Eliminating Absorbing Interference Using The H-point Standard Addition Method: Case of Griess Assay in The Presence of Interferent Heme Enzymes Such As NOS

Standard calibration methods used to determine trace analytes usually yield significant deviations from the actual analyte value in the presence of interferents in the assay media. These deviations become of particular concern when the concentration of the analyte is low, and when the results are used to draw mechanistic or kinetic conclusions, for instance in enzyme structure-function studies. In these circumstances, the H-point standard addition method (HPSAM) provides superior precision and accuracy. This method is developed here for the case of the spectrophotometric Griess assay used to determine nitrite in various enzymology investigations, such as nitrite determination in studies of nitrite reductases (NiR), or when determining nitrite as a breakdown product of nitric oxide synthesized by NOS enzymes. The results obtained by HPSAM are contrasted with those of the traditional calibration method

A Clinicopathological Study of Urinary Bladder Neoplasms in Patients at Three Centers in Khartoum, Sudan

OBJECTIVES: To present the histopathological pattern of urinary bladder neoplasms using the WHO/ISUP classification system and relate it to the outcome.METHODS: This study was conducted in the period from January 2004 through December 2005 at three centres in Khartoum, Sudan. One hundred and six patients with urinary bladder neoplasms were included in the study.RESULTS: The commonest affected age group was 60-80 years with male to female ratio 4.6:1. Urothelial neoplasms were found in 72 (67.9%), Squamous cell carcinoma (SCC) in 26 (24.5%), urothelial neoplasms with Squamous differentiation in 3 (2.8%), and other types in 5 (4.7%) of thepatients. There were 43.4% of the urothelial neoplasms graded as papillary carcinoma of high grade, 52.6% papillary carcinoma of low grade, 1.3% papillary neoplasm of low malignant potential, 1.3% papilloma, and 1.3% was graded as flat neoplasm. Of the SCCs, twelve (42.9%)were poorly differentiated SCCs, nine (32.1%) moderately differentiated, and seven (25%) cases were well differentiated SCCs. Follow-up information was available in 32 patients. At last followup, fifteen (46.9%) patients were dead of the disease, twelve (35.5%) were alive with no evidence of disease, four (12.5%) were alive with disease, and one (3.1%) was alive and terminally ill. CONCLUSION: Histological grade (P: 0.006), and muscle invasion (P: 0.002) were significantly associated with survival. A subset of the cases could not be assessed for muscle invasion due toinadequate sampling; we thus recommend proper trans-urethral bladder biopsy (TUBP) sampling.Key words: Urinary bladder neoplasm; Papillary carcinoma; Squamous cell carcinoma; WHO/ISUP classification system

Reductive Decomposition of A Diazonium Intermediate by Dithiothreitol Affects The Determination of NOS Turnover Rates

Accurate determination of nitrite either as such or as the breakdown product of nitric oxide (NO) is critical in a host of enzymatic reactions in various settings addressing structure–function relationships, as well as mechanisms and kinetics of molecular operation of enzymes. The most common way to quantify nitrite, for instance in nitric oxide synthase (NOS) mechanistic investigations, is the spectrophotometric assay based on the Griess reaction through external standard calibration. This assay is based on a two-step diazotization reaction, in which a cationic diazonium derivative of sulfanilamide is formed as intermediate before the final absorbing azo-product. We show that this intermediate is very sensitive to reducing agents that may be transferred from the reaction media under investigation. The interaction of this vital intermediate with the reducing agent, dithiothreitol (DTT), which is widely used in NOS reactions, is characterized by both electrochemical and spectroscopic means. The effect of DTT on the performance of external calibration, both in sample recovery studies and in actual NOS reactions, is presented. Finally an alternative method of standard additions, which partially compensates for the accuracy and sensitivity problems of external calibration, is proposed and discussed

Electrochemical Carbonylation of Organoiron Methyl Complex: A Study of Reaction Intermediates

The one-electron reduction of CpFe(CO)2CH3 has been investigated by voltammetry and Fourier transform IR spectroelectrochemistry. The reduction initiates the insertion of CO ligand in the FeCH3 bond. The dissociation of a CO group proceeds in a parallel reaction. Reaction intermediates, the acyl derivative and released CO, form the radical anion of a complex CpFe(CO)2(COCH3) which is able to reduce the parent compound. The reversible redox potential − 1.8 V of CpFe(CO)2(COCH3) allows the regeneration of its radical anion which drives a catalytic cycle. The lifetime of intermediates is shortened by side reactions, one of which is the migration of the acyl group from the central atom to the cyclopentadienyl ring. This explains the apparent discrepancy between products observed in preparative scale electrolysis and the absence of catalytic effects in routine voltammetric experiments

Probe Functionalization With A Rhop-3 Antibody: Toward A Rhop-3 Antigen Immunosensor For Detection of Malaria

The antibody specific for the malaria protein, Rhop-3, and FL-Rhop-3, were immobilized on the surface of a gold electrode modified with cysteamine. Colloidal gold was used to enhance the detection signal for Rhop-3 antigens. The Rhop-3 antibody was also immobilized on gold electrodes preactivated with dithiobis(succinimidyl proprionate) (DSP). Immobilization was performed at room temperature and at 37 °C. Cyclic voltammetry (CV) was used to monitor the interaction between the immobilized antibody and its cognate antigen in solution, using ferricyanide, K3Fe(CN)6, as reporting electroactive probe. Tests indicate recognition of Rhop-3 protein by the immobilized antibody. Antigen recognition was enhanced by incubation at 37 °C compared with room-temperature incubation. Our results suggest that an immunosensor can be developed and optimized to aid detection of Rhop-3 antigens in samples from malaria patients. As far as we are aware, this is the first amperometric immunosensor targeting Rhop-3 antigen as a malaria biomarker

Post-surgical follow-up (by ELISA and immunoblotting) of cured versus non-cured cystic echinococcosis in young patients

The study was designed to determine comparatively the prognostic value of immunoblotting and ELISA in the serological follow-up of young cystic echinococcosis (CE) patients exhibiting either a cured or a progredient (non-cured) course of disease after treatment. A total of 54 patients (mean age 9 years, range from 3 to 15 years) with surgically, radiologically and/or histologically proven CE were studied for a period up to 60 months after surgery. Additionally, some of the patients underwent chemotherapy. Based on the clinical course and outcome, as well as on imaging findings, patients were clustered into 2 groups of either cured (CCE), or non-cured (NCCE) CE patients. ELISA showed a high rate of seropositivity 4 to 5 years post-surgery for both CCE (57·1%) and NCCE (100%) patients, the difference found between the two groups was statistically not significant. Immunoblotting based upon recognition of AgB subcomponents (8 and 16kDa bands) showed a decrease of respective antibody reactivities after 4 years post-surgery. Only sera from 14·3% of CCE patients recognized the subcomponents of AgB after 4 years, while none (0%) of these sera was still reactive at 5 years post-surgery. At variance, immunoblotting remained positive for AgB subcomponents in 100% of the NCCE cases as tested between 4 and 5 years after surgical treatment. Immunoblotting therefore proved to be a useful approach for monitoring post-surgical follow-ups of human CCE and NCCE in young patients when based upon the recognition of AgB subcomponent

Rumen fermentation pattern and blood metabolites of lambs from three breeds reared on pasture or feedlot

Publishe

Cholesterol Levels and Activity of Membrane Bound Proteins: Characterization by Thermal and Electrochemical Methods

The long-term goal of this investigation is to study the effects of increased cholesterol levels on the molecular activity of membrane-bound enzymes such as nitric oxide synthase, that are critical in the functioning of the cardiovascular system. In this particular investigation, we used differential scanning calorimetry (DSC) and dielectric thermal analysis (DETA) to study the effect of added cholesterol on melting/recrystallization and dielectric behavior, respectively, of phosphatidylcholine (PC) bilayered thin films. We also used electrochemical methods to investigate the effect of added cholesterol on the redox behavior of the oxygenase domain of nitric oxide synthase as a probe embedded in the PC films. The results show that added cholesterol in the PC films seems to depress the molecular dynamics as indicated by lowered current responses in the presence of cholesterol as well as a slight increase of the transition temperature in the overall two-phase regime behavior observed in PC–cholesterol films. These results are rationalized in the context of the general DSC and DETA behaviors of the PC–chol films