High-Performance Liquid Chromatography Method for Rich Pharmacokinetic Sampling Schemes in Translational Rat Toxicity Models With Vancomycin.

A translational need exists to understand and predict vancomycin-induced kidney toxicity. We describe: (i) a vancomycin high-performance liquid chromatography (HPLC) method for rat plasma and kidney tissue homogenate; (ii) a rat pharmacokinetic (PK) study to demonstrate utility; and (iii) a catheter retention study to enable future preclinical studies. Rat plasma and pup kidney tissue homogenate were analyzed via HPLC for vancomycin concentrations ranging from 3-75 and 15.1-75.5 µg/mL, respectively, using a Kinetex Biphenyl column and gradient elution of water with 0.1% formic acid: acetonitrile (70:30 v/v). Sprague-Dawley rats (n = 10) receiving 150 mg/kg of vancomycin intraperitoneally had plasma sampled for PK. Finally, a catheter retention study was performed on polyurethane catheters to assess adsorption. Precision was <6.1% for all intra-assay and interassay HPLC measurements, with >96.3% analyte recovery. A two-compartment model fit the data well, facilitating PK exposure estimates. Finally, vancomycin was heterogeneously retained by polyurethane catheters.

Evaluation of liposomal nanocarriers loaded with ETB receptor agonist, IRL-1620, using cell-based assays.

One common feature of most neurodegenerative diseases, including Alzheimer's disease (AD) and stroke, is the death of neuronal cells. Neuronal cell death is associated with apoptosis, generation of reactive oxygen species and oxidative stress. Neuronal cell death pathways can be reversed by endothelin B receptor agonist, IRL-1620, which was found to enhance neuroprotection by promoting vascular and neuronal growth in a rodent stroke model. Previous studies conducted at our institution indicated that the treatment with IRL-1620 significantly improved neurological and motor function while reducing oxidative stress and overall infarct area. IRL-1620 is a hydrophilic, 15 amino acid peptide and has a molecular weight of 1820Da. In this study, we have encapsulated IRL-1620 in PEGylated liposomes in order to enhance its efficacy. Each batch of liposomes encapsulating IRL-1620 was evaluated for particle size, polydispersity index, and charge (zeta potential) over a period of time to determine their stability. A dose-response bar graph was plotted based on the effect of neuroprotection by free IRL-1620 on differentiated neuronal PC-12 cells. The 1nM concentration was found to have the highest cell viability. The liposomes loaded with IRL-1620 were tested on differentiated neuronal PC-12 cells for their neuroprotective ability against apoptosis caused by removal of nerve growth factor (NGF) against free (non-encapsulated) IRL-1620. The liposomal IRL-1620 was found to proliferate the growth of serum-deprived differentiated PC-12 cells significantly (p<0.0001). In the western blot analysis, the expression of the anti-apoptotic marker, BCL-2 was found to be increased, and that of pro-apoptotic marker, BAX was found to be decreased with liposomal IRL-1620. The effects were found to be independent of the NGF levels. Finally the free IRL-1620 was found to cause neuronal outgrowth equivalent to the 75ng/ml NGF treatment.

Observer variability of an angiographic grading scale used for the assessment of intracranial aneurysms treated with flow-diverting stents.

BACKGROUND AND PURPOSE: Novel angiographic grading scales for the assessment of intracranial aneurysms treated with flow-diverting stents have been recently developed because previous angiographic grading scales cannot be applied to these aneurysms. The purpose of this study was to evaluate the inter- and intraobserver variability of the novel O'Kelly Marotta grading scale, which was developed specifically for the angiographic assessment of aneurysms treated with flow-diverting stents. MATERIALS AND METHODS: Multiple raters (n = 31) from the disciplines of neuroradiology and neurosurgery were presented with pre- and posttreatment angiographic images of 14 aneurysms treated with intraluminal flow diverters. Raters were asked to classify pre- and posttreatment angiograms by using the OKM grading scale. Statistical analyses were subsequently performed with calculation of a generalized multirater κ statistic for assessment of inter- and intraobserver variability and by performing a Wilcoxon signed rank sum test for assessment of group differences. RESULTS: Variability analysis of the OKM grading scale yielded substantial (κ = 0.74) and almost perfect (κ = 0.99) inter- and intraobserver agreement, respectively, with no statistically significant differences between raters with a background of neuroradiology versus neurosurgery or attending physician versus trainee. CONCLUSIONS: The OKM grading scale for the assessment of intracranial aneurysms treated with flow-diverting stents is a reliable grading scale that can be used equally well by users of varying backgrounds and levels of training. Comparison with interobserver variability of pre-existing angiographic grading scales shows equal or better performance.

Cooperativity of the MUC1 oncoprotein and STAT1 pathway in poor prognosis human breast cancer.

Signal transducer and activator of transcription 1 (STAT1) is activated in the inflammatory response to interferons. The MUC1 oncoprotein is overexpressed in human breast cancers. Analysis of genes differentially expressed in MUC1-transformed cells has identified a network linking MUC1 and STAT1 that is associated with cellular growth and inflammation. The results further show that the MUC1-C subunit associates with STAT1 in cells and the MUC1-C cytoplasmic domain binds directly to the STAT1 DNA-binding domain. The interaction between MUC1-C and STAT1 is inducible by IFNgamma in non-malignant epithelial cells and constitutive in breast cancer cells. Moreover, the MUC1-STAT1 interaction contributes to the activation of STAT1 target genes, including MUC1 itself. Analysis of two independent databases showed that MUC1 and STAT1 are coexpressed in about 15% of primary human breast tumors. Coexpression of MUC1 and the STAT1 pathway was found to be significantly associated with decreased recurrence-free and overall survival. These findings indicate that (i) MUC1 and STAT1 function in an auto-inductive loop, and (ii) activation of both MUC1 and the STAT1 pathway in breast tumors confers a poor prognosis for patients.

Familial clustering of cancer in two tertiary care hospitals in Nairobi, Kenya.

OBJECTIVE: To describe the occurrence of cancers in families of individuals diagnosed cancer. DESIGN: Cross-sectional descriptive study. SETTING: Outpatient cancer clinics at Kenyatta National Hospital (KNH) and Radiotherapy Clinic at Nairobi Hospital. SUBJECTS: Patients with a tissue histological or cytological diagnosis of cancer. MAIN OUTCOME MEASURES: A reported family history of cancer. RESULTS: A total number of 485 cancer patients were recruited, 382, from KNH and 103 from Nairobi Hospital. These index cases had 45 different types of cancer, with the most common being breast and uterine-cervical malignancies. Prevalence of family history of cancer was found to be 18.8% and was highest among 1st degree relatives. Documentary evidence was seen in 48.4% and history of cancer corroborated by medical personnel in an additional 11%. In 18.7% of cases more than one relative was interviewed to confirm the family history of cancer. Educational levels of the index cases correlated with knowledge of family history of cancer, with those of higher educational level having been more informed about their families' medical history. There was a prevalence of familial cancers of 30% at Nairobi Hospital patients and 15.7% at KNH patients. CONCLUSION: We found the prevalence of family history of cancer in our population to be 18.8% and was highest among 1st degree relatives. This has implications for targeted screening and therefore early diagnosis which is beneficial.

Synthesis of alpha-amyrin derivatives and their in vivo antihyperglycemic activity.

Ficus racemosa belongs to the family of Moraceae and is commonly known as 'Gular' in north India. Bio-activity guided isolation work on the fruits of F. racemosa resulted in the identification of antidiabetic active principle, alpha-amyrin acetate 7. Compound 7 lowered the blood glucose levels by 18.4 and 17.0% at 5 and 24 h, respectively, in sucrose challenged streptozotocin induced diabetic rat (STZ-S) model at the dose of 100 mg/kg body weight. Fifteen novel derivatives viz, 9-21, 24, 25 of alpha-amyrin 8 were prepared and their antihyperglycemic activity profile was assessed. The p-chlorobenzoic acid derivative 9 and nicotinic acid derivative 14 showed potent antihyperglycemic activity at 100 mg/kg body weight.

Acute coronary syndromes amongst type 2 diabetics with ischaemic electrocardiograms presenting to accident and emergency department of a Kenyan tertiary institution.

OBJECTIVE: To determine the prevalence of acute coronary syndromes among type 2 diabetic patients presenting to Accident and Emergency department. DESIGN: Prospective cross-sectional study. SETTING: Kenyatta National Hospital, a tertiary teaching and referral hospital. SUBJECTS: Type 2 diabetic patients with ischaemic electrocardiograms (ECG). MAIN OUTCOME MEASURES: Demographics, clinical symptoms, cardiovascular status and risk factors--central obesity, hypertension, dyslipidaemia, smoking. RESULTS: From 12,307 accident and emergency attendees, 400 (33%) diabetics aged > OR =30 years were screened with a resting ECG and 95 (24%) with ischaemic ECG were recruited; age range 41-87 years, 60% were male; diabetes duration ranged 0-30 years with 8.4% being newly diagnosed. The commonest enrolling ECG feature was nonspecific ST-T changes. The commonest presenting complaint were fatigue and dyspnoea. Majority had three coronary artery disease (CAD) risk factors: obesity 86%, elevated LDL 73% and hypertension 60%. Therapy in use was OHA 43%, insulin 42%, insulin and OHA 1%; prophylactic aspirin 14.7% and statins 8.4%. Thirty four (35.8%) were classified as acute coronary syndrome (ACS); 29 (30.5%) acute myocardial infarction (ACS-AMI) and five (5.2%) unstable angina (ACS-UA). Majority (79.4%) of the ACS presented more than six hours after symptom onset and majority had features of acute left ventricular failure. CONCLUSIONS: Acute coronary syndrome accounted for 35% of the morbidity in type 2 diabetics with ischaemic ECG's presenting to KNH accident and emergency department; patients presented late and 80% were not on CAD prophylactic therapy.

High-sensitivity C-reactive protein in type 2 diabetic patients with and without the metabolic syndrome.

OBJECTIVE: To describe the distribution of serum high-sensitivity C-reactive protein (hsCRP) in type 2 diabetes mellitus outpatients, and relate it to cardiovascular disease risk. DESIGN: Cross-sectional descriptive study. SETTING: Kenyatta National Hospital, a tertiary referral hospital. SUBJECTS: One hundred and ninety seven type 2 diabetic outpatients and fifty age- and sex-matched non-diabetic hypertensive outpatients. RESULTS: The distribution of hsCRP in the diabetic population was skewed, with a mean of 4.33 mg/L and a median of 2.53 mg/L. The majority (42%) of diabetics had hsCRP levels in the high-risk category (hsCRP > 3 mg/L). The median hsCRP was non-significantly higher in the diabetic patients with metabolic syndrome compared to those without (2.68 vs 2.30 mg/L, p = 0.433). The median hsCRP was non-significantly higher in the hypertensive group compared to that in matched diabetic non-metabolic syndrome group (2.30 vs 2.23 mg/L, p = 0.297). HsCRP increased with number of metabolic syndrome components, patients with four components having higher hsCRP levels than those with one, though the difference was not statistically significant (3.59 vs 1.57 mg/L, p = 0.095). CONCLUSION: Our study, though cross-sectional in nature, supports the existence of a correlation between hsCRP levels and cardiovascular disease risk. The small difference in CRP levels between diabetic metabolic and non-metabolic groups underpowered the study. Cohort studies are needed to determine the predictive power of hsCRP for cardiovascular disease in our setup.

Prevalence of dyslipidemia and dysglycaemia in HIV infected patients.

BACKGROUND: Highly active antiretroviral therapy (HAART) has dramatically reduced AIDS morbidity and mortality, however long-term metabolic consequences including dysglycaemia and dyslipidemia have raised concern regarding accelerated cardiovascular disease risk. OBJECTIVE: To determine the period prevalence of dyslipidemia and dysglycaemia in HIV-infected patients. DESIGN: Cross-sectional comparative group study. SETTING: Kenyatta National Hospital, a tertiary HIV dedicated out-patient facility. SUBJECTS: Consecutive HIV- positive adult patients. MAIN OUTCOME MEASURES: Dyslipidemia: presence of raised total or LDL cholesterol or low HDL cholesterol, or raised triglycerides. Dysglycaemia: presence of impaired fasting glucose or impaired glucose tolerance, or diabetes mellitus. RESULTS: Between January and April 2006, out of 342 screened patients, 295 were recruited and 58% were females. One hundred and thirty four (45%) were on HAART, 82% of whom were on stavudine, lamivudine and either nevirapine or efavirenz. Overall prevalence of dyslipidemiawas 63.1% and dysglycaemia was 20.7%. High total cholesterol occurred in 39.2% of HAART and 10.0% HAART naive patients (p<0.0001, OR 5.18, CI 3.11-10.86), whereas high LDL cholesterol occurred in 40.8% and in 11.2% respectively (p<0.0001, OR 5.43, CI 2.973-9.917). HDL levels were low in 14.6% and 51.3% among HAART and HAART naive patients, respectively, (p<0.0001, OR 0.16, CI 0.091-0.29) while high triglycerides occurred in 25.6% and 22.5% respectively (p=0.541 OR 1.184 CI 0.688-2.037). Among patients on HAART compared to HAART naive patients, diabetes was found in 1.5% against 1.2% (p=0.85), impaired fasting in 2.2% against 0.6% (p=0.30) and impaired glucose tolerance in 16.4% against 21.1% (p=0.22), respectively. CONCLUSIONS: HIV- infected patients demonstrated a high prevalence of dyslipidemia. HAART use was associated with high levels of total, and LDL cholesterol and high triglyceride levels, an established athrogenic lipid profile. However, HAART was not associated with low HDL cholesterol and had no significant effect on dysglycaemia.

Fertility rate of epileptic women at Kenyatta National Hospital.

BACKGROUND: Reproductive health issues of women with epilepsy (WWE) are complex and multifaceted, and both epilepsy and antiepileptic drug (AEDs) use may alter fertility and pregnancy outcomes in these women. OBJECTIVE: To determine the fertility rate of women with epilepsy at Kenyatta National Hospital (KNH). DESIGN: Cross-sectional study. SETTING: Neurology clinic, KNH, Nairobi, Kenya, between October 2006 and March 2007. SUBJECTS: A total of 191 women with epilepsy (aged 15-49 years) who had been epileptic for at least one year were interviewed regarding their pregnancy and birth histories, and the information validated with medical records where available. RESULTS: The general fertility rate (GFR) for reproductive-aged epileptic women for the three-year period (2003-2006) preceding the study was 46 livebirths per 1000 women-years (95% CI 35.13-63.59). CONCLUSION: Fertility rate in epileptic women is decreased by two thirds (compared to that of general population of women in Kenya). Reasons for this are probably miltifactorial.

Incidence of chemotherapy-related tumour lysis syndrome at Kenyatta National Hospital, Nairobi.

OBJECTIVES: To estimate the magnitude of laboratory defined Tumour Lysis Syndrome (TLS) at Kenyatta National Hospital (KNH), identify its pattern of presentation, resolution, and determine the biochemical outcome of affected patients. DESIGN: Prospective patient-treatment cohort study. SETTING: Kenyatta National Referral and Teaching Hospital, between November 2004 and April 2005. SUBJECTS: One hundred and forty two patients receiving first course chemotherapy. MAIN OUTCOME MEASURE: Laboratory defined Tumour Lysis Syndrome (TLS). RESULTS: One hundred and eleven patients completed the study protocol. Forty two patients (37.8%) developed TLS. The incidence in haematological malignancies was 75.5% while in non-haematological malignancies was 3.6%. Hyperphosphataemia and hyperkalaemia were the most consistent diagnostic parameters while hyperuricaemia occurred in only one patient. No patient developed hypocalcaemia. Ninety five percent of patients developed TLS within the first three days of receiving chemotherapy while 55% resolved in the first week. Two TLS case mortalities occurred. CONCLUSIONS: The incidence of TLS in this cohort study was 38%, and was highest among haematological malignancies. No cases occurred in breast cancer patients. Majority of the cases were diagnosed on the basis of increase in serum phosphate and potassium; uric acid did not rise predominantly due to prophylactic uricosuric therapy. A majority (95%) developed within three days of commencing chemotherapy.

Total lymphocyte count as a surrogate marker for CD4+ t cell count in initiating antiretroviral therapy at Kenyatta National Hospital, Nairobi.

OBJECTIVE: To evaluate the utility of Total Lymphocyte Count (TLC) as a surrogate marker for CD4 + T cell count in antiretroviral (ARV) treatment initiation in a Kenyan population of HIV seropositive patients at Kenyatta National Hospital. DESIGN: Cross-sectional descriptive study. SETTING: Kenyatta National Hospital, HIV treatment and follow-up outpatient facility; Comprehensive Care Centre, Nairobi, Kenya. SUBJECTS: Two hundred and twenty five HIV Elisa positive, ARV naive patients visiting the Comprehensive Care Centre between January 2006 to March 2006. RESULTS: A significant linear correlation was found between TLC and CD4 cell count for the whole group with a Spearman rank correlation of 0.761 (p < 0.01); and was also independently observed in the four WHO clinical stages. The classification utility of TLC 1200 cells/mm3 cut-off was suboptimal; sensitivity 37% specificity of 99% and the NPV of 56%. The receiver operator characteristics (ROC) curve generated an optimal TLC cut-off of 1900 cells/mm3 cut-off to be of greatest utility with a sensitivity of 81.1%, specificity of 90.3%, PPV of 90.8% and NPV of 80.2%. This implies that a TLC cut-off of 1900 cells/mm3 correctly classify eight out of ten HIV positive patients as having a CD4 < 200 cells/mm3 and only misclassify two such patients. Serial CD4 testing can then be performed on the minority of patients who despite a TLC > or = 1900 cells/mm3 are, on basis of clinical data, suspect of more advanced disease warranting ARV therapy. This would reduce the number of patients tested for and focus the application of CD4 testing and thus reduce attendant cost in care provision in CD4 resource poor settings. CONCLUSION: Our data showed a good positive correlation between TLC and CD4 cell count, however the WHO recommended TLC cuto-ff of 1200/mm3 was found to be of low sensitivity in classifying patients as having a CD4 counts < 200 cells/mm3. This would result in underestimation of advanced stage of disease and to withholding ARVs treatment to persons who need treatment. We recommend a TLC cut-off of 1900 cells/mm3 for our population to classify patients as either above or below the CD4 count cut-off of 200 cells/mm3 as an indicator of when to start antiretroviral therapy.

Coronary artery disease and symptoms of depression in a Kenyan population.

BACKGROUND: Depression and heart disease are replacing the traditional enemies of Africa such as infectious diseases and malnutrition as the increasing causes of disability and premature death. Little is known about the co-morbidity of heart disease and depression in Africa. OBJECTIVE: To describe the prevalence of depression in Black Africans with and without Coronary Artery Disease as documented on coronary angiography at the Nairobi Hospital. DESIGN: Prospective comparative study. SETTING: A private not for Profit 210 bed hospital, catering for fee paying middles class clintele. RESULTS: Of the eighteen patients with an abnormal angiogram, the highest score on the BDI was 9 while the average was 2.11. Of the seven with normal angiograms, the highest BDI was 5, and the average was 1.71. There was no statistical significance in these differences. CONCLUSION: While African scientists must continue to concentrate on the urgent medical priorities of today (AIDS, malaria, measles, etc), cognisance has to be made of the other emerging epidemic, of the co-morbidity of coronary artery disease and depression. That no significant difference in depression score between the two groups was found could be due to a number of reasons including the small sample size achieved in this first study of its kind in Kenya.

Platelet functions in patients with meningococcal meningitis at the Kenyatta National Hospital, Nairobi.

OBJECTIVE: To determine platelet abnormalities in patients with menigococcal meningitis. DESIGN: Case control study. SUBJECTS: Fifty seven cases of mennigococcal meningitis based on a cerebrospinal fluid gram stain for gram negative diplococcus or positive culture were recruited. Fifty-seven controls matched for age and sex were also recruited. The following platelet functions tests were performed; platelet counts, platelet adhesiveness, platelet aggregation and clot retraction. RESULTS: Fifty seven patients (41 males and 16 females) with meningococcal meningitis were studied. Their mean age was 25.5 +/- 8.32 years with a range of 15 to 45 years. Five patients had purpura, four peripheral gangrene, eight conjunctival haemorrhages and one was in shock. There was a statistical significant difference in the platelet aggregation and clot retraction between the patients and controls at p-values of 0.0001 and 0.0002 respectively. There was no significant difference in the platelet count and adhesiveness between the patients and the controls at a p-value of 0.203 and 0.22 respectively. No association was found between the platelet functions and the clinical presentations. CONCLUSION: Patients with meningococcal meningitis have abnormalities in the platelet functions mainly in aggregation and adhesiveness.

Dissecting the electrostatic interactions and pH-dependent activity of a family 11 glycosidase.

Previous studies of the low molecular mass family 11 xylanase from Bacillus circulans show that the ionization state of the nucleophile (Glu78, pK(a) 4.6) and the acid/base catalyst (Glu172, pK(a) 6.7) gives rise to its pH-dependent activity profile. Inspection of the crystal structure of BCX reveals that Glu78 and Glu172 are in very similar environments and are surrounded by several chemically equivalent and highly conserved active site residues. Hence, there are no obvious reasons why their apparent pK(a) values are different. To address this question, a mutagenic approach was implemented to determine what features establish the pK(a) values (measured directly by (13)C NMR and indirectly by pH-dependent activity profiles) of these two catalytic carboxylic acids. Analysis of several BCX variants indicates that the ionized form of Glu78 is preferentially stabilized over that of Glu172 in part by stronger hydrogen bonds contributed by two well-ordered residues, namely, Tyr69 and Gln127. In addition, theoretical pK(a) calculations show that Glu78 has a lower pK(a) value than Glu172 due to a smaller desolvation energy and more favorable background interactions with permanent partial charges and ionizable groups within the protein. The pK(a) value of Glu172 is in turn elevated due to electrostatic repulsion from the negatively charged glutamate at position 78. The results also indicate that all of the conserved active site residues act concertedly in establishing the pK(a) values of Glu78 and Glu172, with no particular residue being singly more important than any of the others. In general, residues that contribute positive charges and hydrogen bonds serve to lower the pK(a) values of Glu78 and Glu172. The degree to which a hydrogen bond lowers a pK(a) value is largely dependent on the length of the hydrogen bond (shorter bonds lower pK(a) values more) and the chemical nature of the donor (COOH > OH > CONH(2)). In contrast, neighboring carboxyl groups can either lower or raise the pK(a) values of the catalytic glutamic acids depending upon the electrostatic linkage of the ionization constants of the residues involved in the interaction. While the pH optimum of BCX can be shifted from -1.1 to +0.6 pH units by mutating neighboring residues within the active site, activity is usually compromised due to the loss of important ground and/or transition state interactions. These results suggest that the pH optima of an enzyme might be best engineered by making strategic amino acid substitutions, at positions outside of the "core" active site, that electrostatically influence catalytic residues without perturbing their immediate structural environment.

Hydrogen bonding and catalysis: a novel explanation for how a single amino acid substitution can change the pH optimum of a glycosidase.

The pH optima of family 11 xylanases are well correlated with the nature of the residue adjacent to the acid/base catalyst. In xylanases that function optimally under acidic conditions, this residue is aspartic acid, whereas it is asparagine in those that function under more alkaline conditions. Previous studies of wild-type (WT) Bacillus circulans xylanase (BCX), with an asparagine residue at position 35, demonstrated that its pH-dependent activity follows the ionization states of the nucleophile Glu78 (pKa 4.6) and the acid/base catalyst Glu172 (pKa 6.7). As predicted from sequence comparisons, substitution of this asparagine residue with an aspartic acid residue (N35D BCX) shifts its pH optimum from 5.7 to 4.6, with an approximately 20% increase in activity. The bell-shaped pH-activity profile of this mutant enzyme follows apparent pKa values of 3.5 and 5.8. Based on 13C-NMR titrations, the predominant pKa values of its active-site carboxyl groups are 3.7 (Asp35), 5.7 (Glu78) and 8.4 (Glu172). Thus, in contrast to the WT enzyme, the pH-activity profile of N35D BCX appears to be set by Asp35 and Glu78. Mutational, kinetic, and structural studies of N35D BCX, both in its native and covalently modified 2-fluoro-xylobiosyl glycosyl-enzyme intermediate states, reveal that the xylanase still follows a double-displacement mechanism with Glu78 serving as the nucleophile. We therefore propose that Asp35 and Glu172 function together as the general acid/base catalyst, and that N35D BCX exhibits a "reverse protonation" mechanism in which it is catalytically active when Asp35, with the lower pKa, is protonated, while Glu78, with the higher pKa, is deprotonated. This implies that the mutant enzyme must have an inherent catalytic efficiency at least 100-fold higher than that of the parental WT, because only approximately 1% of its population is in the correct ionization state for catalysis at its pH optimum. The increased efficiency of N35D BCX, and by inference all "acidic" family 11 xylanases, is attributed to the formation of a short (2.7 A) hydrogen bond between Asp35 and Glu172, observed in the crystal structure of the glycosyl-enzyme intermediate of this enzyme, that will substantially stabilize the transition state for glycosyl transfer. Such a mechanism may be much more commonly employed than is generally realized, necessitating careful analysis of the pH-dependence of enzymatic catalysis.

Stability and oligosaccharide binding of the N1 cellulose-binding domain of Cellulomonas fimi endoglucanase CenC.

Differential scanning calorimetry has been used to study the thermal stability and oligosaccharide-binding thermodynamics of the N-terminal cellulose-binding domain of Cellulomonas fimi beta-1,4-glucanase CenC (CBDN1). CBDN1 has a relatively low maximum stability (delta Gmax = 33 kJ/mol = 216 J/residue at 1 degree C and pH 6.1) compared to other small single-domain globular proteins. The unfolding is fully reversible between pH 5.5 and 9 and in accordance with the two-state equilibrium model between pH 5.5 and 11. When the single disulfide bond in CBDN1 is reduced, the protein remains unfolded at all conditions, as judged by NMR spectroscopy. This indicates that the intramolecular cross-link makes a major contribution to the stability of CBDN1. The measured heat capacity change of unfolding (delta Cp = 7.5 kJ mol-1 K-1) agrees well with that calculated from the predicted changes in the solvent accessible nonpolar and polar surface areas upon unfolding. Extrapolation of the specific enthalpy and entropy of unfolding to their respective convergence temperature indicates that per residue unfolding energies for CBDN1, an isolated domain, are in accordance with those found by Privalov (1) for many single-domain globular proteins. DSC thermograms of the unfolding of CBDN1 in the presence of various concentrations of cellopentaose were fit to a thermodynamic model describing the linkage between protein-ligand binding and protein unfolding. A global two-dimensional minimization routine is used to regress the binding enthalpy, binding constant, and unfolding thermodynamics for the CBDN1-cellopentaose system. Extrapolated binding constants are in quantitative agreement with those determined by isothermal titration calorimetry at 35 degrees C.

Basics of epidemiology; Part III: Statistical association and cause-effect relationship

In this; the third part of a series on basics of epidemiology; the theme is statistical association and cause-effect relationship. The primary objective in an epidemiological study is to judge whether an observed association between exposure and disease is real and; indeed; causal. The role of chance and its statistical quantification in explaining an observed association is presented; discussion sampling variation and inference; and interpretation of the frequently quoted P-value. The potential role of bias and confounding is presented; discussing the main categories of bias. Finally; positive criteria useful in supporting a cause and effect judgement; on an observed statistical assoication are enumerated and discussed