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1.  Preparation of (+)-Trans-Isoalliin and Its Isomers by Chemical Synthesis and RP-HPLC Resolution 
Naturally occurring (+)-trans-isoalliin, (RCRS)-(+)-trans-S-1-propenyl-L-cysteine sulfoxide, is a major cysteine sulfoxide in onion. The importance of producing it synthetically to support further research is very well recognized. The (+)-trans-isoalliin is prepared by chemical synthesis and reversed-phase (RP)-HPLC. First, S-2-propenyl-L-cysteine (deoxyalliin) is formed from L-cysteine and allyl bromide, which is then isomerized to S-1-propenyl-L-cysteine (deoxyisoalliin) by a base-catalyzed reaction. A mixture of cis and trans forms of deoxyisoalliin is formed and separated by RP-HPLC. Oxidation of the trans form of deoxyisoalliin by H2O2 produces a mixture of (−)- and (+)-trans-isoalliin. Finally, RP-HPLC is used successfully in separating (−)- and (+)-trans-isoalliin, and hence, (+)-trans-isoalliin is synthesized for the first time in this study. In addition, the (±) diastereomers of cis-isoalliin are also separated and purified by RP-HPLC.
PMCID: PMC3970760
Allium cepa; 1-propenyl-L-cysteine sulfoxide; organosulfur compounds
2.  Profile of Vitiligo in Kumaun Region of Uttarakhand, India 
Vitiligo is a common, acquired, pigmentary disorder characterized by loss of melanocytes resulting in white spots. This disease carries a lot of social stigma in India.
To study the clinico-epidemiological profile of vitiligo patients in Kumaun region of Uttarakhand state in India.
Materials and Methods:
The clinical presentation of vitiligo was examined and analyzed in 762 vitiligo patients attending the Dermatology outdoor of Government Medical College, Haldwani, which is a referral centre for Kumaun region of Uttarakhand state in India.
Male and female patients were found to be affected almost equally. It was observed that onset of vitiligo was most common in 0-10 years age group, as evidenced by 336 cases out of 762 cases. Acrofacial type of vitiligo (339 cases out of 762) was most commonly observed, followed by vitiligo vulgaris, focal, segmental, mucosal, mixed, and universal vitiligo. The most common site of onset was the lower limbs followed by head and neck, upper limbs, trunk, genitalia, and mucasae. Leucotrichia was observed in 33.5%, Koebner's phenomenon in 26.3%, and a positive family history in 19% of the vitiligo patients. The other common conditions associated were thyroid disorders (8.9%), diabetes (5.3%), and atopic dermatitis (4.9%).
The study indicates that acrofacial vitiligo is the most common clinical type observed in Kumaun region of Uttarakhand in India. Onset of vitiligo is most common in first decade of life.
PMCID: PMC3969695  PMID: 24700953
Clinico-epidemiological profile; Uttarakhand; vitiligo
3.  Development of a Novel BAFF Responsive Cell Line Suitable for Detecting Bioactive BAFF and Neutralizing Antibodies against BAFF-Pathway Inhibiting Therapeutics 
Cells  2014;3(1):79-91.
BAFF has a critical role in B-cell survival, maturation and function, which makes its pathway a prime therapeutic target for various autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis and Sjögren’s syndrome. A cell-based assay that measures the functional activity of BAFF is required for many high throughput purposes, such as lead target screening and BAFF quantification. We report here the development of a sensitive BAFF responsive cell line via stable transfection of the BAFFR-TNFR1 hybrid receptor into monkey kidney epithelial COS-1 cells. The cellular response to BAFF can be detected by measuring the secretion of IL-8. This BAFF bioassay is not only reproducible and sensitive, but also responsive to a wide concentration range of BAFF stimulation in sera from various species. This cell line is useful in the development of sensitive bioassays to measure the levels of bioactive BAFF, inhibition of BAFF and neutralizing antibodies against any BAFF pathway-mediated therapeutic proteins.
PMCID: PMC3980743  PMID: 24709903
hybrid receptor; neutralizing antibody detection; BAFF response; serum matrix; transfection; COS-1 cell line
4.  The attitudes and perceptions of medical students towards basic science subjects during their clinical years: A cross-sectional survey 
In the conventional system of medical education, basic subjects are taught in the 1st year with least interdisciplinary interaction. The objective of this study was to explore the students’ perception about content, need and application of basic science subjects during the clinical years of their medical education.
Materials and Methods:
A total of 300 questionnaires were distributed among students randomly after taking their written consent for participation in the study. About 265 completely filled questionnaires were received back and the response was analyzed.
Students identified anatomy as the subject with overloaded syllabus (75.4%) and also with maximum clinical application with 50.1% of them considering it the most important basic subject. Students were satisfied with the practical integration of subjects to impart clinical skills, but considered problem based learning a better method of teaching. According to 37%, 43.8% and 33.2% of respondents respectively; anatomy, biochemistry and physiology curriculum should only cover the general concepts to give the working knowledge of the subject. Approximately, 65% of the respondents were able to recall the knowledge of anatomy and physiology while biochemistry was retained by 40%.
Overall, the attitudes of students toward basic science subjects were positive. The learning experience for them can be improved significantly by better clinical integration of the subjects.
PMCID: PMC3931207  PMID: 24600572
Basic science subjects; medical students; perceptions
5.  Association between Alcohol Screening Scores and Mortality in Black, Hispanic, and White Male Veterans 
AUDIT-C alcohol screening scores are associated with mortality, but whether or how associations vary across race/ethnicity is unknown.
Self-reported black (n=13,068), Hispanic (n=9,466), and white (n=182,688) male VA outpatients completed the AUDIT-C via mailed survey. Logistic regression models evaluated whether race/ethnicity modified the association between AUDIT-C scores (0, 1–4, 5–8, and 9–12) and mortality after 24 months, adjusting for demographics, smoking, and comorbidity.
Adjusted mortality rates were 0.036, 0.033, and 0.054, for black, Hispanic, and white patients with AUDIT-C scores of 1–4, respectively. Race/ethnicity modified the association between AUDIT-C scores and mortality (p=0.0022). Hispanic and white patients with scores of 0, 5–8, and 9–12 had significantly increased risk of death compared to those with scores of 1–4; Hispanic ORs: 1.93, 95% CI 1.50–2.49; 1.57, 1.07–2.30; 1.82, 1.04–3.17, respectively; white ORs: 1.34, 95% CI 1.29–1.40; 1.12, 1.03–1.21; 1.81, 1.59–2.07, respectively. Black patients with scores of 0 and 5–8 had increased risk relative to scores of 1–4 (ORs 1.28, 1.06–1.56 and 1.50, 1.13–1.99), but there was no significant increased risk for scores of 9–12 (ORs 1.27, 0.77–2.09). Post-hoc exploratory analyses suggested an interaction between smoking and AUDIT-C scores might account for some of the observed differences across race/ethnicity.
Among male VA outpatients, associations between alcohol screening scores and mortality varied significantly depending on race/ethnicity. Findings could be integrated into systems with automated risk calculators to provide demographically-tailored feedback regarding medical consequences of drinking.
PMCID: PMC3443543  PMID: 22676340
alcohol; race; ethnicity; mortality; AUDIT-C
6.  Turmeric - A new treatment option for lichen planus: A pilot study 
Turmeric is dried rhizome of the perennial herbs curcumalonga. It is called Haldi in Hindi, turmeric in English, ukon in Japanese. It has been used in Asian Medicine since the second millennium BC. It's utility is referred to in the ancient Hindu script the Ayurveda. Pathogenesis of the OLP should be taken in consideration for the treatment point of view. The Cell mediated immunity to secondary antigenic change in oral mucous membrane is thought to play a major role in its pathogenesis modified keratocyte surface antigens are the primary target for cytotoxic cellular response. Curcumin also been shown to have immune modulatory effect involving activation of host macrophages and natural killer cells and modulation of lymphocytes mediated function.
PMCID: PMC3961895  PMID: 24665176
Antioxidants; lichenplanus; turmeric
7.  Does LDL-C Estimation Using Anandaraja’s Formula Give a Better Agreement with Direct LDL-C Estimation than the Friedewald’s Formula? 
Estimation of low density lipoprotein cholesterol (LDL-C) is crucial in management of coronary artery disease patients. Though a number of homogenous assays are available for estimation of LDL-C, use of calculated LDL-C by Friedewald’s formula (FF) is common in Indian laboratories for logistic reasons. Recently Anandaraja and colleagues have derived a new formula for calculating LDL-C. This formula needs to be evaluated before it is extensively applied in diagnosis. We measured LDL-C by homogenous method (D-LDL-C) in 515 fasting samples. Friedewald’s and Anandaraja’s formulas were used for calculation of LDL-C (F-LDL-C and A-LDL-C, respectively). The mean LDL-C levels were 123.3 ± 53.2, 112.4 ± 50.2 and 109.2 ± 49.8 mg/dl for D-LDL-C, F-LDL-C and A-LDL-C, respectively. There was a statistically significant difference between the results (P > 0.001) obtained by calculation formulas compared to the measured LDL-C. There was underestimation of LDL-C by 10.8 and 14 mg/dl by Friedewald’s and Anandaraja’s formulas respectively. The Pearson’s correlation between F-LDL-C and D-LDL-C was 0.931 and that between A-LDL-C and D-LDL-C was 0.930. Bland–Altman graphs showed a definite agreement between mean and differences of the calculation formulas and direct LDL-C with 95% of values lying with in ±2 SD limits. The mean percentage difference (calculated as {(Calculated LDL-C)-(D-LDL-C)}/D-LDL-C × 100) for F-LDL-C was maximum (−11.6%) at HDL-C ≥ 60 mg/dl and TG levels of 200–300 mg/dl (−10.4%) compared to D-LDL-C. A-LDL-C results gave highest mean percentage difference at total cholesterol concentrations <100 mg/dl (−37.3%) and HDL-C < 40 mg/dl (−17.1%), respectively. The results of our study showed that FF is better in agreement with D-LDL-C than Anandaraja’s formula for estimation of LDL-C by calculation though both lead to its underestimation.
PMCID: PMC3358371  PMID: 23543806
LDL-C; Friedewald’s formula; Anandaraja’s Formula
Hypertension  2012;59(3):665-672.
Cardiotonic steroids signaling through the basolateral sodium pump (Na/KATPase) have been shown to alter renal salt handling in intact animals. As the relationship between renal salt handling and blood pressure is a key determinant of hypertension, and patients with insulin resistance are frequently hypertensive, we chose to examine whether there might be competition for resources necessary for receptor mediated endocytosis.
In LLC-PK1 cells, the Na/K-ATPase-α1 and carcinoembryonic antigen cell adhesion molecule (CEACAM1), a plasma membrane protein that promotes receptor-mediated endocytosis, co-localized in the plasma membranes and translocated to the intracellular region in response to ouabain. Either ouabain or insulin alone caused accumulation of CEACAM1 as well as IRβ, and EGFR in early endosomes, but no synergy was demonstrable. Like ouabain, insulin also caused c-Src activation. When caveolin or Na/K-ATPase-α1 expression was knocked down with siRNA, insulin but not ouabain induced CEACAM1, IRβ, and EGFR endocytosis.
To determine whether this might be relevant to salt handling in vivo, we examined salt loading in mice with null renal CEACAM2 expression (Cc2−/−). The Cc2−/− animals demonstrated greater increases in blood pressure with increases in dietary salt than control animals.
These data demonstrate that cardiotonic steroids and insulin compete for cellular endocytosis resources and suggest that under conditions where circulating insulin concentrations are high, cardiotonic steroid mediated natriuresis could be impaired.
PMCID: PMC3336087  PMID: 22311908
chronic renal insufficiency; renal proximal tubule cell; endocytosis
9.  Lemierre's Syndrome: Rare, but Life Threatening—A Case Report with Streptococcus intermedius 
Case Reports in Medicine  2012;2012:624065.
Lemierre's syndrome (LS) is a rare, but a life-threatening complication of an oropharyngeal infection. Combinations of fever, pharyngitis, dysphagia, odynophagia, or oropharyngeal swelling are common presenting symptoms. Infection of the lateral pharyngeal space may result in thrombosis of the internal jugular vein, subsequent metastatic complications (e.g., lung abscesses, septic arthritis), and significant morbidity and mortality. LS is usually caused by the gram-negative anaerobic bacillus Fusobacterium necrophorum, hence also known as necrobacillosis. We present a case of LS caused by Streptococcus intermedius, likely secondary to gingival scraping, in which the presenting complaint was neck pain. The oropharyngeal examination was normal and an initial CT of the neck was done without contrast, which likely resulted in a diagnostic delay. This syndrome can be easily missed in early phases. However, given the potential severity of LS, early recognition and expedient appropriate antimicrobial treatment are critical. S. intermedius is an unusual cause of LS, with only 2 previous cases being reported in the literature. Therefore, an awareness of the myriad presentations of this syndrome, which in turn will lead to appropriate and timely diagnostic studies, will result in improved outcome for LS.
PMCID: PMC3502875  PMID: 23197986
10.  Isolated aglossia congenita: A rare case of oromandibular limb hypogenesis syndrome type I B 
Aglossia congenita (AC), congenital total absence of the tongue, is a very rare midline developmental anomaly, hypothesized to be associated with vascular disruption between the fourth and eighth week of gestation. It was classified by Hall (1971) as part of oromandibular limb hypogenesis syndrome (OLHS) type I B. Most of the cases reported with OLHS are actually hypoglossia with limb abnormalities whereas isolated aglossia is an extremely rare entity. A case of isolated AC is presented in a 28-year-old Indian male. He had long narrow face, tapering chin, low set ears, and microstomia. Intraorally, he had narrow palatal vault, constricted oropharyngeal isthmus, oligodontia, and maxillo-mandibular hypoplasia. Interestingly, the patient showed a median palatal groove, which has not been reported before. He also had an unusual acquired adaptive mechanism to compensate for aglossia. This report presents the manifestations of this rare syndrome, its complications, differential diagnosis, and rehabilitation strategies.
PMCID: PMC3519220  PMID: 23248477
Adaptation; anomaly; developmental; palate; rehabilitation; tongue
11.  Parenchymal Expression of CD40 Exacerbates Adenovirus-Induced Hepatitis 
Hepatology (Baltimore, Md.)  2011;53(5):1455-1467.
The healthy adult human liver expresses low levels of MHC II and undetectable levels of immune co-stimulatory molecules. However, high levels of MHC class II, CD40 and B7 family molecules are expressed in the activated Kupffer cells and hepatocytes of patients having viral hepatitis. The precise role of these molecules in viral clearance and immune-mediated liver injury is not well understood. We hypothesize that parenchymal CD40 expression enhances T-cell recruitment and effector functions, which may facilitate viral clearance and alleviate liver injury. To test this hypothesis, we generated novel, liver-specific, conditional CD40 transgenic mice, and challenged them i.v. with recombinant replication-deficient adenovirus carrying Cre recombinase (AdCre). Wild-type mice infected with AdCre developed a relatively mild course of viral hepatitis and recovered spontaneously. CD40 expression in the liver of transgenic animals, however, resulted in CD80 and CD86 expression. Dysregulation of population dynamics and effector functions of intrahepatic lymphocytes results in severe lymphocytic infiltration, apoptosis, necroinflammation, and serum alanine transferase (ALT) elevation in a dose-dependent fashion. To our surprise, an early expansion followed by a contraction of intrahepatic lymphocytes, especially CD8+ and NK cells, accompanied by increased granzyme B and IFN-γ production, did not lead to a faster viral clearance in CD40 transgenic mice. Conclusion: Our results demonstrated that hepatic CD40 expression does not accelerate adenoviral clearance, but rather exacerbates liver injury. This study unveils a previously unknown deleterious effect of hepatic CD40 in adenovirus-induced liver inflammation.
PMCID: PMC3082591  PMID: 21360722
liver; animal models; T lymphocytes and co-stimulation
12.  Syndontia with talon cusp 
Teeth are specialized structural components of the craniofacial skeleton. Developmental defects occur either alone or in combination with other birth defects. Macrodontia of anterior teeth may occur as an isolated condition or as a result of fusion or gemination and can occur in the primary or permanent dentition. Fusion is more commonly seen in the anterior maxillary region. This case presentation reports a case of fusion of a supplemental tooth to one in the normal series in conjunction with a talon cusp. This condition is extremely rare and has been reported at fourth occasion in the literature. The etiology, prevalence, clinical features, and management of the aforementioned anomalies have been reviewed in detail. Early diagnosis of this condition is important because it may cause clinical problems, such as esthetic concerns and tooth crowding.
PMCID: PMC3424946  PMID: 22923902
Fusion; gemination; macrodontia; talon cusp
13.  Hypohidrotic ectodermal dysplasia 
Hypohidrotic ectodermal dysplasia (HED) is a rare genetic disorder characterized by the faulty development of the ectodermal structure, resulting in most notably anhydrosis/hypohydrosis, hypotrichosis and hypodontia. The condition is usually an X-linked recessive disorder affecting predominantly males. We are here reporting a classical case of hypohidrotic ectodermal dysplasia with a review of the literature.
PMCID: PMC3481883  PMID: 23130287
Ectodermal dysplasia; genetic disorder; hypohidrotic
14.  Are we using Thyroid Function Tests Appropriately? 
Thyroid function tests are very important for the diagnosis and monitoring of patients with thyroid dysfunction. The guidelines recommend serum thyroid stimulating hormone (TSH) as the single most reliable test to diagnose all common forms of hypothyroidism and hyperthyroidism. The aim of this study was to analyze the ordering pattern for thyroid function tests by physicians and the analysis of results based on the clinical history. The mean age of the patients was 32.5 ± 6.5 years. Majority of samples (87.7% of total) were received from the departments of Medicine and Gynae. Thyroid profiles (47.5%) were ordered more frequently as compared to TSH only (46%). There was no significant difference in the percentage of normal reports for both types of tests. 77.8% of TFT and 76.6% of TSH samples had results within the reference range. The percentage of abnormal results was 13.7% in the patients who were screened for thyroid disorders. There is a need to redefine the case definition for thyroid dysfunction and order the appropriate test in a rational and cost effective manner.
PMCID: PMC3107420  PMID: 22468046
Thyroid function test; TSH; Hypothyroidism; Hyperthyroidism
15.  Risk of future trauma based on alcohol screening scores: A two-year prospective cohort study among US veterans 
Severe alcohol misuse as measured by the Alcohol Use Disorders Identification Test–Consumption (AUDIT-C) is associated with increased risk of future fractures and trauma-related hospitalizations. This study examined the association between AUDIT-C scores and two-year risk of any type of trauma among US Veterans Health Administration (VHA) patients and assessed whether risk varied by age or gender.
Outpatients (215, 924 male and 9168 female) who returned mailed AUDIT-C questionnaires were followed for 24 months in the medical record for any International Statistical Classification of Diseases and Related Health Problems (ICD-9) code related to trauma. The two-year prevalence of trauma was examined as a function of AUDIT-C scores, with low-level drinking (AUDIT-C 1–4) as the reference group. Men and women were examined separately, and age-stratified analyses were performed.
Having an AUDIT-C score of 9–12 (indicating severe alcohol misuse) was associated with increased risk for trauma. Mean (SD) ages for men and women were 68.2 (11.5) and 57.2 (15.8), respectively. Age-stratified analyses showed that, for men ≤50 years, those with AUDIT-C scores ≥9 had an increased risk for trauma compared with those with AUDIT-C scores in the 1–4 range (adjusted prevalence, 25.7% versus 20.8%, respectively; OR = 1.24; 95% confidence interval [CI], 1.03–1.50). For men ≥65 years with average comorbidity and education, those with AUDIT-C scores of 5–8 (adjusted prevalence, 7.9% versus 7.4%; OR = 1.16; 95% CI, 1.02–1.31) and 9–12 (adjusted prevalence 11.1% versus 7.4%; OR = 1.68; 95% CI, 1.30–2.17) were at significantly increased risk for trauma compared with men ≥65 years in the reference group. Higher AUDIT-C scores were not associated with increased risk of trauma among women.
Men with severe alcohol misuse (AUDIT-C 9–12) demonstrate an increased risk of trauma. Men ≥65 showed an increased risk for trauma at all levels of alcohol misuse (AUDIT-C 5–8 and 9–12). These findings may be used as part of an evidence-based brief intervention for alcohol use disorders. More research is needed to understand the relationship between AUDIT-C scores and risk of trauma in women.
PMCID: PMC3414833  PMID: 22966411
Alcohol; Trauma; Fracture; AUDIT-C; Age; Gender; Screening; Women
16.  Immunogenicity of panitumumab in combination chemotherapy clinical trials 
Panitumumab is a fully human antibody against the epidermal growth factor receptor that is indicated for the treatment of metastatic colorectal cancer (mCRC) after disease progression on standard chemotherapy. The purpose of this analysis was to examine the immunogenicity of panitumumab and to evaluate the effect of anti-panitumumab antibodies on pharmacokinetic and safety profiles in patients with mCRC receiving panitumumab in combination with oxaliplatin- or irinotecan-based chemotherapies.
Three validated assays (two screening immunoassays and a neutralizing antibody bioassay) were used to detect the presence of anti-panitumumab antibodies in serum samples collected from patients enrolled in four panitumumab combination chemotherapy clinical trials. The impact of anti-panitumumab antibodies on pharmacokinetic and safety profiles was analyzed using population pharmacokinetic analysis and descriptive statistics, respectively.
Of 1124 patients treated with panitumumab in combination with oxaliplatin- or irinotecan-based chemotherapy with postbaseline samples available for testing, 20 (1.8%) patients developed binding antibodies and 2 (0.2%) developed neutralizing antibodies. The incidence of anti-panitumumab antibodies was similar in patients with tumors expressing wild-type or mutant KRAS and in patients receiving oxaliplatin- or irinotecan-based chemotherapies. No evidence of an altered pharmacokinetic or safety profile was found in patients who tested positive for anti-panitumumab antibodies.
The immunogenicity of panitumumab in the combination chemotherapy setting was infrequent and similar to the immunogenicity observed in the monotherapy setting. Panitumumab immunogenicity did not appear to alter pharmacokinetic or safety profiles. This low rate of immunogenicity may be attributed to the fully human nature of panitumumab.
Trial registration NCT00339183 (study 20050181), NCT00411450 (study 20060277), NCT00332163 (study 20050184), and NCT00364013 (study 20050203).
PMCID: PMC3231982  PMID: 22070868
17.  Analyses of In Vitro Nonenzymatic Glycation of Normal and Variant Hemoglobins by MALDI-TOF Mass Spectrometry 
MALDI-TOF mass spectrometry is used here to differentiate different glycoisoforms of normal and variant hemoglobins (Hbs) in nonenzymatic in vitro glycation. Single, double, and/or multiple glycation of the α-globin, β-globin, and/or γ-globin is observed. Different glycation rates are observed for various Hbs, and the normal Hb A has the slowest rate. Although the Hb A is relatively stable upon condensation with glucose at 37°C, the variants Hb C, Hb E, Hb F, Hb Leiden, and Hb San Diego are less stable. In addition, data reveal that the number of glucose attached/Hb molecule (state of glycation) increases with longer incubation time, higher glucose concentration, and higher temperature. The pH dependence of the state of glycation is more complex and varies for different Hbs. Although pH has little effect on the state of glycation for Hb C, Hb E, and Hb Leiden, it increases for Hb A and Hb F upon changing the pH of the solution from phosphate buffer saline (pH 7.4) to carbonate buffer (pH 10). Results obtained in this study could lead to the inference that the linkage of Hbs with glucose occurs in diabetic conditions in vivo (37°C, ∼neutral pH, ∼0.007 M glucose), and the state of glycation is more severe in the individuals who carry abnormal Hbs.
PMCID: PMC3165857  PMID: 21966256
diabetes mellitus; MS
18.  Spironolactone Attenuates Experimental Uremic Cardiomyopathy by Antagonizing Marinobufagenin 
Hypertension  2009;54(6):1313-1320.
Spironolactone has been noted to attenuate cardiac fibrosis. We have observed that the cardiotonic steroid marinobufagenin plays an important role in the diastolic dysfunction and cardiac fibrosis seen with experimental renal failure. We performed the following studies to determine whether and how spironolactone might ameliorate these changes. First, we studied rats subjected to partial nephrectomy or administration of exogenous marinobufagenin. We found that spironolactone (20 mg/kg per day) attenuated the diastolic dysfunction as assessed by ventricular pressure-volume loops and essentially eliminated cardiac fibrosis as assessed by trichrome staining and Western blot. Next, we examined the effects of spironolactone and its major metabolite, canrenone (both 100 nM), on marinobufagenin stimulation of rat cardiac fibroblasts. Both spironolactone and canrenone prevented the stimulation of collagen production by 1 nM marinobufagenin but not 100 nM marinobufagenin, as assessed by proline incorporation and procollagen 1 expression, as well as signaling through the sodium-potassium-ATPase, as evidenced by protein kinase C isoform δ translocation and extracellular signal regulated kinase 1/2 activation. Both spironolactone and canrenone also altered ouabain binding to cultured porcine cells in a manner consistent with competitive inhibition. Our data suggest that some of the antifibrotic effects of spironolactone may be attributed to antagonism of marinobufagenin signaling through the sodium-potassium-ATPase.
PMCID: PMC2783263  PMID: 19884563
cardiomyopathy; renal failure; cardiotonic steroids; collagen; fibrosis
19.  Two-Dimensional Immobilized Metal Affinity Electrophoresis for Capturing a Phosphoprotein 
A two-dimensional immobilized metal affinity electrophoresis method is described here. In this method, ferric ions are immobilized in the second-dimensional polyacrylamide gel to extract the phosphoprotein β-casein from a mixture containing proteins with a broad range of pI and MW. Native 7.5–15% gradient tris-glycine gel with SDS tris-glycine gel running buffer are used so that proteins can be separated according to their molecular mass in the second dimension.
PMCID: PMC2977962  PMID: 21119927
IMAC; IMAEP; 2-D-PAGE; phosphoproteins; β-casein
20.  Assessment of immunogenicity of romiplostim in clinical studies with ITP subjects 
Annals of Hematology  2010;89(Suppl 1):75-85.
Romiplostim is an Fc-peptide fusion protein that activates intracellular transcriptional pathways via the thrombopoietin (TPO) receptor leading to increased platelet production. Romiplostim has been engineered to have no amino acid sequence homology to endogenous TPO. Recombinant protein therapeutics can be at a risk of development of an antibody response that can impact efficacy and safety. Hence, a strategy to detect potential antibody formation to the drug and to related endogenous molecules can be useful. The immunogenicity assessment strategy involved both the detection and characterization of binding and neutralizing antibodies. The method for detection was based on a surface plasmon resonance biosensor platform using the Biacore 3000. Samples that tested positive for binding antibodies in the Biacore immunoassay were then tested in a neutralization assay. Serum samples from 225 subjects with immune thrombocytopenic purpura (ITP) dosed with romiplostim and 45 ITP subjects dosed with placebo were tested for romiplostim and TPO antibodies. Prior to romiplostim treatment, 17 subjects (7%) tested romiplostim antibody positive and 12 subjects (5%) tested TPO antibody positive for pre-existing binding antibodies. After romiplostim exposure, 11% of the subjects exhibited binding antibodies against romiplostim and 5% of the subjects with ITP showed binding antibodies against TPO. The antibodies against romiplostim did not cross-react with TPO and vice versa. No cases of anti-TPO neutralizing antibodies were detected in romiplostim-treated subjects. The incidence of anti-romiplostim neutralizing antibodies to romiplostim was 0.4% (one subject); this subject tested negative at the time of follow-up 4 months later. No impact on platelet profiles were apparent in subjects that had antibodies to romiplostim to date. In summary, administration of romiplostim in ITP subjects resulted in the development of a binding antibody response against romiplostim and TPO ligand. One subject developed a neutralizing antibody response to romiplostim that impacted the platelet counts of this subject. No neutralizing antibodies to endogenous TPO were observed.
PMCID: PMC2900600  PMID: 19484238
Immune thrombocytopenic purpura (ITP); Romiplostim; Immunogenicity; TPO; Platelet
21.  Partial nephrectomy as a model for uremic cardiomyopathy in the mouse 
Because of the plethora of genetic manipulations available in the mouse, we performed a partial nephrectomy in the mouse and examined whether the phenotypical features of uremic cardiomyopathy described in humans and rats were also present in the murine model. A ⅚ nephrectomy was performed using a combination of electrocautory to decrease renal mass on the left kidney and right surgical nephrectomy. This procedure produced substantial and persistent hypertension as well as increases in circulating concentrations of marinobufagenin. Invasive physiological measurements of cardiac function demonstrated that the ⅚ nephrectomy resulted in impairment of both active and passive left ventricular relaxation at 4 wk whereas tissue Doppler imaging detected changes in diastolic function after 6 wk. Morphologically, hearts demonstrated enlargement and progressive fibrosis, and biochemical measurements demonstrated downregulation of the sarcoplasmic reticulum calcium ATPase as well as increases in collagen-1, fibronectin, and vimentin expression. Our results suggest that partial nephrectomy in the mouse establishes a model of uremic cardiomyopathy which shares phenotypical features with the rat model as well as patients with chronic renal failure.
PMCID: PMC2742580  PMID: 18032546
renal failure; TGF-β; cardiotonic steroids; reactive oxygen species; fibrosis
22.  Immobilized Metal Affinity Electrophoresis: A Novel Method of Capturing Phosphoproteins by Electrophoresis 
An immobilized metal affinity electrophoresis (IMAEP) method is described here. In this method, metal ions are immobilized in a native polyacrylamide gel to capture phosphoproteins. The capture of phosphoproteins by IMAEP is demonstrated with immobilized metals like iron, aluminum, manganese, or titanium. In the case studies, phosphoproteins α-casein, β-casein, and phosvitin are successfully extracted from a protein mixture by IMAEP.
PMCID: PMC2361167  PMID: 19137092
IMAC; immobilized metal affinity electrophoresis; phosphoproteins; α-casein; β-casein; phosvitin
23.  An Improved Protocol for Coupling Synthetic Peptides to Carrier Proteins for Antibody Production Using DMF to Solubilize Peptides 
We present an improved protocol for coupling synthetic peptides to carrier proteins. In this protocol, dimethyl‐formamide is used as the solvent to solubilize peptides instead of phosphate‐buffered saline (PBS) or 6 M guanidine‐HCl/0.01 M phosphate buffer (pH 7). Additionally, the last desalting or dialyzing step to remove uncoupled peptides as in the traditional method is eliminated. Finally, 3 ml of 0.1 M ammonium bicarbonate is added to the carrier protein conjugated peptide solution to help the lyophilization process. Coupling of Cys‐containing synthetic peptides to keyhole limpet hemocyanin or bovine serum albumin using m‐maleimidobenzoyl‐N‐hydroxysuccinimide ester are used as the test cases. This method produces high‐quality antipeptide antibodies. Also, compared to the traditional method, this procedure is simpler and useful for peptides with solubility problems in PBS or 6 M guanidine‐HCl.
PMCID: PMC2062551  PMID: 17595313
peptide; antibody; KLH; BSA; MBS; DMF; MALDI‐TOF MS
24.  Laryngeal chondroma 
Cartilaginous tumors of laryngeal skeleton are a rarity: Laryngeal chondroma is an unusual cause of upper airway obstruction. The present report illustrates a case of a 13 year old male presenting with mass in the subglottis. Histopathology of the mass revealed laryngeal chondroma.
PMCID: PMC3450848  PMID: 23119774
Cartilaginous tumors; Larynx
25.  Evaluation of the changes in serum iron levels in pre-eclampsia 
Serum iron levels were studied in 50 patients with pre-eclampsia and the results were compared with 50 control cases. Their serum iron levels were found to be higher than the controls. Increase in serum iron was directly proportional to the increased levels of uric acid, urea and creatinine. Mean reticulocyte counts, plasma free haemoglobin and unconjugated bilirubin levels were also higher in these patients. It is suggested that haemolysis may be a major contributory factor for the increased levels of serum iron in pre-eclampsia.
PMCID: PMC3454039  PMID: 23100872
Serum iron; Pre-eclampsia

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