Biomarkers for early detection of risk in female patients with coronary artery disease: pilot study.

In women with coronary artery disease (CAD), clinical presentation is different enough from men which leads to missed or delayed diagnosis. Biomarkers can be used for assessment of CAD patients. In case control study, we analyzed blood samples of 30 controls, 30 cases of Unstable Angina (UA) and 30 cases of Myocardial Infarction (MI) for Pro-inflammatory markers (hs-CRP, IL-6, ICAM-1) and Pregnancy Associated Plasma Protein-A (PAPP-A). Based on discriminant analysis, hs-CRP is the potential marker to discriminate cases of UA from controls while PAPP-A is the reliable marker which can discriminate the cases of MI from UA and controls.

Lack of secondary hyperparathyroidism in sub-group of vitamin D deficient postmenopausal women: Is VDR gene polymorphism behind this mystery?

BACKGROUND AND AIMS: We aimed to determine the cause of non-secondary hyperparathyroidism (Non-SHPT) in Indian postmenopausal women. MATERIALS & METHODS: 334 apparently healthy postmenopausal women were assessed for bone mineral homeostaisis including Vitamin D, PTH and VDR polymorphism. RESULTS: 83% of the subjects had vitamin D deficiency further associated with VDR gene polymorphism (P 0.000). A sizable number of subjects (N = 83) did evoke SHPT despite low vitamin D levels. We observe that VDR gene polymorphism was strongly associated in the sub-group of non-SHPT. CONCLUSION: lack of SHPT warrants researchers to study the pathophysiology of non-SHPT in detail to substantiate our findings.

All 25-hydroxyvitamin D-deficient Indian postmenopausal women do not have secondary hyperparathyroidism.

This study shows a high 25-hydroxyvitamin D deficiency among postmenopausal women accompanying secondary hyperparathyroidism. However, a sizable number of subjects did not have secondary hyperparathyroidism despite having low 25-hydroxyvitamin D levels. This condition arises a research question in clinical practice needed to be addressed in the future. PURPOSE: The present study was attempted to determine the prevalence of secondary hyperparathyroidism and also to analyze the mean value (cutoff) of 25-hydroxyvitamin D from where the PTH begins to rise in Indian postmenopausal women. METHODS: A cross-sectional study including 334 postmenopausal women attending the outpatient department (MOPD) of Lok Nayak Hospital, New Delhi, between July 2008 and June 2010. Institutional ethical approval was obtained for this study. The apparently healthy postmenopausal women and attendees of the patients were included in the study. Post-thyroidectomy, thyroid illness, pregnant women, subjects taking drugs that can affect bone mineral metabolism, such as glucocorticoids, antitubercular therapy, antiepileptic, and 25-hydroxyvitamin D supplement were excluded from the study. BMD parameters such as PTH and 25(OH)D were measured by using commercial kits from DiaSorin, USA, and blood chemistry was evaluated by standard methods from the central facility of the center. Dietary calcium was analyzed by applying a food frequency questionnaire by a trained dietician. RESULTS: Mean (SD) age of the subjects was 56.4 ± 7.7 years. The mean BMI was 24.7 ± 5.5 kg/m2. The baseline biochemical investigations such as total bilirubin, liver function test (LFT), kidney function test (KFT), calcium, phosphorous, total protein, and serum albumin were in reference range except alkaline phosphatase (ALP). The mean values of 25(OH)D and PTH were 12.95 ± 8.08 ng/ml and 91.60 ± 75.56 pg/ml respectively. The 24-h dietary calcium intake was 487.06 ± 239.36 mg/24 h. 25-hydroxyvitamin D deficiency was found in 277 subjects (82.93%) and was inversely related to PTH. Forty-three subjects had 25-hydroxyvitamin D levels between 20 and 29 ng/ml (12.87%), and only 14 subjects (4.19%) had optimum 25-hydroxyvitamin D levels. Secondary hyperparathyroidism was found in 235 (70.35%) subjects; however, it was not found in 30%. CONCLUSIONS: Majority of postmenopausal women of India had 25-hydroxyvitamin D deficiency with raised PTH levels. The cutoff point of 25-hydroxyvitamin D at which PTH began to rise was found at 25 ng/ml which seems similar to that of the Caucasians.

Advanced glycosylated end products-mediated activation of polymorphonuclear neutrophils in diabetes mellitus and associated oxidative stress.

Two important consequences of hyperglycemia in diabetes are development of oxidative stress and formation of advanced glycation end products (AGE) which are known to be associated with diabetic complications. Relationship between AGE formation and development of oxidative stress (OS) is yet to be established. In the present study, the involvement of AGE in PMN-mediated ROS generation and the associated OS were investigated in type 2 diabetic mellitus (DM) patients. We assessed OS parameters (serum MDA, FRAP and GSH), PMN oxidative functions (respiratory burst and superoxide production) and total serum AGE in 90 subjects divided equally in three groups--control group, Group I consisting of type 2 diabetic patients without microvascular complications and Group II consisting of type 2 diabetic patients with microvascular complications. PMNs isolated from both groups (I and II) exhibited higher level of respiratory burst (RB) and produced increased amount of superoxide anion as compared to the controls. The increase was more pronounced in diabetes with complications, as compared to those without. Serum malondialdehyde (MDA) level was elevated, whereas glutathione (GSH) and ferric reducing ability of plasma (FRAP) levels were significantly reduced in diabetes as compared to the controls, suggesting the presence of oxidative stress in DM. A positive correlation between PMN oxidative function and OS parameters suggested the involvement of PMN in the development of OS in DM. Serum AGE level was also elevated in diabetic groups as compared to the controls. Further, the positive correlation between serum AGE level and PMN oxidative function suggested the involvement of AGE in increased RB and generation of reactive oxygen species (ROS) by resting diabetic PMN. The results of the study indicate that AGE-PMN interaction possibly upregulates NADPH oxidase, leading to enhanced ROS generation and thus contributes to the pathogenesis in diabetes.

Cloning and sequencing a putative pyrophosphate-dependent phosphofructokinase gene from Entamoeba histolytica.

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) gene from Entamoeba histolytica was cloned from its genomic library and sequenced. The open reading frame has 1149 bp and codes for a protein of 41.5 kDa. The deduced amino acid sequence of E. histolytica PPi-PFK has 25 to 28% identity to the PPi-PFKs from Propionibacterium freudenreichii, Naegleria fowleri and potato. The amino acid residues known to contribute to the active site of PPi-PFK from P. freudenreichii are conserved.

Does Rigid Cystoscopy Affect the Total Serum Prostate-Specific Antigen Levels?

In the present study, we aim to prospectively evaluate the effect of performing rigid cystoscopy (CPE) in urological patients on the total serum prostate-specific antigen (PSA) levels. The study design was a prospective observational study. After giving informed consent, urological patients visiting the outpatient clinic of our institution from November 2010 to March 2012 who satisfied our protocol entry/exclusion criteria were recruited into the present study. Blood sample was withdrawn 1 h prior to CPE for serum PSA estimation, and CPE was performed with a 17-Fr rigid cystoscope. Blood was again withdrawn at 1 and 24 h after CPE for serum PSA estimation. The study used paired samples test (two tailed) for statistical analysis. A total of 50 patients with mean age of 60 years underwent CPE. The baseline, 1-h, and 24-h post CPE mean serum PSA levels were 1.98 ± 2.25 (0.02-12.33), 2.90 ± 2.81 (0.3-14.26), and 3.04 ± 2.95 (0.2-15.03) ng/dl, respectively. The paired samples test (two tailed) revealed that the rise in the baseline PSA versus 1-h PSA and baseline PSA versus 24-h PSA was highly significant at P < 0.001 and P < 0.002, respectively; however, the 1-h PSA versus 24-h PSA rise was not so significant (P < 0.043). The present study demonstrated that rigid cystoscopy may be associated with a variable rise in the serum PSA that may persist for a period of up to or beyond 24 h. Thus, we believe that the history of recent cystoscopy must be taken into consideration while interpreting the serum PSA value in the routine urological setting, as this will reduce unnecessary prostate biopsies in patients with an elevated serum PSA. Therefore, it may be advisable to wait for 24-48 h before withdrawing blood for serum PSA estimation in patients with history of CPE in the previous 24-48 h.

Phagocytic response of leucocytes in secretors and non-secretors of ABH (O) blood group substances.

Secretor status of healthy young volunteers in the age group of 18-22 years and having similar socio-economic background was determined by standard agglutination inhibition test of the saliva. Phagocytic response of leucocyte was worked out by in vitro suspension technique and average number of cocci engulfed by leucocytes calculated in secretors/non-secretors of each blood group. Leucocytes of non-secretors of O and AB seem to have more and highly significant ingestion power as compared to secretors. Non secretors in group A & B also show more phagocytic activity. Relatively different concentration of the components of the blood group substances in secretors/non-secretors seems to affect phagocytic activity of the leucocytes.

Pyrophosphate-dependent phosphofructo-1-kinase complements fructose 1,6-bisphosphatase but not phosphofructokinase deficiency in Escherichia coli.

The gene from Propionibacterium freudenreichii for PPi-dependent phosphofructo-1-kinase, an enzyme that is found in some bacteria, in a number of anaerobic protists, and in plants, complements the absence of fructose 1,6-bisphosphatase in Escherichia coli but does not complement the deficiency of the ATP-dependent phosphofructokinase.

Identification of active site residues in pyrophosphate-dependent phosphofructo-1-kinase by site-directed mutagenesis.

The primary structure of pyrophosphate-dependent phosphofructokinase (PFK) from Propionibacterium freudenreichii exhibits a low but significant level of sequence identity with Escherichia coli ATP-dependent PFK, permitting the tentative assignment of residues that may be involved in catalysis. Based on these assignments, the roles in catalysis of 2 aspartyl residues (Asp151 and Asp153) and 2 lysyl residues (Lys80 and Lys85) were examined. Mutagenesis of the Asp151 to alanine and serine reduced kcat by a factors of 2 x 10(4) and 4 x 10(5), respectively, while showing no change in Km for either substrate in the forward reaction or for metal ion in the back reaction. The kcat for Asp153 was decreased by a factor of 700 with no change in Km for pyrophosphate and an increase of about 20-fold in Km for fructose 6-P and close to 4-fold for magnesium ion. That these changes in the mutants were not the result of global conformational changes was indicated by their identical behavior during substrate-specific elution chromatography, ion-exchange chromatography, and limited proteolysis by trypsin and subtilisin. Mutations of Lys80 and Lys85 showed no significant changes in kinetic parameters, suggesting no involvement in mechanism or substrate binding. These and other results permit preliminary modeling of the active site of pyrophosphate-dependent PFK.

Cloning, sequencing, and expression of pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii.

Pyrophosphate-dependent 6-phosphofructo-1-kinase (PPi-PFK) from Propionibacterium freudenreichii is a non-allosteric enzyme with properties dissimilar to those of other described phosphofructokinases. The enzyme was cloned into pBluescript, sequenced, and expressed in Escherichia coli at levels 15 times higher than those observed in Propionibacterium. The gene consists of 1215 bases which code for a protein of 404 amino acids and a mass of 43,243 daltons. High G + C in the codon usage (66%) of the gene is consistent with the classification of Propionibacterium in the High-G + C subdivision of the Gram-positive bacteria. While showing no sequence identity to the non-allosteric ATP-dependent phosphofructokinase of E. coli, alignments of the amino acid sequence with other PFKs reveal degrees of identities among the amino halves of the proteins, from 26% between the Propionibacterium and potato PPi-PFKs, and 29% between Propionibacterium and E. coli ATP-PFKs. These levels of identities indicate that the amino halves of these proteins are homologous. Identities between the carboxyl half of Propionibacterium PFK and carboxyl halves of other sequences are below 20%, suggesting that the carboxyl half is not homologous. Despite the poor conservation, most of the residues that take part in the binding of fructose-6-P or Mg-PPi could be readily identified by analogy to the structure of the E. coli PFK. Both the fructose-6-P and ATP-binding sites are conserved, indicating that PPi binds to the homologous site of the E. coli ATP-binding site.

Structure and expression of the cDNA for the C isozyme of phosphofructo-1-kinase from rabbit brain.

Rabbit brain contains three phosphofructo-1-kinase (PFK) isozymic subunits designated A, B, and C. The primary structures of the first of these two isozyme types have been determined previously. The isozyme C of rabbit brain was isolated by immunoaffinity chromatography and subjected to proteolytic and chemical digestion. A large number of peptides were sequenced, the total number of amino acids identified being equal to about 80% of the total structure. The sequence of the cDNA derived from brain mRNA for C isozyme was determined from polymerase chain reaction fragments synthesized using oligonucleotides designed on the basis of the peptide sequences. The deduced size of the C isozyme was 86,371 Da, slightly larger than PFKs described previously. The amino acid sequence identity with the rabbit A isozyme was 68.9% and a range of identity to other sequenced mammalian PFKs was 67-69%. Using these data plus previously published data on chemical modification, assignments of the 6 organic ligand binding sites of PFK were inferred. The full-length cDNA was cloned into and expressed in Escherichia coli. Phosphofructokinase C was purified to homogeneity from the bacterial extracts.