Direct Measurement of Single-Molecule Ligand-Receptor Interactions.

Measuring the kinetics that govern ligand-receptor interactions is fundamental to our understanding of pharmacology. For ligand-gated ion channels, binding of an agonist triggers allosteric motions that open an integral ion-permeable pore. By mathematically modeling stochastic electrophysiological responses with high temporal resolution (ms), previous single channel studies have been able to infer the rate constants of ligands binding to these receptors. However, there are no reports of the direct measurement of the single-molecule binding events that are vital to how agonists exert their functional effects. For the first time, we report these direct measurements, the rate constants, and corresponding free energy changes, which describe the transitions between the different binding states. To achieve this, we use the super resolution technique: points accumulation for imaging in nanoscale topography (PAINT) to observe binding of ATP to orthosteric binding sites on the P2X1 receptor. Furthermore, an analysis of time-resolved single-molecule interactions is used to measure elementary rate constants and thermodynamic forces that drive the allosteric motions. These single-molecule measurements unequivocally establish the location of each binding states of the P2X1 receptor and the stochastic nature of the interaction with its native ligand. The analysis leads to the measurement of the forward and reverse rates from a weak ligand-binding state to a strong ligand binding state that is linked to allosteric motion and ion pore formation. These rates (kα = 1.41 sec-1 and kß = 0.32 sec-1) were then used to determine the free energy associated with this critical mechanistic step (3.7 kJ/mol). Importantly, the described methods can be readily applied to all ligand-gated ion channels, and more broadly to the molecular interactions of other classes of membrane proteins.

Impact of diabetes on early and mid-term survival after coronary artery bypass graft surgery in the Hong Kong Chinese population.

OBJECTIVE: To determine the impact of diabetes on early and mid-term survival in the Hong Kong Chinese population undergoing coronary artery bypass graft surgery. DESIGN: Prospective study. SETTING: Regional hospital, Hong Kong. PATIENTS: A total of 904 consecutive patients following coronary artery bypass graft surgery from November 1999 to December 2003 were prospectively analysed. Among them, 377 (42%) diabetic and 527 (58%) non-diabetic patients were evaluated. MAIN OUTCOME MEASURES: Hospital mortality, mid-term mortality, and percutaneous coronary intervention-free survival. RESULTS: The diabetic group had a higher risk score than the non-diabetic group (mean+/-standard deviation: EuroSCORE 4.7+/-3.4 and 3.6+/-3.4, respectively; P<0.001). Hospital mortality was 3.4% in the diabetic group compared to 2.8% in the non-diabetic group (P=0.698). Multiple logistic regression analysis identified left ventricular ejection fraction of less than 30% and preoperative intubation as independent risk factors for early hospital death. There were 81 late deaths and the actuarial survival at 48 months for the diabetic and non-diabetic patients were 86% and 90%, respectively (P=0.298). The angina-free survival and percutaneous coronary intervention-free survival at 48 months for the diabetic and non-diabetic patients yielded no statistically significant difference. CONCLUSIONS: Diabetes mellitus was not a predictor of early and mid-term mortality after coronary artery bypass graft surgery in our Chinese population. Furthermore, diabetes did not affect angina recurrence or intervention free-survival up to 4 years.

Erratum: ZnO-based MIS photodetectors.

[This corrects the article DOI: 10.1016/j.sna.2007.06.006.].

Carbon nanotube Schottky diode: an atomic perspective.

The electron transport properties of semiconducting carbon nanotube (SCNT) Schottky diodes are investigated with atomic models using density functional theory and the non-equilibrium Green's function method. We model the SCNT Schottky diode as a SCNT embedded in the metal electrode, which resembles the experimental set-up. Our study reveals that the rectification behaviour of the diode is mainly due to the asymmetric electron transmission function distribution in the conduction and valence bands and can be improved by changing metal-SCNT contact geometries. The threshold voltage of the diode depends on the electron Schottky barrier height which can be tuned by altering the diameter of the SCNT. Contrary to the traditional perception, the metal-SCNT contact region exhibits better conductivity than the other parts of the diode.

Mortality prediction in adult cardiac surgery patients: comparison of two risk stratification models.

OBJECTIVE: To assess and compare the two commonly applied models--EuroSCORE and Parsonnet--in our local adult cardiac surgery patients, according to risk factor quantification related to mortality using a risk stratification protocol to assess the quality of cardiac surgical care. DESIGN: Prospective study. SETTING: Cardiac surgery centre in a regional hospital in Hong Kong. PATIENTS: All adult patients undergoing coronary artery bypass graft and heart valve surgery at the Grantham Hospital were evaluated prospectively from November 1999 to July 2005. MAIN OUTCOME MEASURES: In-hospital mortality was the defined end-point. Statistical analyses consisted of observed against expected mortality, Hosmer-Lemeshow goodness-of-fit test for calibration accuracy, and receiver operating characteristic curve for discrimination performance. RESULTS: During the study period, 1247 patients underwent coronary artery bypass graft surgery and 1406 underwent heart valve surgery. Observed mortality rates in these two patient groups were 2.9% and 4.8% respectively. The expected mortality rates as predicted by the EuroSCORE were (mean+/-standard deviation) 4.0+/-3.3% and 5.2+/-3.0% respectively, and by the Parsonnet model were 5.9+/-4.2% and 7.3+/-4.4% respectively. EuroSCORE performed better than the Parsonnet model at predicting in-hospital mortality assessed by the Hosmer-Lemeshow goodness-of-fit test. The areas under the receiver operating characteristic curves in coronary artery bypass graft surgery were 0.76 for EuroSCORE and 0.74 for Parsonnet. The receiver operating characteristic curve areas in valve surgery were 0.77 for EuroSCORE and 0.79 for Parsonnet. CONCLUSION: Despite significant geographic and demographic differences between European and Asian patients, in our local adult cardiac surgery patients, the EuroSCORE performed well with good calibration and discrimination in predicting mortality. There was a tendency for both models to over predict. However, the EuroSCORE can serve as a baseline for the development of a local risk model.

Hepatic alpha 2 mu-globulin: a potential metabolic role in the rat proximal tubule.

In the present study, we provide immunohistochemical and immunologic evidence to localize an abundant, 15.5-kDa protein to the soluble protein fraction of the proximal tubule. This 15.5-kDa protein binds fatty acids in vitro and has identity with amino acids 10-117 of alpha 2 mu-globulin (A2 fragment), a 19-kDa protein synthesized predominantly in the male liver. With reverse transcription-polymerase chain reaction, mRNA for A2 was detected in male liver but not in the male kidney. De novo accumulation of the 15.5-kDa protein was observed in the renal cortex of female rats given intravenous injections of purified 19-kDa protein (A2), suggesting intrarenal processing of the larger protein. The potential role of this protein in the proximal tubule, a site that utilizes fatty acids as an important metabolic substrate, was determined in isolated proximal tubule segments. Fatty acid and glucose oxidation rates were measured in three experimental models in which the 15.5-kDa protein was virtually absent: 1) uninephrectomized male rats treated with deoxycorticosterone acetate and salt, 2) male rats subjected to bilateral adrenalectomy, and 3) normal female rats. In the absence of the 15.5-kDa protein, fatty acid oxidation rates decreased by 30-55%, whereas glucose oxidation significantly increased in all three models. In female renal cortex, depletion of the 15.5-kDa protein was associated with a rise in heart fatty acid binding protein, an alternative intracellular transporter of fatty acids. These data support the hypothesis that a proteolytic cleavage product of hepatic alpha 2 mu-globulin may facilitate the oxidation of oleate, a hydrophobic ligand, in the proximal tubule.

Biliary tract invasion and obstruction by hepatocellular carcinoma: report of five cases.

Major biliary tract obstruction caused by tumour invasion is a rare manifestation of hepatocellular carcinoma. The authors had the opportunity to diagnose and treat five such cases, three of whom had features of acute cholangitis. The prevalence of both hepatocellular carcinoma and recurrent pyogenic cholangitis is high in patients from the Far East. The former may first present under the guise of the latter. Gastroenterologists and surgeons should be aware of hepatocellular carcinoma when managing these patients who present with obstructive jaundice, gross hepatomegaly and cholangitis.

Structure and expression of the Xenopus retinoblastoma gene.

We have cloned a Xenopus homology (XRb1) of the human retinoblastoma susceptibility gene. DNA sequence analysis shows that the XRb1 gene product is highly conserved in many regions. The leucine repeat motif and many of the potential cdc2 phosphorylation sites, as well as potential sites for other kinases, are retained. The region of the protein homologous to the SV40 T antigen binding site and the basic region directly C-terminal to the E1A binding site are all conserved. XRb1 gene expression at the RNA level was studied by Northern blot analysis. Transcripts of 4.2 and 10-kb are present as maternal RNA stores in the oocyte. While the 4.2-kb product is stable until at least the mid-blastula stage, the 10-kb transcript is selectively degraded. Between stages 11 and 13 the 10-kb transcript reappears and also a minor product of approximately 11 kb becomes apparent. Both the 4.2- and the 10-kb transcripts remain present until later stages of development and are also present in all adult tissues examined, although at differing levels. Antibodies raised against human p105Rb which recognize the protein product of the XRb1 gene, pXRb1, detect the Xenopus 99-kDa protein prior to the mid-blastula stage, but at lower levels than at later stages in development.

Germ-line mutations of the p53 tumor suppressor gene in patients with high risk for cancer inactivate the p53 protein.

Germ-line mutations in the p53 tumor suppressor gene have been observed in patients with Li-Fraumeni syndrome, brain tumors, second malignancies, and breast cancers. It is unclear whether all of these mutations have inactivated p53 and thereby provide an increased risk for cancer. Therefore, it is necessary to establish the biological significance of these germ-line mutations by the functional and structural analysis of the resulting mutant p53 proteins. We analyzed the ability of seven germ-line mutant proteins observed in patients with Li-Fraumeni syndrome, second primary neoplasms, or familial breast cancer to block the growth of malignant cells and compared the structural properties of the mutant proteins to that of the wild-type protein. Six of seven missense mutations disrupted the growth inhibitory properties and structure of the wild-type protein. One germ-line mutation retained the features of the wild-type p53. Genetic analysis of the breast cancer family in which this mutation was observed indicated that this germ-line mutation was not associated with the development of cancer. These results demonstrate that germ-line p53 mutations observed in patients with Li-Fraumeni syndrome and with second malignancies have inactivated the p53 tumor suppressor gene. The inability of the germ-line p53 mutants to block the growth of malignant cells can explain why patients with these germ-line mutations have an increased risk for cancer. The observation of a functionally silent germ-line mutation indicates that, before associating a germ-line tumor suppressor gene mutation with cancer risk, it is prudent to consider its functional significance.

hsp70 binds specifically to a peptide derived from the highly conserved domain (I) region of p53.

Products of a number of mutant p53 genes bind with high affinity to members of the hsp70 family of chaperonin proteins, whereas wild type p53 lacks this type of association. Examination of the sequences of p53 genes from five different species enabled us to predict domains on p53 which may be involved in the association with hsp70 family members. A synthetic polypeptide (Pro-17-Gly) corresponding to the candidate hsp70 binding domain bound to in vitro translated hsp70 as determined by affinity chromatography and nondenaturing gel mobility shift assays. In addition, the Pro-17-Gly peptide competitively inhibited association between hsp70 and p53, an activity which was determined by immunoprecipitation with anti-p53 monoclonal antibody PAb240. The data indicate that p53 contains a hsp70 binding domain, which is located in a highly conserved region at the amino terminus of the protein, and may participate in the cellular function of wild-type p53 or in the transforming capacity of p53 mutants.

PTCA--the initial 100 cases in the Singapore General Hospital.

Percutaneous transluminal coronary angioplasty (PTCA) is an increasingly important technique for the treatment of patients with coronary artery disease. One hundred cases of PTCA were performed in Singapore General Hospital between 1985 to September 1988. 87% were males and 13% were females. The mean age of these patients were 55 +/- 9 SD years (range from 28 to 79 years). 85% had angina pectoris, 43% had a previous myocardial infarction and 12% received intravenous streptokinase previously. One hundred and twenty-nine lesions were dilated. 51% of the cases had single, 40% had double and 9% triple vessel disease. 83% had single, 16% double and 1% triple vessel dilatation. The distribution of the cases by vessel location was 64% for LAD, 19% for RCA and 17% for CX. Overall lesion success was 86%, with an increment in the success rate from 76% in 1987 to 89% in 1988. Similarly, the clinical success rate was 86% for the whole group; it increased from 74% to 90% from 1987 to 1988. The mean diameter stenosis was decreased from 86 +/- 14% SD predilatation to 28 +/- 28% SD postdilatation (p less than 0.005). The mean transtenotic gradient was reduced from 60 +/- 17 mmHg SD predilatation to 18 +/- 11 mmHg SD postdilatation (p less than 0.005). There were 14 failures (14%), the majority of which were due to inability transversing the lesion with a guide wire or balloon catheter. Fourteen complications (14%) occurred in these 100 cases of PTCA. 3% required emergent/semiemergent surgical revascularization. There was no mortality. PTCA is now an established mode of therapy for coronary artery disease in Singapore General Hospital.

Properties and differential regulation of two fatty acid binding proteins in the rat kidney.

A protein from rat kidney was characterized that had several properties common to a multigene family of fatty acid binding proteins identified in other tissues. The putative kidney fatty acid binding protein (FABP) was purified from the soluble fraction of kidney homogenates using gel filtration and ion exchange chromatography. It was relatively abundant, had an apparent molecular mass of 15.5 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, bound equimolar amounts of oleic acid, and could be distinguished from other FABPs on the basis of size, amino acid composition, and tissue distribution. Polyclonal antibodies to kidney FABP were obtained and used to show that only kidney contained the 15.5-kDa protein, although the antibodies also recognized a slightly larger and less abundant protein in kidney that also was present in bladder. Rat kidney also contained heart FABP, and the properties of both FABPs in rat kidney were compared. The distribution of both proteins within the kidney differed, with kidney FABP being localized almost exclusively within the cortex, whereas heart FABP was found both in cortex and medulla. Kidney FABP was expressed developmentally after the neonatal period, whereas heart FABP was present in both neonatal and adult kidney at comparable amounts. Hypertension induced by mineralocorticoids or infusion of angiotensin II caused a marked suppression of kidney FABP expression, whereas amounts of heart FABP in kidney were unchanged. The studies showed that rat kidney contains at least two FABPs, and that these proteins are differentially regulated, suggesting that functional differences between the proteins may exist.

Sucralfate compared with ranitidine in the short-term healing of duodenal ulcers.

The efficacy of sucralfate (1 g four times daily) and ranitidine (150 mg twice daily) in the short-term healing of duodenal ulcers has been assessed in a randomized single-blind trial involving 104 patients with three drop-outs. The healing rate at 3 weeks of 83% for sucralfate and 84% for ranitidine improved to 96% and 92%, respectively, at 6 weeks. The difference between the two treatment groups was not statistically significant. Smoking and male sex had no influence on the healing rates. Constipation was a prominent symptom in sucralfate treatment which was otherwise a safe and effective therapy.

The Ultra-Flo 100 platelet counter: a new approach to platelet counting.

The Clay Adams Ultra-Flo 100 whole blood platelet counter is a semiautomated instrument. The count is made on dilute whole blood by the detection of comparatively small current changes induced by the cells suspended in a conducting diluent as they flow thrugh an orifice. Alarm systems are incorporated in the instrument to detect sample irregularities due to microcytosis, and large and small platelets. The results of this evaluation are given, and confirm that the results using the Ultra-Flo 100 compare very favourably with those obtained using phase contrast microscopy.

Prolonged apnoea due to suxamethonium in Chinese.

A Chinese patient was hypersensitive to a therapeutic dose of suxamethonium because of a genetically determined quantitative and qualitative abnormality of plasma pseudocholinesterase.