Membrane topology and glycosylation of the human multidrug resistance-associated protein

The membrane topology of the human multidrug resistance-associated protein (MRP) was examined by flow cytometry phenotyping, immunoblotting, and limited proteolysis in drug-resistant human and baculovirus-infected insect cells, expressing either the glycosylated or the underglycosylated forms of this protein. Inhibition of N-linked glycosylation in human cells by tunicamycin did not inhibit the transport function or the antibody recognition of MRP, although its apparent molecular mass was reduced from 180 kDa to 150 kDa. Extracellular addition of trypsin or chymotrypsin had no effect either on the function or on the molecular mass of MRP, while in isolated membranes limited proteolysis produced three large membrane-bound fragments. These experiments and the alignment of the MRP sequence with the human cystic fibrosis transmembrane conductance regulator (CFTR) suggest that human MRP, similarly to CFTR, contains a tandem repeat of six transmembrane helices, each followed by a nucleotide binding domain, and that the C-terminal membrane-bound region is glycosylated. However, the N-terminal region of MRP contains an additional membrane-bound, glycosylated area with four or five transmembrane helices, which seems to be a characteristic feature of MRP-like ATP-binding cassette transporters.     

Validation of the three-dimensional acquisition mode in positron emission tomography for the quantitation of [18F]fluoro-DOPA uptake in the human striata

Three-dimensional (3D) positron emission tomography (PET) is attractive for [18F]fluoro-DOPA studies, since the sensitivity improvement is maximal for radioactive sources located in central planes, which is usually the case for the human striata. However, the image quantitation in that mode must be assessed because of the nearly threefold increase in scattered coincidences. We report the results of [18F]fluoro-DOPA studies performed on six normal volunteers. Each one was scanned in the 3D and two-dimensional (2D) modes on the same tomograph. The quantitation in the 3D and 2D modes was compared for a Patlak graphical analysis with the occipital counts as the input function (Ki) and a striatooccipital ratio analysis. We find that, in 3D PET, a scatter correction is required to preserve the same quantitation as in 2D PET. When the 3D data sets are corrected for scatter, the quantitation of the [18F]fluoro-DOPA uptake, using the Patlak analysis, is similar in the 2D and 3D acquisition modes. Conversely, analysis of the striatooccipital ratio leads to higher values in 3D PET because of a better in-plane resolution. Finally, using the 3D mode, the dose injected to the subjects can be reduced by a factor greater than 1.5 without any loss in accuracy compared to the 2D mode.     

Identification of an opioid kappa receptor subtype-selective N-substituent for (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine

A three-component library of compounds was prepared in parallel using multiple simultaneous solution-phase synthetic methodology. The compounds were biased toward opioid receptor antagonist activity by incorporating (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (a potent, nonselective opioid pure antagonist) as one of the monomers. The other two monomers, which included N-substituted or unsubstituted Boc-protected amino acids and a range of substituted aryl carboxylic acids, were selected to add chemical diversity. Screening of these compounds in competitive binding experiments with the kappa opioid receptor selective ligand [3H]U69,593 led to the discovery of a novel kappa opioid receptor selective ligand, N-?(2'S)-[3-(4-hydroxyphenyl)propanamido]-3'-methylbutyl?-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (8, RTI-5989-29). Additional structure-activity relationship studies suggested that 8 possesses lipophilic and hydrogen-bonding sites that are important to its opioid receptor potency and selectivity. These sites appear to exist predominantly within the kappa receptor since the selectivity arises from a 530-fold loss of affinity of 8 for the mu receptor and an 18-fold increase in affinity for the kappa receptor relative to the mu-selective ligand, (+)-N-[trans-4-phenyl-2-butenyl]-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (5a). The degree of selectivity observed in the radioligand binding experiments was not observed in the functional assay. According to its ability to inhibit agonist stimulated binding of [35S]GTPgammaS at all three opioid receptors, compound 8 behaves as a mu/kappa opioid receptor pure antagonist with negligible affinity for the delta receptor.     

The apolipoprotein E phenotype has a strong influence on tracking of serum cholesterol and lipoprotein levels in children: a follow-up study from birth to the age of 11 years

Fatty acid uptake is partly controlled by the FATP gene family, of which at least five members are known in mice. Using the mmFATP1 cDNA as hybridization probe, a 1.6 kb partial cDNA clone was isolated from a human heart cDNA library. With 5' and 3' RACE procedures, the complete cDNA was isolated. Sequence comparisons with its mouse homologues identified this clone as hsFATP4.     

Geographic variations in the composition of ivory of the African elephant (Loxodonta africana)

OBJECTIVES: The purpose of the present study was to compare the inclination of the occlusal plane with occlusal guidance as a contributing factor to masticatory movement. METHODS: Masticatory movements of 41 young adults were measured using a 3-D mandibular movement analysing system. The inclination of the occlusal plane was measured in the sagittal plane using a 3-D digitizer. The contribution of the occlusal guidance and the inclination of the occlusal plane to the direction of the masticatory path of closure was evaluated at various closing levels. RESULTS: The masticatory path of closure outside the intercuspal range was influenced mainly by the inclination of the occlusal plane, and the masticatory path of closure near the intercuspal range was only influenced by occlusal guidance. The so-called gliding type masticatory pattern was observed predominantly in subjects with a posteriorly inclined occlusal plane. In contrast, a chopping type masticatory pattern was observed predominantly in subjects with an anteriorly inclined occlusal plane. CONCLUSIONS: The contribution of the inclination of the occlusal plane to masticatory movement was greater than that of occlusal guidance throughout the closing phase except near the intercuspal range.     

The effect of endothelin and its antagonist Bosentan on hemodynamics and microvascular exchange in cirrhotic rat liver

BACKGROUND/AIMS: To characterize the effects of endothelin-1 and of Bosentan, a mixed endothelin antagonist, on hepatic hemodynamics in cirrhotic animals in vivo and on hepatic microvascular exchange in the perfused rat liver. METHODS: Biliary cirrhosis was induced by bile duct ligation, and micronodular cirrhosis by chronic exposure to phenobarbital/CCl4 in male rats. Hepatic hemodynamics were studied under basal conditions and after administration of Bosentan (3-30 mg/kg) by the microsphere technique. Microvascular exchange was assessed in the in situ perfused rat liver by the multiple indicator dilution technique. RESULTS: Bosentan lowered portal pressure in a dose-dependent fashion; at the highest dose tested, this decrease averaged -29+/-11 and -26+/-8% in biliary and micronodular cirrhosis, respectively (p<0.01). This was achieved mainly via a marked decrease in hepatic arterial flow. In the perfused liver, endothelin-1 induced a dose-dependent vasoconstriction; up to 10(-9) mol/l; this was not associated with any effect on viability. At this dose, endothelin-1 markedly decreased extravascular albumin space in both controls and micronodular cirrhosis; this could be antagonized by Bosentan 10(-5) mol/l. CONCLUSIONS: Endothelin-1 affects hepatic microvascular exchange, presumably by a direct effect on hepatic sinusoidal endothelial cells. A mixed endothelin antagonist lowers portal pressure in vivo, presumably by acting on hepatic stellate cells, and counteracts the microvascular effects of endothelin-1 in vitro. These properties could prove useful in treatment of portal hypertension.     

Lack of familial clustering of hepatitis C virus infection

BACKGROUND: Although transmission of hepatitis C virus (HCV) through parental exposure is well documented, it is still controversial whether familial clustering of HCV occurs. METHODS: To investigate risk factors for HCV infection, 109 cases and 84 non-infected controls were studied. In addition, 250 family members (104 men, 146 women) of cases and 170 family members of controls (64 men, 106 women) were tested for HCV infection using an anti-HCV antibody, alanine aminotransferase (ALT), and reverse transcribed polymerase chain reaction (RT-PCR). RESULTS: In the case-control analysis, people aged > or =60 were almost three times more likely to be infected by HCV than those aged <40. Risk of HCV infection was most strongly related to a history of blood transfusion (OR = 12.6, 95% CI: 4.3-36.5) followed by a history of jaundice (OR = 4.1, 95% CI: 1.3-12.6). Only one family member of cases and no-one related to the controls had HCV infection. CONCLUSIONS: These results suggest that, in Korea, age and parenteral exposure, such as a blood transfusion, are risk factors for HCV infection and familial clustering of HCV infection, if it occurs, is rare.     

Bedside diagnostic laparoscopy and peritoneal lavage in the intensive care unit

BACKGROUND: Early diagnosis and treatment of intra-abdominal pathology in critically ill intensive care unit (ICU) patients remains a clinical challenge. The objective of this study is to assess the feasibility of portable, bedside diagnostic laparoscopy (DL) in the ICU for patients suspected of intra-abdominal pathology, and to contrast its accuracy with diagnostic peritoneal lavage (DPL). METHODS: All adult ICU patients for whom a general surgery consultation was requested were eligible. Patients with a recent laparotomy or obvious peritonitis were excluded. All procedures were performed in the ICU. RESULTS: Over a consecutive 16-month period, 12 patients underwent DPL/DL. Ages ranged from 28 to 88 (mean, 72) years. Causative findings were disclosed by DL in five patients, (42%) including intestinal ischemia in two. Perforated diverticulitis, thickened terminal ileum, and nonpurulent peritonitis were found in one patient each. All patients with findings by DL had a positive DPL (WBC > 200 cells/mm3), and one negative laparoscopy was positive by lavage. The average length of time to perform DPL was 14 min, and to complete DL 19 min. One patient underwent laparotomy based on DPL/DL and survived along with three others with negative DPL/DL. Eight patients died (67%), four from their surgically untreated intra-abdominal pathology. One patient sustained a procedure-related complication of bradycardia and high ventilatory airway pressures. Peak airway pressures increased an average of 8 mmHg and were significantly higher (p < 0. 001) than pre-DL pressures without any significant change in end-tidal CO2 or pCO2. There were no statistically significant hemodynamic changes based on mean arterial pressure (MAP), central venous pressure (CVP), or pulmonary artery diastolic pressure (PADP). CONCLUSIONS: Bedside laparoscopy can be performed rapidly and safely in the ICU. In predicting the need for laparotomy, DL was more accurate than DPL.     

Randomised trial of educational visits to enhance use of systematic reviews in 25 obstetric units

OBJECTIVE: To evaluate the effectiveness of an educational visit to help obstetricians and midwives select and use evidence from a Cochrane database containing 600 systematic reviews. DESIGN: Randomised single blind controlled trial with obstetric units allocated to an educational visit or control group. SETTING: 25 of the 26 district general obstetric units in two former NHS regions. SUBJECTS: The senior obstetrician and midwife from each intervention unit participated in educational visits. Clinical practices of all staff were assessed in 4508 pregnancies. INTERVENTION: Single informal educational visit by a respected obstetrician including discussion of evidence based obstetrics, guidance on implementation, and donation of Cochrane database and other materials. MAIN OUTCOME MEASURES: Rates of perineal suturing with polyglycolic acid, ventouse delivery, prophylactic antibiotics in caesarean section, and steroids in preterm delivery, before and 9 months after visits, and concordance of guidelines with review evidence for same marker practices before and after visits. RESULTS: Rates varied greatly, but the overall baseline mean of 43% (986/2312) increased to 54% (1189/2196) 9 months later. Rates of ventouse delivery increased significantly in intervention units but not in control units; there was no difference between the two types of units in uptake of other practices. Pooling rates from all 25 units, use of antibiotics in caesarean section and use of polyglycolic acid sutures increased significantly over the period, but use of steroids in preterm delivery was unchanged. Labour ward guidelines seldom agreed with evidence at baseline; this hardly improved after visits. Educational visits cost pound860 each (at 1995 prices). CONCLUSIONS: There was considerable uptake of evidence into practice in both control and intervention units between 1994 and 1995. Our educational visits added little to this, despite the informal setting, targeting of senior staff from two disciplines, and donation of educational materials. Further work is needed to define cost effective methods to enhance the uptake of evidence from systematic reviews and to clarify leadership and roles of senior obstetric staff in implementing the evidence.     

Detection of optic disc changes with Glaucoma-Scope probability maps

Before use of cardiovascular surgical techniques and procedures in humans, many experiments, e.g., hypothermic circulatory arrest and cardiopulmonary bypass using the heart-lung machine, have been performed in the dog. As a consequence experimental canine cardiovascular surgery is highly developed. This has not resulted in the routine performance of open heart surgery in veterinary medicine, probably because of the high costs. Cardiovascular surgery in the dog is generally limited to interventions not depending on hypothermic circulatory arrest or cardiopulmonary bypass. The clinical cardiovascular surgery in dogs can be divided into routine and more specialized interventions. The first category includes correction of peritoneopericardial diaphragmatic hernia, pericardial fenestration in dogs with pericardial effusion, treatment of persistent right aortic arch, and patent ductus closure. The specialized interventions include dilation of pulmonic and aortic stenoses and pacemaker implantation. The diagnosis and surgical treatment of such diseases is described. New developments in cardiovascular surgical treatment that can be expected include catheter techniques for occlusion of shunts and dilations using balloons, because the financial costs of these procedures are not prohibitive.