Identification and functional characterization of putative ligand binding domain(s) of JlpA protein of Campylobacter jejuni.

Among the major Surface Exposed Colonization Proteins (SECPs) of Campylobacter jejuni (C. jejuni), Jejuni lipoprotein A (JlpA) plays a crucial role in host cell adhesion specifically by binding to the N-terminal domain of the human heat shock protein 90α (Hsp90α-NTD). Although the JlpA binding to Hsp90α activates NF-κB and p38 MAP kinase pathways, the underlying mechanism of JlpA association with the cellular receptor remains unclear. To this end, we predicted two potential receptor binding sites within the C-terminal domain of JlpA: one spanning from amino acid residues Q332-A354 and the other from S258-T295; however, the latter exhibited weaker binding. To assess the functional attributes of these predicted sequences, we generated two JlpA mutants (JlpAΔ1: S258-T295; JlpAΔ2: Q332-A354) and assessed the Hsp90α-binding affinity-kinetics by in vitro and ex vivo experiments. Our findings confirmed that the residues Q332-A354 are of greater importance in host cell adhesion with a measurable impact on cellular responses. Further, thermal denaturation by circular dichroism (CD) confirmed that the reduced binding affinity of the JlpAΔ2 to Hsp90α is not associated with protein folding or stability. Together, this study provides a possible framework for determining the molecular function of designing rational inhibitors efficiently targeting JlpA.

Serum sonic hedgehog (SHH) and interleukin-(IL-6) as dual prognostic biomarkers in progressive metastatic breast cancer.

Serum from one hundred and ten breast cancer patients and thirty healthy female volunteers, were prospectively collected and evaluated for serum levels of Shh and IL-6 using human Shh and IL-6 specific enzyme-linked immunoassays. All patients were regularly monitored for event free survival (EFS) and overall survival (OS). Overall outcome analysis was based on serum Shh and IL-6 levels. In patients with progressive metastatic BC, both serum Shh and IL-6 concentrations were elevated in 44% (29 of 65) and 63% (41 of 65) of patients, respectively, at a statistically significant level [Shh (p = 0.0001) and IL-6 (p = 0.0001)] compared to the low levels in healthy volunteers. Serum levels tended to increase with metastatic progression and lymph node positivity. High serum Shh and IL-6 levels were associated with poor EFS and OS opposite to the negative or lower levels in serum Shh and IL-6. The elevated levels of both serum Shh and IL-6 were mainly observed in BC patients who had a significantly higher risk of early recurrence and bone metastasis, and associated with a worse survival for patients with progressive metastatic BC. Further studies are warranted for validating these biomarkers as prognostic tools in a larger patient cohort and in a longer follow-up study.

Overexpression of sonic hedgehog in the triple negative breast cancer: clinicopathological characteristics of high burden breast cancer patients from Bangladesh.

Dysregulation of Hedgehog (Hh) signaling pathway has been documented in mammary gland development and breast cancer (BC) progression. Despite the remarkable progress in therapeutic interventions, BC related mortality in Bangladesh increased in the last decade. Triple negative breast cancer (TNBC) still presents a critical therapeutic challenge. Thus effective targeted therapy is urgently needed. In this study, we report the clinicopathological characteristics and prognosis of BC patients from Bangladesh. Routine immunohistochemical analysis and high throughput RNA-Seq data from the TCGA library were used to analyze the expression pattern and association of high and low level of Shh expression in a collection of BC patients with a long-term follow-up. High levels of Shh were observed in a subset of BC tumors with poor prognostic pathological features. Higher level of Shh expression correlated with a significantly poorer overall survival of patients compared with patients whose tumors expressed a low level of Shh. These data support the contention that Shh could be a novel biomarker for breast cancer that is involved in mediating the aggressive phenotype of BC. We propose that BC patients exhibiting a higher level of Shh expression, representing a subset of BC patients, would be amenable to Shh targeted therapy.

Effect of ibandronate therapy on serum homocysteine and leptin in postmenopausal osteoporotic females.

UNLABELLED: A significant change in serum leptin level and no change in homocysteine were observed with ibandronate treatment. No correlation of homocysteine and leptin was found with bone mass density (BMD). Results indicate that ibandronate reduces serum leptin levels but how does it help in reducing the osteoporosis. It needs to be explored. INTRODUCTION: The current study was planned to determine the effects of ibandronate on serum homocysteine and leptin levels in postmenopausal osteoporotic females and to correlate these with BMD. METHODS: Forty-two newly diagnosed and untreated postmenopausal osteoporotic females were selected on the basis of their BMD (BMD < -2.5) from Orthopaedic Out Patient Department of Shaikh Zayed Hospital, Lahore, Pakistan, and 36, age and BMI matched non-osteoporotic postmenopausal females, were also selected as a control group. Baseline physical and biochemical parameters were compared. In osteoporotic patients, changes in circulating leptin and homocysteine levels were studied after 6 months of therapy with ibandronate (150 mg). The collected data were analyzed on SPSS 16. RESULTS: There was no significant difference observed in the mean value of all baseline parameters except BMD in both groups. After 6 months of treatment with ibandronate (150 mg), a significant change was observed in serum leptin levels (19.48 ± 1.60 ng/ml vs. 14.09 ± 0.85 ng/ml, p < 0.002), while no considerable change observed in serum homocysteine levels (16.22 ± 0.95 µmol/l vs. 16.80 ± 1.03 µmol/l, p < 0.63). Serum leptin was found significantly correlated with anthropometric parameters. No correlation of serum leptin and homocysteine was found with BMD (r = 0.09, p value = 0.54; r = -0.17, p value = 0.27). CONCLUSION: Our results show that ibandronate reduces serum leptin levels while it has no effect on serum homocysteine levels. Further studies are needed to explain how the decrease in serum leptin level may help in reducing the progression of osteoporosis.

High-dose thiamine therapy for patients with type 2 diabetes and microalbuminuria: a randomised, double-blind placebo-controlled pilot study.

AIMS/HYPOTHESIS: High-dose supplements of thiamine prevent the development of microalbuminuria in experimental diabetes. The aim of this pilot study was to assess whether oral supplements of thiamine could reverse microalbuminuria in patients with type 2 diabetes. METHODS: Type 2 diabetic patients (21 male, 19 female) with microalbuminuria were recruited at the Diabetes Clinic, Sheikh Zayed Hospital, Lahore, Pakistan, and randomised to placebo and treatment arms. Randomisation was by central office in sequentially numbered opaque, sealed envelopes. Participants, caregivers and those assessing the outcomes were blinded to group assignment. Patients were given 3 x 100 mg capsules of thiamine or placebo per day for 3 months with a 2 month follow-up washout period. The primary endpoint was change in urinary albumin excretion (UAE). Other markers of renal and vascular dysfunction and plasma concentrations of thiamine were determined. RESULTS: UAE was decreased in patients receiving thiamine therapy for 3 months with respect to baseline (median -17.7 mg/24 h; p < 0.001, n = 20). There was no significant decrease in UAE in patients receiving placebo after 3 months of therapy (n = 20). UAE was significantly lower in patients who had received thiamine therapy compared with those who had received placebo (30.1 vs 35.5 mg/24 h, p < 0.01) but not at baseline. UAE continued to decrease in the 2 month washout period in both groups, but not significantly. There was no effect of thiamine treatment on glycaemic control, dyslipidaemia or BP. There were no adverse effects of therapy. CONCLUSIONS/INTERPRETATION: In this pilot study, high-dose thiamine therapy produced a regression of UAE in type 2 diabetic patients with microalbuminuria. Thiamine supplements at high dose may provide improved therapy for early-stage diabetic nephropathy. TRIAL REGISTRATION: CTRI (India) CTRI/2008/091/000112. FUNDING: Pakistan Higher Education Commission.

Adsorption and photocatalytic decolorization of a synthetic dye erythrosine on anatase TiO2 and ZnO surfaces.

The UV radiation assisted photocatalytic decolorization/degradation kinetics of an anionic dye erythrosine (ER), has been studied over TiO2 and ZnO surfaces. Since adsorption is the prerequisite condition for decolorization/degradation of dye molecules in presence of heterogeneous catalysis, the Langmuir and Freundlich isotherms were examined to verify the adsorption intensity. Standard adsorption free energy measurement implies that the adsorption of ER on both TiO2 and ZnO surfaces is spontaneous endothermic process. The effect of catalyst loading (TiO2/ZnO) revealed the fact that the maximum decolorization rate is obtained under an optimized catalyst loading condition. The decolorization efficiency was also investigated over the pH range of 5.0-10.0 indicating that increasing pH enhances decolorization efficiency. The influence of H2O2 on decolorization efficiency was found noticeable since it is a hydroxyl radical provider. The kinetic study of this degradation indicates that under the experimental condition, the decolorization mechanism follows zero order kinetics on the basis of Langmuir-Hinshelwood (L-H) heterogeneous reaction mechanism.

Genomic structure of the rat major AP endonuclease gene (Apex) with an adjacent putative O-sialoglycoprotease gene (Prsmg1/Gcpl1) and a processed Apex pseudogene (Apexp1).

Genomic sequencing and chromosomal assignment of the gene encoding rat APEX nuclease, a multifunctional DNA repair enzyme, were performed. An active Apex gene and a processed pseudogene were isolated from a rat genomic library. The active Apex gene consists of 5 exons and 4 introns spanning 2.1 kb. The putative promoter region of the Apex gene lacks the typical TATA box, but contains CAAT boxes and a CpG island having putative binding sites for several transcription factors, such as Sp1, AP-2, GATA-1 and ATF. A putative O-sialoglycoprotease (a homologue of Pasteurella haemolytica glycoprotease, gcp; abbreviated as Prsmg1/Gcpl1) gene consisting of 11 exons and 10 introns spanning 7.3 kb lies immediately adjacent to the Apex gene in a 5'-to-5' orientation. The Apex gene locus was mapped to rat chromosome 15p12 using in situ hybridization. The processed pseudogene (designated as rat Apexp1) has a nucleotide sequence 87.1% identical to that of the rat Apex cDNA, although several stop codons interrupting the coding sequences and multiple nucleotide deletions were observed. The Apexp1 is located in an inactive LINE sequence. Calculation of nucleotide substitution rates suggests that the immediate, active progenitor of Apexp1 arose 23 million years ago and that the non-functionalization occurred 15 million years ago.

Enhancement of cholesteryl ester metabolism in cultured human monocyte-derived macrophages by verapamil.

The effect of the Ca2+ entry blocker, verapamil, on the biosynthesis of cholesterol and the metabolism of low-density lipoprotein (LDL) was studied in cultured human monocyte-derived macrophages. Addition of verapamil (50 microM) of monocyte-derived macrophages enhanced 125I-LDL and 125I-labelled acetyl-LDL binding and internalization, and increased [2-14C]acetate incorporation into cholesterol. Since higher levels of LDL and modified lipoproteins may be implicated in atherogenesis, the more efficient processing of these lipoproteins by monocyte-derived macrophages in the presence of Ca2+ blocker warrants further assessment for its potential as an antiatherogenic agent.

Regulation of cholesterol synthesis by plasma lipoproteins from patients with abetalipoproteinemia.

Despite a complete lack of apoprotein B-containing lipoproteins from the plasma of patients with abetalipoproteinemia, rates of cholesterol synthesis measured in vivo or in freshly isolated cells in vitro are not markedly elevated. These observations suggest that other lipoprotein particles present in the plasma of patients with abetalipoproteinemia may regulate cellular cholesterol synthesis in this disorder. In the present report we have studied the effects of lipoprotein fractions from plasma of normal subjects, patients with abetalipoproteinemia, and a patient with Type III hyperlipoproteinemia on cholesterol synthesis in cultured human fibroblasts. LDL from normal subjects or the HDL2 fraction from the plasma of patients with abetalipoproteinemia were effective inhibitors of cholesterol synthesis (greater than 75% inhibition at 20 micrograms protein/ml) whereas HDL3 from normal or abetalipoproteinemia plasma stimulated cholesterol synthesis. Rates of cholesterol synthesis in fibroblasts from a patient with receptor-negative homozygous familial hypercholesterolemia were only minimally reduced by prior incubation in media containing either normal LDL or HDL2 from the plasma of a patient with abetalipoproteinemia. We conclude that lipoproteins present in the HDL2 fraction of plasma from patients with abetalipoproteinemia (which are relatively rich in apoprotein E) are effective regulators of cholesterol synthesis in normal human fibroblasts and that this regulation is mediated by an interaction of these lipoproteins with the LDL (B, E) receptor. These in vitro findings may explain why rates of cholesterol synthesis are not markedly elevated in patients with abetalipoproteinemia studied in vivo.

Lipoprotein lipase and hepatic lipase activity after heparin administration in abetalipoproteinemia and hypobetalipoproteinemia.

The purpose of this study was to examine whether an absence of triglyceride-rich lipoproteins (chylomicrons and very-low-density lipoproteins) in plasma is associated with any changes in the enzyme activity of lipoprotein lipase or hepatic lipase after heparin administration. To study this, the activities of hepatic lipase and lipoprotein lipase were determined in control subjects, in two patients with heterozygous hypobetalipoproteinemia, and in three patients with phenotypic abetalipoproteinemia after administration of heparin. Both enzymes showed normal activity in the patients with hypobetalipoproteinemia, but showed consistently reduced activity in the patients with abetalipoproteinemia. Hepatic lipase activity in plasma samples from these three patients obtained 15 minutes after intravenous injection of heparin was 55%, 87%, and 46% of that of the controls, whereas corresponding values in plasma samples obtained 30 minutes after heparin were 47%, 70%, and 57%, respectively. Lipoprotein lipase activity in the three patients with abetalipoproteinemia was 46%, 29%, and 34% of that of the controls in the samples obtained 15 minutes after heparin injection, whereas the values obtained after 30 minutes were 53%, 64%, and 47% of that of the controls. We conclude that an inherent absence of triglyceride-rich lipoproteins, as occurs in abetalipoproteinemia, is associated with reduced enzyme activity of both hepatic lipase and lipoprotein lipase in plasma after heparin administration.

Dimethyl sulfoxide as a cholesterol-lowering agent in cultured fibroblasts exposed to low density lipoproteins.

Confluent cultures of human skin fibroblasts were exposed to medium containing high levels of low density lipoproteins (LDL-cholesterol equivalent to 400 micrograms per ml) and 0 or 2% dimethyl sulfoxide (DMSO). The uptake and accumulation of cellular cholesterol from LDL were reduced significantly (30%) in the DMSO-treated cells as compared to the controls. The reduction in cellular sterol was due almost exclusively to a significant decrease (50%) in cholesterol ester accumulation. Incubation of cells with 125I-labelled LDL showed clearly that DMSO did not act by increasing the secretion of cholesterol from the cell, but rather by significantly decreasing the binding, internalization and degradation of exogenous LDL. De novo synthesis of cholesterol from [14C]acetate was measured and found to correlate inversely with cellular sterol levels in either control or DMSO-treated cells.

Effects of long-term protein deficiency on plasma lipoprotein concentrations and metabolism in rhesus monkeys.

Lipoprotein concentrations and metabolism were studied in 5- and 9-year-old rhesus monkeys (Macaca mulatta). Both age groups had been divided into control (13.8% of the calories as protein) and low-protein (3.7% protein) subgroups at birth. All were tested before and after their dietary lipid was changed from corn oil to butter plus cholesterol. The concentrations of very-low-density and high density2 lipoproteins (VLDL and HDL2) tended to be higher in monkeys of the low-protein group, and butter plus cholesterol accentuated the difference. All monkeys of the low protein group had elevated levels of at least one of these two classes of lipoproteins. The secretion of nascent VLDL (after intravenous Triton WR-1339) was greater in the low protein than in the control group when both were fed butter plus cholesterol, and rates of VLDL secretion showed a strong positive correlation with the fasting levels of VLDL. Thirty minutes after the intravenous administration of heparin, VLDL triglyceride was almost completely removed, and the apolipoprotein was reduced by 40%; VLDL cholesteryl esters were unchanged. There was a simultaneous decrease in the intermediate density lipoproteins (IDL) and an increase in HDL2, but the low density lipoproteins (LDL) and HDL2 did not change. The rate of VLDL protein removal from plasma was greater in the low protein than in the control group, and the rates for individual monkeys correlated with the levels of VLDL and HDL2 prior to heparin administration. Hepatic postheparin lipase measured in vitro with saturating levels of substrate was significantly higher in the plasma of control than in that of low protein monkeys.

The mass uptake of cholesterol ester from low density lipoproteins by cultured smooth muscle and adventitial cells of human aortas.

Cultured human smooth muscle and adventitial cells were incubated with human serum and low density lipoprotein (LDL) to study the uptake and accumulation of cholesterol ester from exogenous LDL. The cellular total cholesterol varied with the amount of LDL cholesterol in the medium. The cholesterol ester content increased 4-fold after 2 hours of incubation. A 6-fold rise occurred by 24 hours and continued to 72 hours. The cholesterol ester of the adventitial cells was markedly depleted by incubation with abetalipoproteinemic serum or with a lipid-depleted plasma fraction. By the use of 14C-labeled LDL free cholesterol in the incubation medium, we calculated that some 70-80% of the total accumulated cholesterol ester after 24 hours of incubation was derived from LDL cholesterol ester, and only 20-30% was synthesized by the cells. These studies demonstrated conclusively that human cells greatly increase their cholesterol ester mass after incubation with LDL.