Avian influenza A H7N9 virus infects human astrocytes and neuronal cells and induces inflammatory immune responses.

Seasonal, pandemic, and avian influenza virus infections may be associated with central nervous system pathology, albeit with varying frequency and different mechanisms. Here, we demonstrate that differentiated human astrocytic (T98G) and neuronal (SH-SY5Y) cells can be infected by avian H7N9 and pandemic H1N1 viruses. However, infectious progeny viruses can only be detected in H7N9 virus infected human neuronal cells. Neither of these viral strains can generate infectious progeny virus in human astrocytes despite replication of viral genome was observed. Furthermore, H7N9 virus triggered high pro-inflammatory cytokine expression, while pandemic H1N1 virus induced only low cytokine expression in either brain cell type. The experimental finding here is the first data to demonstrate that avian H7N9 virus can infect, transcribe, and replicate its viral genome; induce cytokine upregulation; and cause cytopathic effects in human brain cells, which may potentially lead to profound central nervous system injury. Observation for neurological problems due to H7N9 virus infection deserves further attention when managing these patients.

Clopidogrel, prasugrel or ticagrelor in patients with acute coronary syndromes undergoing percutaneous coronary intervention.

BACKGROUND: Guidelines recommend prasugrel or ticagrelor instead of clopidogrel in patients with acute coronary syndromes (ACS) undergoing percutaneous coronary interventions (PCI). AIM: We sought to describe the trends in uptake of the newer agents and analyse the clinical characteristics and short-term outcomes of patients treated with clopidogrel, prasugrel or ticagrelor. METHODS: We analysed the temporal trends of antiplatelet use since the availability of prasugrel (2009-2013) in patients with ACS from the Melbourne Interventional Group registry. To assess clinical characteristics and outcomes, we included 1850 patients from 2012 to 2013, corresponding to the time all three agents were available. The primary outcome was major adverse cardiovascular events (MACE). The safety end-point was in-hospital bleeding. RESULTS: For the period of 2009-2013, the majority of patients were treated with clopidogrel (72%) compared with prasugrel (14%) or ticagrelor (14%). There was a clear trend towards ticagrelor by the end of 2013. Patients treated with clopidogrel were more likely to present with non-ST-elevation ACS, be older, and have more comorbidities. There was no difference in unadjusted 30-day mortality (0.9 vs 0.5 vs 1.0%, P = 0.76), myocardial infarction (2 vs 1 vs 2%, P = 0.52) or MACE (3 vs 3 vs 4%, P = 0.57) between the three agents. There was no difference in in-hospital bleeding (3 vs 2 vs 2%, P = 0.64). CONCLUSION: Prasugrel and ticagrelor are increasingly used in ACS patients treated with PCI, predominantly in a younger cohort with less comorbidity. Although antiplatelet therapy should still be individualised based on the thrombotic and bleeding risk, our study highlights the safety of the new P2Y12 inhibitors in contemporary Australian practice.

SCN1A variations and response to multiple antiepileptic drugs.

In the current study, we have used the haplotype-tagging single-nucleotide polymorphisms (SNPs) to determine associations between genetic variants in SCN1A and treatment response in 519 Caucasian patients with known response status for epilepsy treated with antiepileptic drugs (AEDs) with sodium channel blocking effects. Nine SNPs within SCN1A were genotyped in this cohort. The only association observed was for rs10188577. A greater proportion of drug-resistant patients were heterozygous compared with drug responsive patients (48.3% vs 35.4%, P=0.014). After correction for potential confounding factors, the association for rs10188577 was only marginally significant (P=0.049). In light of our findings, it seems unlikely that rs10188577 could be a major determinant of response to AEDs. However, looking at the influence of rs10188577 on the expressed quantitative trait association patterns within the immediate vicinity of SCN1A, we found significant associations with neighbouring sodium channel genes, SCN7A and SCN9A (P<0.025), which warrants further studies.

Ion exchange and structural aging in the layered perovskite phases H(1-x)Li(x)LaTiO4.

Grinding together the solid acid HLaTiO4 with stoichiometric quantities of lithium hydroxide monohydrate gives the solid solution H(1-x)Li(x)LaTiO4. The structures of these crystalline phases have been refined against neutron powder diffraction data to show that all of these compounds crystallize in the centrosymmetric space group P4/nmm. The protons and lithium cations occupy sites between the perovskite layers; the former in hydroxide groups that hydrogen-bond to adjacent layers while Li(+) is in four-coordinate sites that bridge the perovskite slabs with a geometry intermediate between square-planar and tetrahedral. The reaction proceeds rapidly, but the unit cell size continues to evolve over the course of days with a gradual compression along the interlayer direction that can be modeled using a power law dependence reminiscent of an Ostwald ripening process. On heating, these materials undergo a mass loss because of dehydration but retain the layered Ruddlesden-Popper structure up to 480 °C before a substantial loss of crystallinity on further heating to 600 °C. Impedance spectroscopy studies of the dehydrated materials shows that Li(+) mobility in these materials is lower than the LiLaTiO4 end member, possibly because of microstructural effects causing large intergrain resistance through the defective phases.

Vitamins B-12 and C Supplementation Improves Arterial Reactivity and Structure in Passive Smokers: Implication in Prevention of Smoking-Related Atherosclerosis.

BACKGROUND: Atherosclerosis is an important medical problem of modern society. High environmental tobacco smoke in casino is associated with an accelerated atherogenic process. We have previously shown vitamin B12 and C supplementation improves vascular reactivity and may be beneficial in vascular protection. OBJECTIVE: To evaluate the impact of vitamin supplementation on atherosclerosis (brachial artery reactivity FMD and carotid intima-media thickness IMT) in subjects exposed to high environmental tobacco smoke. DESIGN: Double-blind 2x2 factorial design fashion. SETTING: Computer randomization in 4 treatment groups: placebo (n=24), vitamin B12 (n=21), vitamin C (n=23) and vitamin B12+C (n=23) groups. PARTICIPANTS: 91 passive-smoking casino employees (19.2% male, mean age 45.0±8.2 years). INTERVENTION: Subjects were randomized to receive vitamin B12 (500µg daily), vitamin C (200mg daily), vitamin B12+C or image-matched placebo capsules for 1 year. MEASUREMENT: Brachial FMD and carotid IMT (surrogate atherosclerotic markers) were measured by ultrasound at baseline and on completion at 12 months. METHODS: 91 passive smoking casino employees (19.2% male, mean age 45.0±8.2 years) were randomized to receive vitamin B12 (500µg daily), vitamin C (200mg daily), vitamin B12+C or image-matched placebo capsules in double-blind 2 x 2 factorial design fashion for 1 year. Brachial FMD and carotid IMT (surrogate atherosclerotic markers) were measured by ultrasound at baseline and 12 months. RESULTS: Of the 78 (85.7%) passive-smoking employees completed the study, 11.5% had hypertension, 5.1% diabetes mellitus and 15.4% hypercholesterolemia. There were no significant changes in their blood pressures, lipid profiles, glucose and body mass index after supplementation for 1 year, but mild decrease in DBP (p<0.001) and blood creatinine (p<0.01) after combined vitamin B12 and C, and significant increase in blood B12 after vitamin B12 (p<0.01) and vitamin B12+C supplementations (p<0.001). Brachial FMD and cartotid IMT improved after the 3 vitamin supplementations (p<0.001), but not after placebo, being more significant after combined vitamin supplementations (p<0.0001). No adverse effects were reported. CONCLUSION: Vitamin B12 or C supplementation in passive smokers improved vascular reactivity and structures at 1 year, with implication in long term atherosclerosis prevention.

Serum amyloid A is elevated in the serum of lung cancer patients with poor prognosis.

BACKGROUND: Lung cancer is known as the top cancer killer in most developed countries. However, there is currently no promising diagnostic or prognostic biomarker for lung cancer. This study aims to discover non-invasive differential markers in the serum of lung cancer patients, to determine the protein identity of the candidate biomarker(s), and to investigate any clinical implication of the biomarker(s) concerned. METHODS: Blood specimens were collected from 154 pre-operative patients with lung cancer and 35 healthy blood donors with no evidence of lung cancer. Fractionated serum samples were processed by surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (MS). Candidate biomarker was identified using sodium dodecyl sulphate polyacrylamide gel electrophoresis and tryptic digestion followed by tandem MS fragmentation analysis, which was subsequently validated with immunoassay. RESULTS: A differential protein with m/z 11.6 kDa was detected and identified as an isoform of human serum amyloid A (SAA). It was significantly increased by 1822% in lung cancer patients when compared with the healthy controls, which gave an area under the receiver operator characteristic curve of 0.88. In addition, the protein was also significantly elevated by 77% in lung cancer patients with survival <5 years when compared with patients with survival > or =5 years. CONCLUSION: There are several functions of the SAA protein, described in the context of inflammation, that are compatible with the mechanism of tumour invasion and metastasis. Our study not only detected increased SAA level in the serum of lung cancer patients but also identified that elevated SAA level may be a non-invasive biomarker useful for the prediction of lung cancer prognosis.

White light sources based on multiple precision selective micro-filling of structured optical waveguides.

Multiple precision selective micro-filling of a structured optical fibre using three luminescent dyes enables the simultaneous capture of red, blue and green luminescence within the core to generate white light. The technology opens up a new approach to integration and superposition of the properties of multiple materials to create unique composite properties within structured waveguides.

Cadherin- and Rigidity-Dependent Growth of Lung Cancer Cells in a Partially Confined Microenvironment.

During tumor development, cancer cells constantly confront different types of extracellular barriers. However, fundamental questions like whether tumor cells will continue to grow against confinement or away from it and what key factors govern this process remain poorly understood. To address these issues, here we examined the growth dynamics of human lung epithelial carcinoma A549 cells partially confined in micrometer-sized cylindrical pores with precisely controlled wall stiffness. It was found that, after reaching confluency, the cell monolayer enclosed by a compliant wall was able to keep growing and pushing the boundary, eventually leading to a markedly enlarged pore. In contrast, a much reduced in-plane growth and elevated strain level among cells were observed when the confining wall becomes stiff. Furthermore, under such circumstance, cells switched their growth from within the monolayer to along the out-of-plane direction, resulting in cell stacking. We showed that these observations can be well explained by a simple model taking into account the deformability of the wall and the threshold stress for inhibiting cell growth. Interestingly, cadherins were found to play an important role in the proliferation and stress buildup within the cell monolayer by aggregating at cell-cell junctions. The stiff confinement led to an elevated expression level of cadherins. Furthermore, inhibition of N-cadherin resulted in a significantly suppressed cell growth under the same confining conditions.

Clinical experience of open carpal tunnel release and Camitz operation in elderly Chinese patients.

OBJECTIVE: To report preliminary experience on the Camitz operation for elderly Chinese patients in a Hong Kong public hospital. DESIGN: Retrospective study. SETTING: Tertiary referral hospital with hand surgery service in Hong Kong. PATIENTS: Between January 2000 and January 2004, patients with carpal tunnel syndrome having the Camitz operation were recruited. They were assessed using the measurements of pinch and grip power, sensation, the Kapandji score, and functional grading as well as complications encountered during the subsequent follow-up. INTERVENTION: The Camitz operation. RESULTS: A total of 21 patients (8 male and 13 female; mean age, 70 years) were recruited. The mean duration of follow-up was 15 months. There was significant improvement in pinch power, grip power, and hand functions, as well as a positive correlation between the functional score and the Kapandji score. No major complication was recorded. One patient with pre-existing osteoarthritis of the ring finger developed contracture of the proximal interphalangeal joint. CONCLUSION: The Camitz operation is a simple, effective, and safe means of improving hand function in elderly Chinese patients with long-standing carpal tunnel syndrome and thenar muscle atrophy. Newly acquired strength in antepulsion of thumb resulted in improved pinch, grip, and hand function in this patient group.

Long-term use of oral nucleos(t)ide analogues for chronic hepatitis B does not increase cancer risk - a cohort study of 44 494 subjects.

BACKGROUND: Patients with chronic hepatitis B (CHB) need long-term antiviral treatment with nucleos(t)ide analogues (NA). Animal studies suggest that some NA may increase cancer risk, but human data are lacking. AIM: To investigate cancer risks in patients with or without NA treatment. METHODS: We conducted a territory-wide cohort study using the database from Hospital Authority in Hong Kong. The diagnosis of CHB and various malignancies was based on the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) diagnosis codes between 2000 and 2012. Patients exposed to any of the oral NA for CHB were included. The primary outcome was incident cancers. A 3-year landmark analysis, with follow-up up to 7 years, was used to evaluate the relative risk of cancers in treated and untreated patients. RESULTS: A total of 44 494 patients (39 712 untreated and 4782 treated) were included in the analysis. During 194 890 patient-years of follow-up, hepatocellular carcinoma developed in 402 (1.0%) untreated patients and 179 (3.7%) treated patients, while other cancers developed in 528 (1.3%) and 128 (2.7%) patients respectively. After propensity score weighting, treated patients had similar risks of all malignancies [weighted hazard ratio (wHR): 1.01, 95% CI: 0.82-1.25, P = 0.899], lung/pleural cancers (wHR: 0.82, 95% CI: 0.52-1.31, P = 0.409) and urinary/renal malignancies (wHR: 1.04, 95% CI: 0.38-2.81, P = 0.944) when compared with untreated patients. CONCLUSIONS: Oral nucleos(t)ide analogue treatment does not appear to increase cancer risk in patients with chronic hepatitis B. Given the beneficial effect on liver outcomes, our data support the current practice of long-term anti-viral therapy.

Statins reduce the risk of liver decompensation and death in chronic viral hepatitis: a propensity score weighted landmark analysis.

BACKGROUND: Decompensated liver disease due to portal hypertension leads to significant morbidity and mortality. Statins can modulate intrahepatic vascular tone, but the clinical significance remains uncertain. AIM: To determine the effects of statin use on the risk of liver decompensation and death among patients with chronic viral hepatitis. METHODS: We conducted a population wide cohort study using a hospital based database from the Hong Kong Hospital Authority. Adults with chronic viral hepatitis without prior liver decompensation were identified from 2000 to 2012 by International Classification of Diseases, Ninth Revision, Clinical Modification, diagnostic codes. Statin use was defined as a cumulative defined daily dose of >28. Landmark analysis was used to overcome immortal time bias. Propensity score weighting was further performed to minimise baseline confounders. Primary outcome was a composite of portal hypertension related liver decompensation events, with adjustment for death as a competing risk. RESULTS: A total of 69 184 patients with chronic viral hepatitis (2053 statin users and 67 131 statin non-users) were identified for the 2-year landmark analysis. After propensity score weighting of 23 baseline covariates, statin use was associated with a significant reduction in composite liver decompensation events (HR: 0.55; 95% CI: 0.36-0.83; P = .005), ascites (HR: 0.57; 95% CI: 0.36-0.92; P = .02), and a dose-dependent decrease in death (HR: 0.87; 95% CI: 0.76-0.99; P = .035) relative to no statin use. CONCLUSIONS: Patients with chronic viral hepatitis who used statins have a reduced risk of liver decompensation and death compared to non-users in this propensity score weighted landmark analysis.

Laboratory parameter-based machine learning model for excluding non-alcoholic fatty liver disease (NAFLD) in the general population.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) affects 20%-40% of the general population in developed countries and is an increasingly important cause of hepatocellular carcinoma. Electronic medical records facilitate large-scale epidemiological studies, existing NAFLD scores often require clinical and anthropometric parameters that may not be captured in those databases. AIM: To develop and validate a laboratory parameter-based machine learning model to detect NAFLD for the general population. METHODS: We randomly divided 922 subjects from a population screening study into training and validation groups; NAFLD was diagnosed by proton-magnetic resonance spectroscopy. On the basis of machine learning from 23 routine clinical and laboratory parameters after elastic net regulation, we evaluated the logistic regression, ridge regression, AdaBoost and decision tree models. The areas under receiver-operating characteristic curve (AUROC) of models in validation group were compared. RESULTS: Six predictors including alanine aminotransferase, high-density lipoprotein cholesterol, triglyceride, haemoglobin A1c , white blood cell count and the presence of hypertension were selected. The NAFLD ridge score achieved AUROC of 0.87 (95% CI 0.83-0.90) and 0.88 (0.84-0.91) in the training and validation groups respectively. Using dual cut-offs of 0.24 and 0.44, NAFLD ridge score achieved 92% (86%-96%) sensitivity and 90% (86%-93%) specificity with corresponding negative and positive predictive values of 96% (91%-98%) and 69% (59%-78%), and 87% of overall accuracy among 70% of classifiable subjects in the validation group; 30% of subjects remained indeterminate. CONCLUSIONS: NAFLD ridge score is a simple and robust reference comparable to existing NAFLD scores to exclude NAFLD patients in epidemiological studies.

Oral nucleos(t)ide analogues reduce recurrence and death in chronic hepatitis B-related hepatocellular carcinoma.

BACKGROUND: In patients with chronic hepatitis B (CHB)-related hepatocellular carcinoma (HCC), high viral load was associated with tumour recurrence and deaths. AIMS: To investigate the effect of nucleos(t)ide analogues (NA) on the clinical outcomes after different HCC treatments. METHODS: A territory-wide cohort study was conducted using the database from Hospital Authority. We identified CHB patients with HCC by International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) diagnosis codes in 2000-2012. HCC treatments, NA use and laboratory parameters were retrieved. The primary endpoint was HCC recurrence and death. A 3-month landmark analysis was used to evaluate the primary outcome in patients with or without NA treatment. RESULTS: A total of 2198 CHB patients (1230 NA-untreated and 968 NA-treated) with HCC, receiving at least one type of HCC treatment were included in the analysis. At a median follow-up of 2.8 (IQR 1.4-4.9) years, tumour recurrence and death occurred in 451 (36.7%) and 578 (47.0%) untreated patients; and in 216 (22.3%) and 301 (31.1%) NA-treated patients respectively. NA therapy reduced the risk of overall HCC recurrence [adjusted sub-hazard ratio (SHR) 0.63, 95% confidence interval (CI) 0.49-0.80; P < 0.001]. The effect was most obvious in patients undergoing resection (SHR = 0.58, 95% CI = 0.37-0.91, P = 0.018). The possibility of NA therapy reducing the risk of death (HR = 0.82, 95% CI = 0.64-1.03, P = 0.092), is most obvious in resection subgroup (HR = 0.64, 95% CI = 0.41-0.99, P = 0.050) but insignificant in the other treatment groups. CONCLUSION: Our findings show that nucleos(t)ide analogues treatment reduces the risk of HCC recurrence in patients with chronic hepatitis B treated by surgical resection.

Rapid purification of porcine colostral whey lactoferrin by affinity chromatography on single-stranded DNA-agarose. Characterization, amino acid composition and N-terminal amino acid sequence.

We have determined that the major iron-binding and DNA-binding protein in porcine colostral whey is lactoferrin. This lactoferrin was purified to homogeneity in one chromatographic step using immobilized single-stranded DNA-agarose. Although different in chromatographic behavior from human lactoferrin, the porcine lactoferrin purified in this manner was shown to be homogeneous by high-performance ion-exchange chromatography (Mono-S), immobilized metal ion (Cu2+) affinity chromatography, size-exclusion chromatography (TSK-4000SW), and reverse-phase (phenyl) chromatography. Electrophoresis on SDS-polyacrylamide gradient (10-20%) gels under reducing conditions showed the purified lactoferrin to be a single protein (silver-stained) of 78 kDa. Apolactoferrin purified in this manner bound iron and displayed a UV/VIS absorption spectrum indistinguishable from that of human lactoferrin. The molar absorption coefficient of hololactoferrin was 3.86 x 10(3) M-1 at 465 nm and 1.08 x 10(5) M-1 at 280 nm. Affinity elution analyses of the purified lactoferrin on immobilized DNA revealed that the affinity of this protein for DNA was independent of bound iron. Porcine lactoferrin was recognized by antibodies directed against human lactoferrin and bovine lactoferrin. The amino acid composition and N-terminal amino acid sequence analysis (30 residues) revealed a high degree of sequence homology with human, equine and bovine lactoferrin. These results demonstrate the effectiveness of immobilized DNA as a rapid and simple lactoferrin purification procedure and demonstrate the presence of a lactoferrin in porcine colostral whey with a high degree of sequence homology to human lactoferrin.

Direct evidence of the generation in human stomach of an antimicrobial peptide domain (lactoferricin) from ingested lactoferrin.

The ability to define specific alterations in the structure and function of proteins as they are introduced and processed in vivo remains an important goal. We have evaluated the generation, in vivo, of an antimicrobial peptide (lactoferricin) derived from ingested bovine lactoferrin by surface-enhanced laser desorption/ionization (SELDI). SELDI was used in the affinity mass spectrometry operational mode to detect and quantify lactoferricin directly from unfractionated gastric contents using a chemically defined ligand with a terminal n-butyl group as the lactoferricin affinity capture device. By this method, we were able to detect and quantify lactoferricin directly upon examination of unfractionated gastric contents recovered from an adult subject 10 min after ingestion of bovine lactoferrin (200 ml of 10 mg/ml (1.2 x 10(-4) mol/l) solution). Lactoferricin produced in vivo was directly captured by a surface-enhanced affinity capture (SEAC) device composed of molecules with a terminal n-butyl group and analyzed by laser desorption/ionization time-of-flight mass spectrometry. The recovery of standard lactoferricin or lactoferrin added to an aliquot of the gastric contents was determined to be nearly 100%, confirming the efficiency of this method. The amount of lactoferricin detected in the gastric contents was 16.9+/-2.7 microg/ml (5.4+/-0.8 x 10(-6) mol/l). However, a large proportion of ingested lactoferrin was found to be incompletely hydrolyzed. Lactoferrin fragments containing the lactoferricin region were analyzed by in situ pepsin hydrolysis after being captured on the SEAC device. Partially degraded lactoferrin fragments containing the lactoferricin region, including fragments corresponding to positions 17-43, 17-44, 12-44, 9-58 and 16-79 of the bovine lactoferrin sequence, were found to be present at concentrations as high as 5.7+/-0.7 x 10(-5) mol/l. These results suggest that significant amounts of bovine lactoferricin would be produced in the human stomach following ingestion of food, such as infant formula, supplemented with bovine lactoferrin. We propose that physiologically functional quantities of human lactoferricin could be generated in the stomach of breast-fed infants, and possibly, in the case of adults, from lactoferrin secreted into saliva.

Phosphorylation of human progesterone receptor by cyclin-dependent kinase 2 on three sites that are authentic basal phosphorylation sites in vivo.

The human progesterone receptor (hPR) in T47D breast cancer cells is phosphorylated on at least nine different serine residues. We have previously reported the identification of five sites; three are hormone inducible (Ser102, Ser294 and Ser345), and their phosphorylation correlates with the timing of the change in receptor mobility on gel electrophoresis in response to hormone treatment. The other two sites, Ser81 and Ser162, along with the remaining sites, are basally phosphorylated and exhibit a general increase in phosphorylation in response to hormone. With the exception of Ser81, all of these sites are in Ser-Pro motifs, suggesting that proline-directed kinases are responsible for their phosphorylation. We now report that cyclin A-cyclin-dependent kinase-2 complexes phosphorylate hPR-B in vitro with a high stoichiometry on three sites that are authentic basal sites in vivo. One of these is Ser162, which has been described previously. The other two sites are identified here as Ser190 and Ser400. The specificity and stoichiometry of the in vitro phosphorylation suggest that hPR phosphorylation may be regulated in a cell cycle-dependent manner in vivo.

Unique molecular properties of a urea- and salt-stable DNA-binding estrogen receptor dimer covalently labeled with the antiestrogen [3H]desmethylnafoxidine aziridine. A comparison with the estrogen-receptor complex.

A new antiestrogen affinity ligand for the covalent labeling of estrogen receptors, [3H]desmethylnafoxidine aziridine, has been used to investigate the salt- and temperature-independent formation of DNA-binding estrogen receptor forms from untransformed (300 kilodaltons) receptor. Calf uterine estrogen receptor proteins labeled with [3H]estradiol or [3H]desmethylnafoxidine aziridine were quantitatively transformed (greater than 90%) to their DNA-binding configuration in low ionic strength buffers by brief exposure to 3 M urea at 0 C. The urea effect was hormone-dependent and partially reversible. The transformed receptors were purified (ca 250-fold) by affinity chromatography on single-stranded DNA-agarose in the continued presence of 3 M urea to prevent transformation reversal. Scatchard analyses revealed a single class of high affinity radioligand binding sites (Kd = 0.34 nM) unchanged by urea-induced transformation and purification. The DNA-binding receptor form labeled with [3H]desmethylnafoxidine aziridine was stable as a probable dimer in 3 M urea with 0.4 M KCl and displayed no evidence of size (Stokes radius 7.3 to 7.5 nm; 4.2 to 4.3 S; Mr = 136,800) heterogeneity. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis indicated the presence of an intact 67 kDa steroid-binding receptor subunit. Reverse-phase chromatography of the covalently labeled receptor on C4 and phenyl stationary phases revealed no evidence of structural heterogeneity. The surface charge of the estrogen- and antiestrogen-receptor complexes, however, was distinctly different in both the presence and absence of 3 M urea. Thus, exposure to urea was an effective salt- and temperature-independent means for achieving the complete transformation of receptor to its stable DNA-binding dimer configuration. The ligand-induced differences in receptor surface charge and the urea effects on DNA-binding (but not hormone-binding) suggest that both electrostatic and hydrophobic or hydrogen bonding receptor domains are influenced by ligand binding.

Carpal ligament injuries with acute scaphoid fractures - a combined wrist injury.

Fifty-two consecutive patients with acute scaphoid fractures were treated by closed reduction and percutaneous screw fixation. In addition, all underwent a wrist arthrogram and 22 patients also underwent a wrist arthroscopy. Eighteen patients (34%) had an associated carpal ligament injury (four scapholunate ligament, eight lunotriquetral ligament, two combined, three TFCC and one minor leak from the distal carpal row). These patients had worse Mayo wrist scores (no ligament injury median score=95: ligament injury median score=85) at a mean final follow-up of 61 weeks. We advocate that scaphoid fractures with associated carpal ligament injuries should be defined as a combined wrist injury.

Mapping and sequence-specific identification of phosphopeptides in unfractionated protein digest mixtures by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

We have demonstrated a procedure for the rapid (minutes), sensitive (less than pmol), and sequence-specific identification of phosphopeptides in unfractionated digests of phosphoproteins using matrix-assisted UV laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry. The mass-dependent identification of one specific 13-residue phosphopeptide (S105-K117), observed among the 153 possible trypsin digest fragments of human beta-casein (211 residues), was confirmed by amino acid sequence analysis of the 33P-labeled peptide after isolation by reverse-phase HPLC. MALDI-TOF was also used to monitor the rate and extent to which an 18-residue N-terminal beta-casein peptide (R1-K18) was phosphorylated in vitro. These results demonstrate that MALDI-TOF may be used (i) to facilitate the identification of sequence-specific sites of protein phosphorylation and dephosphorylation, (ii) to monitor protein and peptide phosphorylation and dephosphorylation reaction rates, even in complex unfractionated mixtures, (iii) to determine the minimum primary structure necessary for the phosphorylation of specific protein surface domains, and (iv) to evaluate the effects of intact protein phosphorylation and dephosphorylation on susceptibility to subsequent proteolytic events.

Recognition of transition metal ions by peptides. Identification of specific metal-binding peptides in proteolytic digest maps by UV laser desorption time-of-flight mass spectrometry.

Metal-binding peptides in proteolytic digest maps have been identified by matrix-assisted UV laser desorption time-of-flight mass spectrometry (LDTOF-MS). The plasma and milk metal transport protein chosen to demonstrate this process, histidine-rich glycoprotein (HRG), was purified and then digested with trypsin; the cleavage products were analyzed by LDTOF-MS with dihydroxybenzoic acid as the matrix. The selective interaction of specific peptides with one or more Cu atoms was observed when Cu(II) ions were added to the digest mixture. At least one specific metal-binding peptide was identified by computerized sequence analysis using the molecular mass data and available cDNA sequence. These results demonstrate the first direct observation by mass spectrometry of differential peptide-metal ion interactions in protein digest maps. The ability to evaluate peptide-metal ion interactions, including stoichiometry, with less than 1 pmol of sample improves significantly our ability to identify metal binding domains in metal-binding proteins.