Unusual metal ion cofactor requirement of Entamoeba histolytica choline and ethanolamine kinase isoforms.

The de novo biosynthesis of phosphatidylcholine and phosphatidylethanolamine in Entamoeba histolytica is largely dependent on the CDP-choline and CDP-ethanolamine pathways. Although the first enzymes of these pathways, EhCK1 and EhCK2, have been previously characterized, their enzymatic activity was found to be low and undetectable, respectively. This study aimed to identify the unusual characteristics of these enzymes in this deadly parasite. The discovery that EhCKs prefer Mn2+ over the typical Mg2+ as a metal ion cofactor is intriguing for CK/EK family of enzymes. In the presence of Mn2+, the activity of EhCK1 increased by approximately 108-fold compared to that in Mg2+. Specifically, in Mg2+, EhCK1 exhibited a Vmax and K0.5 of 3.5 ± 0.1 U/mg and 13.9 ± 0.2 mM, respectively. However, in Mn2+, it displayed a Vmax of 149.1 ± 2.5 U/mg and a K0.5 of 9.5 ± 0.1 mM. Moreover, when Mg2+ was present at a constant concentration of 12 mM, the K0.5 value for Mn2+ was ~ 2.4-fold lower than that in Mn2+ alone, without affecting its Vmax. Although the enzyme efficiency of EhCK1 was significantly improved by about 25-fold in Mn2+, it is worth noting that its Km for choline and ATP were higher than in equimolar of Mg2+ in a previous study. In contrast, EhCK2 showed specific activity towards ethanolamine in Mn2+, exhibiting Michaelis-Menten kinetic with ethanolamine (Km = 312 ± 27 µM) and cooperativity with ATP (K0.5 = 2.1 ± 0.2 mM). Additionally, we investigated the effect of metal ions on the substrate recognition of human choline and ethanolamine kinase isoforms. Human choline kinase α2 was found to absolutely require Mg2+, while choline kinase ß differentially recognized choline and ethanolamine in Mg2+ and Mn2+, respectively. Finally, mutagenesis studies revealed that EhCK1 Tyr129 was critical for Mn2+ binding, while Lys233 was essential for substrate catalysis but not metal ion binding. Overall, these findings provide insight into the unique characteristics of the EhCKs and highlight the potential for new approaches to treating amoebiasis. Amoebiasis is a challenging disease for clinicians to diagnose and treat, as many patients are asymptomatic. However, by studying the enzymes involved in the CDP-choline and CDP-ethanolamine pathways, which are crucial for de novo biosynthesis of phosphatidylcholine and phosphatidylethanolamine in Entamoeba histolytica, there is great potential to discover new therapeutic approaches to combat this disease.

Macrophage polarization in THP-1 cell line and primary monocytes: A systematic review.

Macrophages derived from human monocytic leukemia THP-1 cell line are often used as the alternative of human primary macrophage. However, the polarization method of THP-1 to macrophages varies between different laboratories, which may unknowingly affect the relevance of research output across research groups. In this regard, a systematic search was developed in Pubmed, BioOne, Scopus, and Science Direct to identify articles focusing on THP-1 polarization into M1 and M2 macrophages. All selected articles were read and discussed by two independent reviewers. The selection process was based on selected keywords on the title, abstract and full-text level. A total of 85 articles were selected and categorized based on the field of studies, method of THP-1 differentiation, and markers or genes expressed upon differentiation. THP-1 derived macrophages were mainly used together with primary monocyte-derived macrophages in cellular inflammation studies, while it was commonly employed alone in cancer research. THP-1 derived macrophages are also of paramount importance in biomaterials studies to prevent unfavorable immune responses in-vivo. We explored various methods of THP-1 differentiation and suggested several common genes encountered to characterize M1 and M2 macrophages differentiated from THP-1. The systematic review highlights the relevance of using THP-1 derived macrophage as a useful alternative to primary macrophage. Although it is not possible to derive a standard method of THP-1 polarization into M1 and M2 macrophages from this review, it may lead researchers to obtain reproducible polarization protocol based on commonly used stimulants and markers of differentiation.

Differential polarization and the expression of efferocytosis receptor MerTK on M1 and M2 macrophages isolated from coronary artery disease patients.

BACKGROUND: Differential polarization of macrophage into M1 and M2 mediates atherosclerotic plaque clearance through efferocytosis. Higher expression of Mer proto-oncogene tyrosine kinase (MerTK) on M2 macrophage helps in maintaining macrophage efferocytic efficiency. In healthy individuals, macrophage polarization into M1 and M2 occurs in tissues in concomitance with the acquisition of functional phenotypes depending on specific microenvironment stimuli. However, whether the macrophage differential polarization and MerTK expression vary in coronary artery disease (CAD) patients remain unknown. OBJECTIVE: This study aimed to elucidate the polarization of M1 and M2 macrophage from CAD patients as well as to investigate the expression of MerTK in these macrophage phenotypes. METHODS: A total of 14 (n) CAD patients were recruited and subsequently grouped into "no apparent CAD", "non-obstructive CAD" and "obstructive CAD" according to the degree of stenosis. Thirty ml of venous blood was withdrawn to obtain monocyte from the patients. The M1 macrophage was generated by treating the monocyte with GMCSF, LPS and IFN-γ while MCSF, IL-4 and IL-13 were employed to differentiate monocyte into M2 macrophage. After 7 days of polarization, analysis of cell surface differentiation markers (CD86+/CD80+ for M1 and CD206+/CD200R+ for M2) and measurement of MerTK expression were performed using flow cytometry. RESULTS: Both M1 and M2 macrophage expressed similar level of CD86, CD80 and CD206 in all groups of CAD patients. MerTK expression in no apparent CAD patients was significantly higher in M2 macrophage compared to M1 macrophage [12.58 ± 4.40 vs. 6.58 ± 1.37, p = 0.040]. CONCLUSION: Differential polarization of macrophage into M1 and M2 was highly dynamic and can be varied due to the microenvironment stimuli in atherosclerotic plaque. Besides, higher expression of MerTK in patients with the least coronary obstructive suggest its vital involvement in efferocytosis.

Pilot study and bioinformatics analysis of differentially expressed genes in adipose tissues of rats with excess dietary intake.

Excessive adipose tissue accumulation is an increasing health problem worldwide. The present study aimed to determine differentially expressed genes (DEGs) that are associated with the excessive accumulation of adipose tissues by PCR arrays in an excess dietary intake animal model. For this purpose, male Sprague Dawley rats were randomly assigned to 2 groups: Control (given an ordinary diet) and experimental (given twice the amount of the ordinary diet). After 2 months of feeding, the abdominal cavities of the rats from each group were opened, then subcutaneous and visceral adipose tissues were removed. The adipose tissues collected were then used for total RNA extraction and then reverse transcribed to cDNA, which was then used as a template to identify the DEGs of 84 transcripts for rat obesity by RT2 Profiler PCR Arrays. The results showed significant downregulation of bombesin­like receptor 3 (BRS3) and uncoupling protein 1 (UCP1) in visceral adipose tissues of experimental rats compared with those of the control rats, and differential gene expression analysis showed an association with fat cell differentiation and regulation of triglyceride sequestration, as well as fatty acid binding. The gene expression patterns observed in the present study, which may be associated with peroxisome proliferator­activated receptor­Î³ (PPARG) on excessive visceral adipose tissue accumulation, may be useful in identifying a group of surrogate biomarkers for the early diet­induced accumulation of visceral adipose tissue detection in humans. The biomarkers can also be the specific targets for drug development to reduce excessive visceral adipose tissue accumulation in the body and its associated diseases.

Systemic and coronary levels of CRP, MPO, sCD40L and PlGF in patients with coronary artery disease.

BACKGROUND: Biomarkers play a pivotal role in the diagnosis and management of patients with acute coronary syndrome. This study aimed to investigate the differences in level of several biomarkers, i.e. C-reactive protein, myeloperoxidase, soluble CD40 ligand and placental growth factor, between acute coronary syndrome and chronic stable angina patients. The relationship between these biomarkers in the coronary circulation and systemic circulation was also investigated. METHODS: A total of 79 patients were recruited in this study. The coronary blood was sampled from occluded coronary artery, while the peripheral venous blood was withdrawn from antecubital fossa. The serum concentrations of C-reactive protein, soluble CD40 ligand and placental growth factor and plasma concentration of myeloperoxidase were measured using ELISA method. RESULTS: The systemic level of the markers measured in the peripheral venous blood was significantly increased in acute coronary syndrome compared to chronic stable angina patients. The concentrations of the C-reactive protein, myeloperoxidase and soluble CD40 ligand taken from peripheral vein were closely similar to the concentration found in coronary blood of ACS patients. The level of placental growth factor was significantly higher in coronary circulation than its systemic level. CONCLUSION: The concentration of these C-reactive protein, myeloperoxidase, soluble CD40 ligand and placental growth factor were significantly increased in acute coronary syndrome patients. The concentration of the markers measured in the systemic circulation directly reflected those in the local coronary circulation. Thus, these markers have potential to become a useful tool in predicting plaque vulnerability in the future.

Method optimization on the use of postocclusive hyperemia model to assess microvascular function.

INTRODUCTION: Recent development had allowed non-invasive assessment of microvascular function in vivo; however, the method has not been fully optimized and standardized. In this study, we aimed to characterize the "effective" occlusion duration needed to elicit sufficient postocclusive hyperemia (PORH) responses in forearm skin using laser Doppler fluximetry (LDF), in subjects with differing age, gender and menstrual phases. MATERIALS AND METHODS: A total of 120 healthy subjects were studied (20 subjects each in the age ranges of 21-30, 31-40, 41-50 for both genders). Male subjects were randomized to receive 1, 2 or 3 min occlusion on three study days. Females attended six study days: the first three days (with different occlusion times) were performed during low estrogenic phase of menstrual cycle and subsequent three visits were done during high estrogenic phase. Skin perfusion was measured before, during and after occlusion using LDF. The magnitude and temporal courses of PORH were expressed as PORH max (absolute maximal increase in hyperemia perfusion) and Tp (time-to-peak), respectively. RESULTS: For PORH max analysis, the occlusion duration should be applied based on one's age, gender and menstrual phase. The PORH responses were more consistent during high estrogenic phase with 2 min found as the "effective" occlusion duration in all female groups. For Tp analysis, 3 min occlusion produced the significant change in all age ranges for both genders irrespective of menstrual phase. CONCLUSION: This study revealed that for assessment of microvascular function using PORH+LDF model, the occlusion duration for PORH max is influenced by age, gender and menstrual phase. Measurement based on Tp is however independent of these factors.

Noninvasive assessment of cutaneous vascular function in vivo using capillaroscopy, plethysmography and laser-Doppler instruments: its strengths and weaknesses.

Given that functional abnormalities of the microcirculation are one of the primary abnormalities in cardiovascular disease pathogenesis, various noninvasive clinical tools have been developed recently to assess the microvascular function, particularly at the skin. The common techniques used to assess cutaneous microvascular function in vivo include capillaroscopy, venous occlusion plethysmography, and laser-Doppler instruments (laser-Doppler fluximetry and laser-Doppler imaging). These noninvasive techniques can be used as an early measure of functional abnormalities within the microvascular tree, predominantly in population at high risk for cardiovascular events. This review discusses some underlying application principle of these techniques, including its clinical significance, method reproducibility and limitations.

Reproducibility of different laser Doppler fluximetry parameters of postocclusive reactive hyperemia in human forearm skin.

INTRODUCTION: Postocclusive reactive hyperemia in forearm skin is a commonly used model for studying microvascular reactivity function, particularly in the assessment of vascular effect of topically applied pharmacological substances. In this study, we investigated the reproducibility of several different laser-Doppler-derived parameters in the measurement of postocclusive reactive hyperemia at forearm skin in healthy subjects. METHODS: Eighteen young healthy male volunteers were recruited and studied in a supine position while fasted. Forearm blood flow was occluded at suprasystolic pressure for 3 min. Microvascular perfusion was measured continuously using laser Doppler fluximetry. Parameters studied were maximum increase in hyperemia perfusion (PORHmax), time-to-peak (Tp), amplitude of peak perfusion (PORHpeak), percentage of hyperemic response (PORH%) and mean velocity of the hyperemia increase (PORHmax/Tp). Measurement was performed twice within each study day for 2 study days. Coefficient of variation and intraclass correlation coefficient (ICC; with 95% confidence interval) were calculated for each parameter. An ICC value above 0.75 was interpreted as "excellent reproducibility". RESULTS: ICC analysis showed that all studied parameters, except for PORH%, demonstrated excellent reproducibility for both within- and between-day measurements. Satisfactory intraday and interday coefficients of variation (<10%) were also obtained for these parameters. CONCLUSION: Laser-Doppler-derived PORHmax, Tp, PORHpeak and PORHmax/Tp were highly reproducible parameters for measuring microvascular reactivity during reactive hyperemia, with PORHmax shown as the most reproducible index. PORH% is, however, less reproducible. These findings have implications for the use of laser Doppler fluximetry coupled with 3-min-occlusion PORHmax as a useful and reliable noninvasive clinical measurement index of microvascular function.