliorated systemic pressor and cardiodepressant effects of terlipressin, but it also attenuated the latter's portal hypotensive effects.

Alkaloids↗

PubMed2001

Structural characterization and intramolecular aliphatic C-H oxidation ability of M(III)(mu-O)2M(III) complexes of Ni and Co with the hydrotris-(3,5-dialkyl-4-X-pyrazolyl)borate ligands TpMe2,X (X = Me, H, Br) and TpiPr2.

Reaction of the dinuclear M(II)-bis(mu-hydroxo) complexes of nickel and cobalt, [(M(II)(TpR)]2(mu-OH)2] (M = Ni; 3Ni M = Co: 3Co), with one equivalent of H2O2 yields the corresponding M(III)-bis(mu-oxo) complexes, [[M(III)(TpR)]2-(mu-O)2] (M=Ni; 2Ni, M=Co: 2Co). The employment of a series of TpMe2,X (TpMe2,X = hydrotris(3,5-dimethyl-4-X-1-pyrazolyl)borate; X = Me, H, Br) as a metal supporting ligand makes it possible to isolate and structurally characterize the thermally unstable M(III)-bis-(mu-oxo) complexes 2Ni and 2Co. Both the starting (3Ni and 3Co) and resulting complexes (2Ni and 2Co) contain five-coordinate metal centers with a slightly distorted square-pyramidal geometry. Characteristic features of the nickel complexes 2Ni, such as the two intense absorptions around 400 and 300 nm in the UV-visible spectra and the apparent diamagnetism, are very similar to those of the previously reported bis(mu-oxo) species of Cu(III) and Ni(III) with ligands other than TpR, whereas the spectroscopic properties of the cobalt complexes 2Co (i.e., paramagnetically shifted NMR signals and a single intense absorption appearing at 350 nm) are clearly distinct from those of the isostructural nickel compounds 2Ni. Thermal decomposition of 2Ni and 2Co results in oxidation of the inner saturated hydrocarbyl substituents of the TpR ligand. Large kH/kD values obtained from the first-order decomposition rates of the TpMe3 and Tp(CD3)2,Me derivatives of 2 evidently indicate that the rate-determining step is an hydrogen abstraction from the primary C-H bond of the methyl substituents. mediated by the M(III)2-(mu-O)2 species. The nickel complex 2Ni shows reactivity about 10(3) times greater than that of the cobalt analogue 2Co. The oxidation ability of the M(III)(mu-O)2M(III) core should be affected by the hindered TpR ligand system, which can stabilize the +2 oxidation state of the metal centers.

Cobalt↗

PubMed1993

Combining independent studies of a diagnostic test into a summary ROC curve: data-analytic approaches and some additional considerations.

We consider how to combine several independent studies of the same diagnostic test, where each study reports an estimated false positive rate (FPR) and an estimated true positive rate (TPR). We propose constructing a summary receiver operating characteristic (ROC) curve by the following steps. (i) Convert each FPR to its logistic transform U and each TPR to its logistic transform V after increasing each observed frequency by adding 1/2. (ii) For each study calculate D = V - U, which is the log odds ratio of TPR and FPR, and S = V + U, an implied function of test threshold; then plot each study's point (Si, Di). (iii) Fit a robust-resistant regression line to these points (or an equally weighted least-squares regression line), with V - U as the dependent variable. (iv) Back-transform the line to ROC space. To avoid model-dependent extrapolation from irrelevant regions of ROC space we propose defining a priori a value of FPR so large that the test simply would not be used at that FPR, and a value of TPR so low that the test would not be used at that TPR. Then (a) only data points lying in the thus defined north-west rectangle of the unit square are used in the data analysis, and (b) the estimated summary ROC is depicted only within that subregion of the unit square. We illustrate the methods using simulated and real data sets, and we point to ways of comparing different tests and of taking into account the effects of covariates.

Carcinoma, Non-Small-Cell Lung↗

PubMed1976

Regional distribution of vascular resistance in two models of experimental renovascular hypertension.

The regional distribution of the peripheral vascular resistance was studied in normotensive and hypertensive Wistar rats. Two models of experimental hypertension were investigated: (I) in 32 animals the right renal artery was constricted by a silver clip (two-kidney Goldblatt hypertension); (II) in 46 animals the left kidney was removed and the right renal artery was clipped as in the first group (one-kidney Goldblatt hypertension). The normotensive control group comprised 61 untreated animals of the same strain and age. The distribution of cardiac output to 14 tissues was determined by means of the particle distribution technique. The resistance was increased in all regions investigated, a decreased or unchanged resistance was not observed. For most of the investigated tissues the regional resistance was increased exactly in proportion to the total peripheral resistance (TPR). Exceptions to this were found in 2 regions where the change of local resistance deviated from that of TPR: the splanchnic area and the skeletal muscle. In both cases the 2 models differed from each other. In the two-kidney model the increase of resistance in the splanchnic circulation was more intense than in other organs. In contrast, in the one-kidney model the local change of resistance was less than that of TPR. The change of skeletal muscle resistance was not significantly different from the change of TPR in the two-kidney model, while in the one-kidney model the increase of local resistance was significantly higher than that of TPR. It is concluded that the etiology of the abnormal resistance is different in the 2 models investigated and that known extrinsinc pressor factors may play a role in the two-kidney, but not in the one-kidney Goldblatt hypertension.

Abdomen↗

PubMed1986

Pressure-flow relationships of the canine iliac periphery and systemic hemodynamics: effects of sodiumnitroprusside and adenosine.

It has been reported that sodiumnitroprusside (SNP) decreases mean systemic pressure and simultaneously increases pressure pulse amplification towards the iliac periphery (Kenner and van Zwieten 1982). This unexpected finding was suggested to be due to a decrease in iliac peripheral resistance but an increase in iliac differential resistance. In order to investigate this apparent contradiction, the iliac periphery was hemodynamically isolated from the rest of the circulation and perfused with the dog's own blood by means of a pump. Perfusion pressure (P) and flow (F), femoral venous pressure (Pv), systemic pressure (Ps) and cardiac output (CO) were measured. Steady state pressure-flow relations of the isolated bed were obtained during control and during various i.v. infusion rates of SNP and adenosine (ADS) and were found to be straight (mean r = 0.99). Their slope (delta P/delta F) was defined as differential resistance (Rd). Peripheral resistance (Rp) of the iliac bed was defined as Rp = (P-Pv)/F, calculated at the flow value where perfusion pressure equalled the prevailing systemic pressure. Total peripheral resistance (TPR) was defined as TPR = Ps/CO. The changes of Rd, Rp, Ps, CO and TPR with respect to control show that during low SNP infusion rates Rd and Rp were both increased while TPR was decreased. During all infusion rates of SNP CO did not change while Ps decreased. During low infusion rates of adenosine CO increased while Ps, Rd and Rp did not change and TPR decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗

PubMed1998

Pressor and vasoconstrictor effects of methylene blue in endotoxaemic rats.

Nitric oxide (NO) is a primary mediator of hypotension in sepsis. We examined the effects of methylene blue (MB), an inhibitor of the NO/cGMP pathway, on mean arterial pressure (MAP), cardiac output (CO), total peripheral resistance (TPR), mesenteric blood flow (MBF) and renal blood flow (RBF) in pentobarbitone-anaesthetised rats injected with lipopolysaccharide (LPS, 7.5 mg/kg). MB (1, 3 or 10 mg/kg x h) or vehicle was i.v. infused into four groups at 2.5 h after i.v. injection of LPS. Two other groups received MB or vehicle at 2.5 h after receiving saline. LPS reduced MAP, CO, RBF as well as MBF at 2.5 and 4 h, and increased TPR at 2.5 but not 4 h. Whereas MB alone had no effects on measured variables in control rats at 4 h, in LPS-treated rats, it elevated TPR at all doses and attenuated the fall in MAP at the two low doses. CO was unaltered by low doses of MB but reduced by the high dose. MBF was unaltered, but RBF was increased by the lowest dose but decreased by the highest dose of MB. Therefore, in endotoxaemia, a low dose of MB increases MAP and TPR but does not alter CO; a high dose of MB does not raise MAP but increases TPR and reduces CO.

Animals↗

PubMed2001

Transient pleural effusion in norepinephrine-stimulated rats.

Transient pleural effusions occurred in rats receiving continuous intravenous infusion of norepinephrine (NE, 0.1 mg/kg/h). We hypothesized that these pleural effusions result from a NE-induced increase in right ventricular systolic pressure (RVSP) and total peripheral resistance (TPR). NE was administered over time intervals between 20 min and 72 h. It induced an immediate doubling in RVSP whereas LVSP remained at the control level. TPR increased with a delay of 6 h. At this time, pleural effusions occurred in NE-treated animals, reached their maximum after 8h and disappeared after 24 h of NE stimulation. Combining NE with the alpha-blocker prazosin normalized TPR and prevented pleural effusions. Therefore, we interpret the pleural effusion as a consequence of pulmonary venous congestion, mainly caused by an increased TPR. LV hypertrophy which developed after 24 h of NE stimulation is considered to compensate for the hemodynamic disturbance due to the NE-induced elevation in TPR. This is reflected in the disappearance of pleural effusion.

Animals↗

PubMed2001

The tetratricopeptide repeat domains of human, tobacco, and nematode PEX5 proteins are functionally interchangeable with the analogous native domain for peroxisomal import of PTS1-terminated proteins in yeast.

In the yeast Saccharomyces cerevisiae, beta-oxidation of fatty acids is compartmentalised in peroxisomes. Most yeast peroxisomal matrix proteins contain a type 1C-terminal peroxisomal targeting signal (PTS1) consisting of the tripeptide SKL or a conservative variant thereof. PTS1-terminated proteins are imported by Pex5p, which interacts with the targeting signal via a tetratricopeptide repeat (TPR) domain. Yeast cells devoid of Pex5p are unable to import PTS1-containing proteins and cannot degrade fatty acids. Here, the PEX5-TPR domains from human, tobacco, and nematode were inserted into a TPR-less yeast Pex5p construct to generate Pex5p chimaeras. These hybrid proteins were examined for functional complementation of the pex5delta mutant phenotype. Expression of the Pex5p chimaeras in pex5delta mutant cells restored peroxisomal import of PTS1-terminated proteins. Chimaera expression also re-established degradation of oleic acid, allowing growth on this fatty acid as a sole carbon source. We conclude that, in the context of Pex5p chimaeras, the human, tobacco, and nematode Pex5p-TPR domains are functionally interchangeable with the native domain for the peroxisomal import of yeast proteins terminating with canonical PTS1s. Non-conserved yeast PTS1s, such as HRL and HKL, did not interact with the tobacco PEX5-TPR domain in the two-hybrid system. HRL occurs at the C-terminus of the peroxisomal protein Eci1p, which is required for growth on unsaturated fatty acids. Although mutant pex5delta cells expressing a yeast/tobacco Pex5p chimaera failed to import a GFP-Eci1p reporter protein, they were able to grow on oleic acid. We reason that this is due to a cryptic PTS in native Eci1p that can function in a redundant system with the C-terminal HRL.

Animals↗

PubMed2000

The isolation and characterization of missense mutants in the general repressor protein Ssn6 of Saccharomyces cerevisiae.

Ssn6, a TPR repeat-containing protein, associates with the Tup1 protein to form a general transcriptional repression complex in Saccharomyces cerevisiae. As part of a genetic analysis of this complex, we targeted mutations to the TPR repeat-coding region of the SSN6 gene, and applied selection for constitutive expression of the hypoxic gene ANB1. All but one of the resulting mutants failed to express full-length Ssn6 protein, indicating that they harbored deletion, frameshift, or nonsense mutations. The one missense mutation encoded a protein with three amino acid substitutions, and the combination was required for the mutant phenotype. One mutation, a proline substitution for a serine at codon 51, was used in a second round of mutagenesis in which six further multiple-substitution alleles were obtained. These were separated into their component mutations, and again, all but one of the single substitutions displayed the wild-type phenotype. The single and multiple mutants were characterized in terms of their effects on the repression of the glucose-repressible SUC2 gene and the a mating-type gene STE2. The mutant Ssn6 proteins were also tested for their ability to associate with Tup1. The S5 P mutation, despite its lack of a mutant phenotype, had lost the ability to fully associate with Tupl in vitro. In general, those single substitutions that fell within the first two TPR repeats impaired Tup1-associating activity, while the two that fell in TPR repeats five and eight retained this activity. Overexpression of TUP1 partially suppressed the mutant phenotype in only some of the multiple mutants. The results are discussed in terms of the current models of Ssn6 function and the structure of TPR repeats.

Amino Acid Sequence↗

PubMed2005

Blood pressure and total peripheral resistance in children with chronic kidney disease.

We performed a study to assess cardiac output (CO) and total peripheral resistance (TPR) at rest and during peak exercise with the goal to better define the role of these parameters in the development of hypertension in children with chronic kidney disease (CKD) stage 2-4. Fifty-two pediatric patients with CKD (mean age 12.7+/-3.7 years) and 28 healthy individuals of comparable age and sex participated in the study. At rest, children with CKD had a significantly higher systolic and diastolic blood pressure (BP) and calculated mean arterial pressure (MAP) than healthy controls. Total peripheral resistance was significantly higher in children with CKD than in controls (1627.7+/-534.6 vs 1354.6+/-338.9 dynexsxcm(-5), p =0.02). There was no significant difference in heart rate or CO between the two groups. Children taking antihypertensive medications had lower TPR than children without BP medications (1514.6+/-439.6 vs 1788.2+/-505.4 dynexsxcm(-5), respectively, p =0.06). At peak exercise, children with CKD had a significant increase in MAP, heart rate and CO and had a significant decrease in TPR (difference between rest and peak exercise: -782.4+/-375.9 dynexsxcm(-5), p <0.001). Children taking BP medications had blunted MAP and CO responses when compared to controls (Delta CO: 6.2+/-2.8 l/min vs 9.8+/-4.5 l/min, respectively, p =0.01; MAP: 13.9+/-10.2 mmHg vs 21.5+/-11.7 mmHg, respectively, p =0.01). Children without BP medications had a similar to controls response to exercise in respect to CO, MAP and TPR. We conclude that increased TPR is a major contributor to elevated blood pressure in children with CKD and suggest that BP medications decreasing vascular resistance should be used as a first line of antihypertensive therapy in these patients.

Adolescent↗

PubMed1993

The pharmacokinetics and pharmacodynamics of the prostacyclin analog 15AU81 in the anesthetized beagle dog.

15AU81, a tricyclic benzindene analog of prostacyclin, is currently under preclinical evaluation as a potential treatment for congestive heart failure. The cardiovascular effects of 15AU81 were evaluated in anesthetized beagle dogs given 4-h infusions at rates of either 0.1, 0.3, 1.0, or 3.0 micrograms/kg/min. Plasma samples taken from these dogs prior to, during, and after the infusion, were analyzed for 15AU81 by a radioimmunoassay (RIA). This report integrates the vasodilatory effects and plasma concentration data from the 15AU81 infusion study. Pharmacokinetic analysis of mean data indicated a biphasic decay of 15AU81 in plasma, with an initial half-life of approximately 2 min, and a terminal half-life of approximately 20 min. Visual inspection of plots of drug effect and drug concentration against time indicated a close relationship between plasma concentration of 15AU81 and the onset of decreases in total peripheral resistance (TPR) and pulmonary vascular resistance (PVR). In general, the decreases in TPR and PVR induced by 15AU81 were maintained during infusion. Concentration-effect plots indicated some hysteresis in TPR vs plasma concentrations of 15AU81 after termination of the infusion; possible explanations for this hysteresis include the presence of saturating concentrations of 15AU81 at the effect site, with a delay in the clearance of 15AU81 from the effect site compared to its clearance from plasma, and/or the presence of active metabolites at the effect site. A fit of the TPR and PVR data to the Emax pharmacodynamic model predicted that the maximum decrease in TPR achievable with 15AU81 in anesthetized dogs was 66%, and that the concentration of 15AU81 producing 50% of the maximum effect (EC50) was 8.6 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2004

Sociotropic cognition moderates stress-induced cardiovascular responsiveness in women through effects on total peripheral resistance, but not cardiac output.

Although cardiovascular disease (CVD) remains the leading cause of mortality in women, few studies have examined the role of psychosocial factors in its development. This study examined the moderating effects of sociotropic cognition (SC), a need for social acceptance and approval, on psychosocial stress-induced cardiovascular responsiveness (CVR) and affect reactivity in women. Sixty-eight normotensive, college-aged females were randomly assigned to a low or high social threat condition. Measures of systolic, diastolic and mean arterial blood pressures (SBP, DBP and MAP, respectively), heart rate (HR), cardiac output (CO), total peripheral resistance (TPR) and negative affect were collected during rest, and under conditions of high vs. low interpersonal threat. A two-step hierarchical regression analysis was performed to predict all response variables (BPs, HR, CO, TPR and affect). Increases in SBP, DBP, MAP, TPR and negative affect were greater in the high threat than low threat condition. Changes in SBP, MAP and TPR positively covaried with SC under conditions of high interpersonal threat, but showed no significant covariation in the low threat condition. The data suggest that an excessive need for social acceptance may contribute to rises in BP through an increase in TPR, but not CO under conditions of high social threat.

Adolescent↗

PubMed1998

Combined effects of angiotensin converting enzyme inhibition and angiotensin II receptor antagonism in conscious pigs with congestive heart failure.

OBJECTIVE: The goal of this study was to determine if the hemodynamic effects of the combined administration of an angiotensin converting enzyme (ACE) inhibitor and angiotensin II type 1 (AT1) receptor antagonist are greater than those produced by either of these agents administered individually during heart failure. METHODS: Ten farm pigs were chronically instrumented with aortic, left atrial and right atrial catheters, a left ventricular (LV) pressure gauge, LV dimension crystals, coronary occluders, an ascending aortic flow probe and pacing leads. Heart failure was induced by serial myocardial infarctions followed by repeated rapid ventricular pacing. RESULTS: Heart failure was manifested by significant (p < 0.01) decreases in LV dP/dt (-38 +/- 5%, from 2943 +/- 107 mmHg/s) and cardiac output (-27 +/- 4%, from 4.1 +/- 0.2 l/min) and increases in left atrial pressure (+18 +/- 1 mmHg, from 4 +/- 1 mmHg) and total peripheral resistance (TPR)(+40 +/- 8%, from 23 +/- 2 mmHg/l/min). The effects of an ACE inhibitor (enalaprilat) and an AT1 receptor antagonist (L-158,809), administered in maximally effective doses, either individually or concomitantly, were examined on different days in conscious pigs with heart failure. There were no differences in any of the baseline hemodynamic measurements among the groups studied. Thirty minutes after administration, enalaprilat (4 mg/kg i.v.) increased (p < 0.05) cardiac output by 8 +/- 2% and reduced (p < 0.05) mean arterial pressure and TPR by 5 +/- 1 and 12 +/- 1%, respectively, while the changes in LV dP/dt (0 +/- 2%), LV fractional shortening (+4 +/- 3%) and heart rate (+1 +/- 1%) were not statistically significant. Similarly, L-158,809 (4 mg/kg, i.v.) increased cardiac output by 9 +/- 2% and reduced mean arterial pressure and TPR by 4 +/- 1 and 11 +/- 3%, respectively, while the changes in LV dP/dt (+3 +/- 3%), LV fractional shortening (+3 +/- 1%) and heart rate (0 +/- 1%) were not significant. However, enalaprilat (1 mg/kg, i.v.) and L-158,809 (1 mg/kg, i.v.), administered concomitantly, reduced TPR by 21 +/- 3%, an effect greater (p < 0.05) than when either of these agents was administered individually at a dose of 4 mg/kg, i.v. The changes in mean arterial pressure (-9 +/- 2%), cardiac output (+15 +/- 4%) and LV fractional shortening (+11 +/- 3%) also tended to be greater with concomitant administration. In addition, in a sequential dosing protocol, when L-158,809 (1 mg/kg, i.v.) was administered 30 min after enalaprilat (1 mg/kg, i.v.), TPR was reduced by 20 +/- 4% compared to only a 6 +/- 3% reduction (p < 0.05) when the enalaprilat was followed 30 min later by a second dose of enalaprilat (1 mg/kg, i.v.). The changes in mean arterial pressure and cardiac output for the combined treatment group also tended to be greater than those for the group given two sequential doses of enalaprilat. CONCLUSION: In conscious pigs with heart failure, the combined vasodilatory effects of an ACE inhibitor and AT1 receptor antagonist are greater than those produced when only one of these agents is administered, suggesting that independent mechanisms of ACE inhibition and AT1 receptor antagonism could be partly responsible for the improved vascular dynamics during heart failure.

Analysis of Variance↗

PubMed2003

A conserved insertion in protein-primed DNA polymerases is involved in primer terminus stabilisation.

Protein-primed DNA polymerases form a subgroup of the eukaryotic-type DNA polymerases family, also called family B or alpha-like. A multiple amino acid sequence alignment of this subgroup of DNA polymerases led to the identification of two insertions, TPR-1 and TPR-2, in the polymerisation domain. We showed previously that Asp332 of the TPR-1 insertion of phi29 DNA polymerase is involved in the correct orientation of the terminal protein (TP) for the initiation of replication. In this work, the functional role of two other conserved residues from TPR-1, Lys305 and Tyr315, has been analysed. The four mutant derivatives constructed, K305I, K305R, Y315A and Y315F, displayed a wild-type 3'-5' exonuclease activity on single-stranded DNA. However, when assayed on double-stranded DNA such activity was higher than that of the wild-type enzyme. This activity led to a reduced pol/exo ratio, suggesting a defect in stabilising the primer terminus at the polymerase active site. On the other hand, although mutant polymerases K305I and Y315A were able to couple processive DNA polymerisation to strand displacement, they were severely impaired in phi29 TP-DNA replication. The possible role of the TPR-1 insertion in the set of interactions with the nascent chain during the first steps of TP-DNA replication is discussed.

Amino Acid Sequence↗

PubMed1999

Chronic hypertension in ANP knockout mice: contribution of peripheral resistance.

Atrial Natriuretic Peptide (ANP) exerts a chronic hypotensive effect which is mediated by a reduction in total peripheral resistance (TPR). Mice with a homozygous disruption of the pro-ANP gene (-/-) fail to synthesize ANP and develop chronic hypertension in comparison to their normotensive wild-type (+/+) siblings. In order to determine whether alterations in basal hemodynamics underlie the hypertension associated with lack of endogenous ANP activity, we used anesthetized mice to measure arterial blood pressure (ABP) and heart rate (HR), as well as cardiac output (CO) by thermodilution technique. -/- (n = 7) and +/+ (n = 10) mice of comparable weight and age were used. Stroke volume (SV) and TPR were derived from CO, HR, and ABP by a standard formula. ABP (mm Hg) was significantly higher in -/- (132+/-4) (P < 0.0001) than in +/+ mice (95+/-2). CO (ml min(-1)), HR(beats min(-1))and SV (microl beat(-1)) did not differ significantly between -/- and +/+ mice (CO -/- = 7.3+/-0.5, +/+ = 8.3+/-0.6; HR -/- = 407+/-22, +/+ = 462+/-21; SV -/- = 17.6+/-1.1, +/+ = 17.6+/-1.7). However, TPR (mm Hg ml(-1) min(-1)) was significantly elevated in -/- mice (18.4+/-0.7) compared to +/+ mice (12.3+/-1) (P = 0.0003). Autonomic ganglion blockade with a mixture of hexamethonium and pentolinium was followed by comparable percent reductions in CO (-/- = 28+/-4, +/+ = 29+/-3), HR (-/- = 9+/-4, +/+ = 16+/-4) and SV(-/- = 21+/-4, +/+ = 15+/-6) in both genotypes. However, the concomitant decrease in ABP (%) in -/- (41+/-2) was significantly greater than in +/+ (23+/-4) mice (P = 0.0009) and was accompanied by a significant reduction in TPR. We conclude that the hypertension associated with lack of endogenous ANP is due to elevated TPR, which is determined by an increase in cardiovascular autonomic tone.

Animals↗

PubMed1996

Effect of human variability on independent double reading in screening mammography.

RATIONALE AND OBJECTIVES: To demonstrate the range of gains and losses that radiologists might experience from independent double reading in screening mammography. METHODS: From a national random sample of radiologists, the authors formed 131 pairs. For each radiologist, the authors analyzed the increase relative to his or her individual true-positive rate (TPR) or false-positive rate (FPR), number of additional cancers detected, and change to negative biopsy rate that would result from independent double reading after pairing. RESULTS: The average radiologist can expect an 8%-14% gain in TPR and a 4%-10% increase in FPR with pairing. For some radiologists, double reading increased the TPR with a small concomitant increase in FPR. Other radiologists, however, realized small gains in TPR with large increases in FPR. Adding the reading from a more experienced radiologist did not necessarily improve the TPR of a radiologist with less experience. CONCLUSION: Radiologists can form complementary and noncomplementary pairs for double reading. Use of this procedure must be decided on an individual basis.

Breast Neoplasms↗

PubMed2001

Identification of amino acids in the tetratricopeptide repeat and C-terminal domains of protein phosphatase 5 involved in autoinhibition and lipid activation.

Protein phosphatase 5 (PP5) exhibits low basal activity due to the autoinhibitory properties of its N-terminal and C-terminal domains but can be activated approximately 40-fold in vitro by polyunsaturated fatty acids. To identify residues involved in regulating PP5 activity, we performed scanning mutagenesis of its N-terminal tetratricopeptide repeat (TPR) domain and deletion mutagenesis of its C-terminal domain. Mutating residues in a groove of the TPR domain that binds to heat shock protein 90 had no effect on basal phosphatase activity. Mutation of Glu-76, however, whose side chain projects away from this groove, resulted in a 10-fold elevation of basal activity without affecting arachidonic acid-stimulated activity. Thus, the interface of the TPR domain involved in PP5 autoinhibition appears to be different from that involved in heat shock protein 90 binding. We also observed a 10-fold elevation of basal phosphatase activity upon removing the C-terminal 13 amino acids of PP5, with a concomitant 50% decrease in arachidonic acid-stimulated activity. These two effects were accounted for by two distinct amino acid deletions: deleting the four C-terminal residues (496-499) of PP5 had no effect on its activity, but removing Gln-495 elevated basal activity 10-fold. Removal of a further three amino acids had no additional effect, but deleting Asn-491 resulted in a 50% reduction in arachidonic acid-stimulated activity. Thus, Glu-76 in the TPR domain and Gln-495 at the C-terminus were implicated in maintaining the low basal activity of PP5. While the TPR domain alone has been thought to mediate fatty acid activation of PP5, our data suggest that Asn-491, near its C-terminus, may also be involved in this process.

Amino Acids↗