A myeloid leukemia factor homolog involved in encystation-induced protein metabolism in Giardia lamblia.

BACKGROUND: Giardia lamblia differentiates into resistant cysts as an established model for dormancy. Myeloid leukemia factor (MLF) proteins are important regulators of cell differentiation. Giardia possesses a MLF homolog which was up-regulated during encystation and localized to unknown cytosolic vesicles named MLF vesicles (MLFVs). METHODS: We used double staining for visualization of potential factors with role in protein metabolism pathway and a strategy that employed a deletion mutant, CDK2m3, to test the protein degradation pathway. We also explored whether autophagy or proteasomal degradation are regulators of Giardia encystation by treatment with MG132, rapamycin, or chloroquine. RESULTS: Double staining of MLF and ISCU or CWP1 revealed no overlap between their vesicles. The aberrant CDK2m3 colocalized with MLFVs and formed complexes with MLF. MG132 increased the number of CDK2m3-localized vesicles and its protein level. We further found that MLF colocalized and interacted with a FYVE protein and an ATG8-like (ATG8L) protein, which were up-regulated during encystation and their expression induced Giardia encystation. The addition of MG132, rapamycin, or chloroquine, increased their levels and the number of their vesicles, and inhibited the cyst formation. MLF and FYVE were detected in exosomes released from culture. CONCLUSIONS: The MLFVs are not mitosomes or encystation-specific vesicles, but are related with degradative pathway for CDK2m3. MLF, FYVE, and ATG8L play a positive role in encystation and function in protein clearance pathway, which is important for encystation and coordinated with Exosomes. GENERAL SIGNIFICANCE: MLF, FYVE, and ATG8L may be involved an encystation-induced protein metabolism during Giardia differentiation.

Effects of doxazosin mesylate versus nifedipine on blood pressure variability in hypertensive patients: a randomized crossover study (SIMILAR).

OBJECTIVE: Blood pressure variability (BPV) is a powerful predictor of end-organ damage, cardiovascular events and mortality independently of the BP level. Calcium channel blockers may offer an advantage over other first-line antihypertensive drugs by preventing increased BPV. But the effect of alpha-receptor blockers on BPV in hypertensive patients is still unclear. METHODS: In this crossover trial, 36 hypertensive patients were randomly assigned to two groups, receiving doxazosin mesylate gastrointestinal therapeutic system (GITS) (4 mg/day) or nifedipine GITS (30 mg/day) for 12 weeks, followed by a 2-week washout period then a 12-week crossover phase. At baseline and after 12-week treatment, 24-hour ambulatory BP monitoring was performed. BPV was evaluated through standard deviation (SD), coefficient of variation (CV), and average real variability (ARV) of systolic BP (SBP) and diastolic BP (DBP) during daytime, nighttime and over 24 hours. RESULTS: After 12-week treatment, both doxazosin and nifedipine significantly decreased SBP and DBP (P < 0.05), whereas no between-group differences were shown (P>0.05). Systolic BPV (24-hour SD, CV, and ARV; daytime SD; nighttime SD and CV) and diastolic BPV (24-hour SD and ARV) were significantly lowered by nifedipine (P < 0.05); doxazosin resulted in significant reductions of systolic BPV (24-hour SD, CV and ARV; daytime SD; nighttime SD) and diastolic BPV (nighttime SD and CV) (P < 0.05). Doxazosin was revealed to be as effective as nifedipine for reducing BPV (P > 0.05) except for 24-hour SBP ARV. CONCLUSIONS: Doxazosin mesylate GITS had similar therapeutic effects on BP, BP SD, and BP CV lowering as nifedipine GITS in patients with mild-to-moderate essential hypertension.

Effects of different doses of ticagrelor on platelet aggregation and endothelial function in diabetic patients with stable coronary artery disease.

We performed this study to observe the effects of different doses of ticagrelor and standard-dose clopidogrel on platelet reactivity and endothelial function in diabetic patients with stable coronary artery disease (CAD). Sixty type 2 diabetic patients were assigned to one-quarter standard-dose ticagrelor, half standard-dose ticagrelor, standard-dose ticagrelor and standard-dose clopidogrel groups. Light transmission aggregometry (LTA) and VerifyNow assay were used to measure platelet function. Endothelial function was assessed by measurement of flow-mediated vasodilation (FMD) and plasma von Willebrand factor (VWF) levels were detected. Enzyme-linked immunosorbent assay (ELISA) examined the Interleukin-8(IL-8) and IL-10. The results suggested that the one-quarter dose (34.0%± 14.7%), half-dose (26.9%± 11.6%) and standard-dose (17.3%± 10.3%) ticagrelor showed lower platelet aggregation rate than clopidogrel (52.8%± 18.3%; P ï¼œ0.0001). PRU values in three ticagrelor groups were lower than that in clopidogrel group (102 (76-184)；75 (33-88)；38 (11-52) versus 194 (138-271) and；P ï¼œ0.0001). FMD levels were higher in ticagrelor groups compared with baseline levels while lower in clopidogrel group after treatment. However, no significant differences were found in the percentage increase in the FMD between ticagrelor groups and clopidogrel group. The levels of VWF after treatment were lower than the baseline levels, but there was no statistically significant difference between ticagrelor group and clopidogrel group after treatment. The concentration of IL-8 and IL-10 were decreased in patients with half and standard-dose ticagrelor group. In conclusion, one-quarter standard-dose ticagrelor produced similar inhibitory effects on platelet aggregation as standard-dose clopidogrel in diabetic patients with stable CAD. The half standard-dose ticagrelor had a similar inhibitory effect on platelet inhibition as standard-dose ticagrelor, which was stronger than that of clopidogrel. Moreover, the half-dose ticagrelor had equal protection of endothelial function and inhibition of inflammatory factor as standard-dose ticagrelor.

Metoprolol has a similar therapeutic effect as amlodipine on BP lowering in hypertensive patients with obstructive sleep apnea.

PURPOSE: ß-Blocker use has been controversial for a long time in the management of hypertensive patients with obstructive sleep apnea (OSA). The aim of present study was to compare the effects of metoprolol on BP lowering with amlodipine in hypertensive OSA patients. METHODS: Hypertensive subjects with OSA were randomly assigned to metoprolol and amlodipine groups, receiving 12 weeks of oral either metoprolol (47.5 mg once daily) or amlodipine (5 mg once daily) treatment. At baseline and after the 12-week treatment period, 24-h ambulatory blood pressure monitoring was performed in both groups. RESULTS: Both of metoprolol and amlodipine treatments significantly lowered 24-h blood pressure (BP) (from 143/88 to 132.3/81.6 mmHg; from 141.3/84.5 to 133.7/80.8 mmHg), daytime BP (from 146/90.2 to 136.4/84.6 mmHg; from 145.1/87.6 to 138.2/84.1 mmHg), and nighttime BP (from 139.1/83.9 to 125.7/76.2 mmHg; from 134.5/78.5 to 125.8/74.1 mmHg) (all P < 0.05). But there were no significant differences between the groups in BP variability (P > 0.05). Besides, metoprolol significantly reduced daytime heart rate (HR) (P < 0.05), while 24-h and nighttime HR values had no remarkable changes compared with baseline (P > 0.05). CONCLUSIONS: Metoprolol had similar therapeutic effects on BP lowering as amlodipine and could not decrease HR during the nighttime in hypertensive patients with OSA.

Obstructive sleep apnea increases systolic and diastolic blood pressure variability in hypertensive patients.

OBJECTIVE: Obstructive sleep apnea (OSA) has been identified as the most common secondary contributing factor for the development and worsening of hypertension. However, the underlying relationships between blood pressure variability (BPV) and OSA are still not very clear. Therefore, we investigated the influences of OSA on BPV in hypertensive patients and explored the potential pathophysiologic mechanisms. PARTICIPANTS AND METHODS: Ambulatory blood pressure (BP) monitoring was carried out and polysomnography was performed to detect sleep apnea. A total of 86 hypertensive individuals were divided into patients without OSA (n=43) and patients with severe OSA (n=43). Systolic and diastolic BPV were obtained by calculating the SD, coefficient of variation, and average real variability during day-time, night-time, and over 24 h. The relationship between OSA and BPV was assessed after adjustment for potential confounding variables (age, sex, BMI, neck circumference, heart rate, and snoring history). RESULTS: Compared with participants without OSA, nocturnal systolic BPV and 24-h systolic BP average real variability from OSA participants were obviously increased (P<0.05), but there were no statistically significant differences in day-time and 24-h systolic BP SD and coefficient of variation (P>0.05). Compared with participants without OSA, 24-h diastolic BPV and day-time diastolic BP SD from OSA participants were markedly increased (P<0.05), but nocturnal indices showed no significant differences between the two groups. CONCLUSION: OSA mainly increases night-time systolic and 24-h diastolic BPV in hypertensive patients. This may provide a plausible explanation for OSA remaining a major risk determinant for cardiovascular diseases.

Correction: DNA Topoisomerase II Is Involved in Regulation of Cyst Wall Protein Genes and Differentiation in Giardia lamblia.

[This corrects the article DOI: 10.1371/journal.pntd.0002218.].

One-quarter standard-dose ticagrelor better than standard-dose clopidogrel in Chinese patients with stable coronary artery disease: A randomized, single-blind, crossover clinical study.

BACKGROUND: Ticagrelor has been demonstrated to provide a more rapid and powerful inhibition of platelet aggregation compared with clopidogrel in coronary artery disease (CAD) patients. In our previous study, we found that half-dose ticagrelor produced similar inhibitory effects on platelet aggregation as standard-dose ticagrelor and exerted significantly stronger effects than clopidogrel in Chinese patients with non-ST-elevation ACS. Therefore, we performed this study to observe the efficacy of one-quarter standard-dose ticagrelor in comparison to standard-dose clopidogrel in Chinese patients with stable CAD. METHODS: In a randomized, single-blind, crossover trial, 30 patients with stable CAD were randomized to one-quarter standard-dose ticagrelor (22.5mg BID for 7days) or standard-dose clopidogrel (75mg QD for 7days). Following a 2-week washout period, patients switched regimens. Light transmission aggregometry (LTA) and VerifyNow assay were used to measure platelet function. RESULTS: The platelet aggregation rate (PAgR) was obviously lower with ticagrelor than clopidogrel (17.70%±12.67% versus 27.63%±13.10%, P<0.05). The % inhibition levels in the ticagrelor group exhibited significantly greater than that in the clopidogrel group (65.33%±21.31% versus 36.23%±23.01%, P<0.01). PRU values in the ticagrelor group were dramatically lower than that in the clopidogrel group (87.03±51.38 versus 163.77±58.66, P<0.01). High-platelet reactivity (HPR) (≥208 PRU) was 0% with ticagrelor and 16.67% with clopidogrel. CONCLUSIONS: One-quarter standard-dose ticagrelor provided greater degree of platelet inhibition than standard-dose clopidogrel in Chinese patients with stable CAD.

DNA topoisomerase II is involved in regulation of cyst wall protein genes and differentiation in Giardia lamblia.

The protozoan Giardia lamblia differentiates into infectious cysts within the human intestinal tract for disease transmission. Expression of the cyst wall protein (cwp) genes increases with similar kinetics during encystation. However, little is known how their gene regulation shares common mechanisms. DNA topoisomerases maintain normal topology of genomic DNA. They are necessary for cell proliferation and tissue development as they are involved in transcription, DNA replication, and chromosome condensation. A putative topoisomerase II (topo II) gene has been identified in the G. lamblia genome. We asked whether Topo II could regulate Giardia encystation. We found that Topo II was present in cell nuclei and its gene was up-regulated during encystation. Topo II has typical ATPase and DNA cleavage activity of type II topoisomerases. Mutation analysis revealed that the catalytic important Tyr residue and cleavage domain are important for Topo II function. We used etoposide-mediated topoisomerase immunoprecipitation assays to confirm the binding of Topo II to the cwp promoters in vivo. Interestingly, Topo II overexpression increased the levels of cwp gene expression and cyst formation. Microarray analysis identified up-regulation of cwp and specific vsp genes by Topo II. We also found that the type II topoisomerase inhibitor etoposide has growth inhibition effect on Giardia. Addition of etoposide significantly decreased the levels of cwp gene expression and cyst formation. Our results suggest that Topo II has been functionally conserved during evolution and that Topo II plays important roles in induction of the cwp genes, which is key to Giardia differentiation into cysts.

Functional redundancy of two Pax-like proteins in transcriptional activation of cyst wall protein genes in Giardia lamblia.

The protozoan Giardia lamblia differentiates from a pathogenic trophozoite into an infectious cyst to survive outside of the host. During encystation, genes encoding cyst wall proteins (CWPs) are coordinately induced. Pax family transcription factors are involved in a variety of developmental processes in animals. Nine Pax proteins have been found to play an important role in tissue and organ development in humans. To understand the progression from primitive to more complex eukaryotic cells, we tried to identify putative pax genes in the G. lamblia genome and found two genes, pax1 and pax2, with limited similarity. We found that Pax1 may transactivate the encystation-induced cwp genes and interact with AT-rich initiatior elements that are essential for promoter activity and transcription start site selection. In this study, we further characterized Pax2 and found that, like Pax1, Pax2 was present in Giardia nuclei and it may specifically bind to the AT-rich initiator elements of the encystation-induced cwp1-3 and myb2 genes. Interestingly, overexpression of Pax2 increased the cwp1-3 and myb2 gene expression and cyst formation. Deletion of the C-terminal paired domain or mutation of the basic amino acids of the paired domain resulted in a decrease of nuclear localization, DNA-binding activity, and transactivation activity of Pax2. These results are similar to those found in the previous Pax1 study. In addition, the profiles of gene expression in the Pax2 and Pax1 overexpressing cells significantly overlap in the same direction and ERK1 associated complexes may phosphorylate Pax2 and Pax1, suggesting that Pax2 and Pax1 may be downstream components of a MAPK/ERK1 signaling pathway. Our results reveal functional redundancy between Pax2 and Pax1 in up-regulation of the key encystation-induced genes. These results illustrate functional redundancy of a gene family can occur in order to increase maintenance of important gene function in the protozoan organism G. lamblia.

Regulation of a Myb transcription factor by cyclin-dependent kinase 2 in Giardia lamblia.

The protozoan Giardia lamblia parasitizes the human small intestine to cause diseases. It undergoes differentiation into infectious cysts by responding to intestinal stimulation. How the activated signal transduction pathways relate to encystation stimulation remain largely unknown. During encystation, genes encoding cyst wall proteins (CWPs) are coordinately up-regulated by a Myb2 transcription factor. Because cell differentiation is linked to cell cycle regulation, we tried to understand the role of cell cycle regulators, cyclin-dependent kinases (Cdks), in encystation. We found that the recombinant Myb2 was phosphorylated by Cdk-associated complexes and the levels of phosphorylation increased significantly during encystation. We have identified a putative cdk gene (cdk2) by searching the Giardia genome database. Cdk2 was found to localize in the cytoplasm with higher expression during encystation. Interestingly, overexpression of Cdk2 resulted in a significant increase of the levels of cwp gene expression and cyst formation. In addition, the Cdk2-associated complexes can phosphorylate Myb2 and the levels of phosphorylation increased significantly during encystation. Mutations of important catalytic residues of Cdk2 resulted in a significant decrease of kinase activity and ability of inducing cyst formation. Addition of a Cdk inhibitor, purvalanol A, significantly decreased the Cdk2 kinase activity and the levels of cwp gene expression and cyst formation. Our results suggest that the Cdk2 pathway may be involved in phosphorylation of Myb2, leading to activation of the Myb2 function and up-regulation of cwp genes during encystation. The results provide insights into the use of Cdk inhibitory drugs in disruption of Giardia differentiation into cysts.

A novel E2F-like protein involved in transcriptional activation of cyst wall protein genes in Giardia lamblia.

Giardia lamblia differentiates into resistant walled cysts for survival outside the host and transmission. During encystation, synthesis of cyst wall proteins is coordinately induced. The E2F family of transcription factors in higher eukaryotes is involved in cell cycle progression and cell differentiation. We asked whether Giardia has E2F-like genes and whether they influence gene expression during Giardia encystation. Blast searches of the Giardia genome database identified one gene (e2f1) encoding a putative E2F protein with two putative DNA-binding domains. We found that the e2f1 gene expression levels increased significantly during encystation. Epitope-tagged E2F1 was found to localize to nuclei. Recombinant E2F1 specifically bound to the thymidine kinase and cwp1-3 gene promoters. E2F1 contains several key residues for DNA binding, and mutation analysis revealed that its binding sequence is similar to those of the known E2F family proteins. The E2F1-binding sequences were positive cis-acting elements of the thymidine kinase and cwp1 promoters. We also found that E2F1 transactivated the thymidine kinase and cwp1 promoters through its binding sequences in vivo. Interestingly, E2F1 overexpression resulted in a significant increase of the levels of CWP1 protein, cwp1-3 gene mRNA, and cyst formation. We also found E2F1 can interact with Myb2, a transcription factor that coordinate up-regulates the cwp1-3 genes during encystation. Our results suggest that E2F family has been conserved during evolution and that E2F1 is an important transcription factor in regulation of the Giardia cwp genes, which are key to Giardia differentiation into cysts.

A novel pax-like protein involved in transcriptional activation of cyst wall protein genes in Giardia lamblia.

Giardia lamblia differentiates into infectious cysts to survive outside of the host. It is of interest to identify factors involved in up-regulation of cyst wall proteins (CWPs) during this differentiation. Pax proteins are important regulators of development and cell differentiation in Drosophila and vertebrates. No member of this gene family has been reported to date in yeast, plants, or protozoan parasites. We have identified a pax-like gene (pax1) encoding a putative paired domain in the G. lamblia genome. Epitope-tagged Pax1 localized to nuclei during both vegetative growth and encystation. Recombinant Pax1 specifically bound to the AT-rich initiator elements of the encystation-induced cwp1 to -3 and myb2 genes. Interestingly, overexpression of Pax1 increased cwp1 to -3 and myb2 gene expression and cyst formation. Deletion of the C-terminal paired domain or mutation of the basic amino acids of the paired domain resulted in a decrease of the transactivation function of Pax1. Our results indicate that the Pax family has been conserved during evolution, and Pax1 could up-regulate the key encystation-induced genes to regulate differentiation of the protozoan eukaryote, G. lamblia.

A novel family of cyst proteins with epidermal growth factor repeats in Giardia lamblia.

BACKGROUND: Giardia lamblia parasitizes the human small intestine to cause diarrhea and malabsorption. It undergoes differentiation from a pathogenic trophozoite form into a resistant walled cyst form. Few cyst proteins have been identified to date, including three cyst wall proteins (CWPs) and one High Cysteine Non-variant Cyst protein (HCNCp). They are highly expressed during encystation and are mainly targeted to the cyst wall. METHODOLOGY AND PRINCIPAL FINDINGS: To identify new cyst wall proteins, we searched the G. lamblia genome data base with the sequence of the Cryptosporidium parvum oocyst wall protein as a query and found an Epidermal Growth Factor (EGF)-like Cyst Protein (EGFCP1). Sequence analysis revealed that the EGF-like repeats of the EGFCP1 are similar to those of the tenascin family of extracellular matrix glycoproteins. EGFCP1 and HCNCp have a higher percentage of cysteine than CWPs, but EGFCP1 has no C-terminal transmembrane region found in HCNCp. Like CWPs and HCNCp, the EGFCP1 protein (but not transcript) was expressed at higher levels during encystation and it was localized to encystation-specific vesicles in encysting trophozoites. Like HCNCp, EGFCP1 was localized to the encystation-specific vesicles, cyst wall and cell body of cysts, suggesting that they may share a common trafficking pathway. Interestingly, overexpression of EGFCP1 induced cyst formation and deletion of the signal peptide from EGFCP1 reduced its protein levels and cyst formation, suggesting that EGFCP1 may help mediate cyst wall synthesis. We also found that five other putative EGFCPs have similar expression profiles and similar locations and that the cyst formation was induced upon their overexpression. CONCLUSIONS AND SIGNIFICANCE: Our results suggest that EGFCPs may function like cyst wall proteins, involved in differentiation of G. lamblia trophozoites into cysts. The results lead to greater understanding of parasite cyst walls and provide valuable information that helps develop ways to interrupt the G. lamblia life cycle.

A novel WRKY-like protein involved in transcriptional activation of cyst wall protein genes in Giardia lamblia.

Synthesis of a protective cyst wall is required for survival outside of the host and for infection of Giardia lamblia. Little is known of gene regulation of the cyst wall proteins (CWPs) during differentiation into dormant cysts. WRKY homologues constitute a large family of DNA-binding proteins in plants that are involved in several key cellular functions, including disease resistance, stress response, dormancy, and development. A putative wrky gene has been identified in the G. lamblia genome. We found that wrky expression levels increased significantly during encystation. The epitope-tagged WRKY was translocated into the nuclei during encystation. Recombinant WRKY specifically bound to its own promoter and the encystation-induced cwp1 and cwp2 promoters. WRKY contains several key residues for DNA binding, and mutation analysis revealed that its binding sequences are similar to those of the known plant WRKY proteins and that two of them are positive cis-acting elements of the wrky and cwp2 promoters. Overexpression of WRKY increased the cwp1-2 and myb2 mRNA levels, and these gene promoters were bound by WRKY in vivo. Interestingly, the wrky and cwp1-2 genes were up-regulated by ERK1 (extracellular signal-related kinase 1) overexpression, suggesting that WRKY may be a downstream component of the ERK1 pathway. In addition, a WRKY mutant that cannot enter nuclei and an ERK1 mutant lacking the predicted kinase domain showed decreased cwp1-2 gene expression. Our results suggest that the WRKY family has been conserved during evolution and that WRKY is an important transactivator of the cwp1-2 genes during G. lamblia differentiation into dormant cysts.