Interaction of integrin-linked kinase with the kidney chloride/bicarbonate exchanger, kAE1

Kidney anion exchanger 1 (kAE1) mediates chloride/bicarbonate exchange at the basolateral membrane of kidney alpha-intercalated cells, thereby facilitating bicarbonate reabsorption into the blood. Human kAE1 lacks the N-terminal 65 residues of the erythroid form (AE1, band 3), which are essential for binding of cytoskeletal and cytosolic proteins. Yeast two-hybrid screening identified integrin-linked kinase (ILK), a serine/threonine kinase, and an actin-binding protein as an interacting partner with the N-terminal domain of kAE1. Interaction between kAE1 and ILK was confirmed in co-expression experiments in HEK 293 cells and is mediated by a previously unidentified calponin homology domain in the kAE1 N-terminal region. The calponin homology domain of kAE1 binds the C-terminal catalytic domain of ILK to enhance association of kAE1 with the actin cytoskeleton. Overexpression of ILK increased kAE1 levels at the cell surface as shown by flow cytometry, cell surface biotinylation, and anion transport activity assays. Pulse-chase experiments revealed that ILK associates with kAE1 early in biosynthesis, likely in the endoplasmic reticulum. ILK co-localized with kAE1 at the basolateral membrane of polarized Madin-Darby canine kidney cells and in alpha-intercalated cells of human kidneys. Taken together these results suggest that ILK and kAE1 traffic together from the endoplasmic reticulum to the basolateral membrane. ILK may provide a linkage between kAE1 and the underlying actin cytoskeleton to stabilize kAE1 at the basolateral membrane, resulting in higher levels of cell surface expression.     

Specific serum IgG, but not IgA, antibody against purified Opisthorchis viverrini antigen associated with hepatobiliary disease and cholangiocarcinoma

Opisthorchiasis caused by Opisthorchis viverrini infection induces hepatobiliary disease (HBD)-associated cholangiocarcinoma (CCA) via a chronic inflammatory immune response. Here, we evaluated specific IgG and IgA antibodies against different fractions of O. viverrini antigen in residents from an endemic community in Northeast Thailand with varying hepatobiliary abnormalities. Crude somatic O. viverrini antigen was purified into three fractions (viz., P1, P2 and P3) by gel infiltration chromatography and these served as antigens for detection of fluke-specific IgG and IgA antibodies by enzyme-linked immunosorbent assay (ELISA). The results revealed fluke-specific IgG and IgA antibody levels—against these antigens from subjects with O. viverrini-positive HBD—higher than in subjects with O. viverrini-negative HBD. Interestingly, the rank of fluke-specific IgG (and not IgA) antibody levels against crude extract and P1 antigens was CCA > severe HBD > mild HBD > healthy individuals. Purified antigens reduced cross-reactivity with other parasites compared to the crude antigen. Multiple linear regression analysis showed that HBD status was significantly associated with the liver fluke-specific IgG antibody against purified antigens. These results suggest that purified O. viverrini-antigen improves serodiagnosis for the evaluation of opisthorchiasis-associated HBD, and may be useful in the screening of opisthorchiasis in subjects at risk of developing CCA.     

Long-term cisplatin exposure impairs autophagy and causes cisplatin resistance in human lung cancer cells

Cisplatin-based chemotherapy frequently resulted in acquired resistance of cancer cells. The underlying mechanism of such resistance is not fully understood especially the involvement of autophagy and autophagic cell death. This study thus investigated whether an alteration in autophagy could be responsible for cisplatin resistance in the long-term exposure lung carcinoma cells. The cisplatin resistant clone (H460/cis) of H460 cells was established by exposing the cells with gradually increasing concentrations of cisplatin until chemoresistance acquisition was elucidated by MTT, Hoechst 33342 staining and comet assays. Degree of autophagosome formation and level of LC3 marker were evaluated by acridine orange and western blot analysis, respectively. H460/cis cells exhibited irregular shape with ~3-fold resistant to cisplatin-induced apoptosis compared with H460 cells. Proteins analysis for LC3 indicated that the levels of LC3 in resistant cells were significantly lower than those in H460 cells. Moreover, autophagosome formation detected by acridine orange staining was dramatically reduced in the resistant cells, suggesting the role of autophagy in attenuating of cisplatin-induced cell death. Further, co-treatment of cisplatin with autophagy inducer, trifluorperazine, could resensitize H460/cis cells to cisplatin-induced cell death. Our findings reveal the novel mechanisms causing cisplatin resistance in lung carcinoma cells after long-term drug exposure regarding autophagy.     

Human kanadaptin and kidney anion exchanger 1 (kAE1) do not interact in transfected HEK 293 cells

Kanadaptin (kidney anion exchanger adaptor protein) is a widely expressed protein, shown previously to interact with the cytosolic domain of mouse Cl-/HCO3- anion exchanger 1 (kAE1) but not erythroid AE1 (eAE1) by a yeast-two hybrid assay. Kanadaptin was co-localized with kAE1 in intracellular membranes but not at the plasma membrane in alpha-intercalated cells of rabbit kidney. It was suggested that kanadaptin is an adaptor protein or chaperone involved in targeting kAE1 to the plasma membrane. To test this hypothesis, the interaction of human kanadaptin with human kAE1 was studied in co-transfected HEK293 cells. Human kanadaptin contains 796 amino acids and was immuno-detected as a 90 kDa protein in transfected cells. Pulse-chase experiments showed that it has a half-life (t1/2) of 7 h. Human kanadaptin was localized predominantly to the nucleus, whereas kAE1 was present intracellularly and at the plasma membrane. Trafficking of kAE1 from its site of synthesis in the endoplasmic reticulum to the plasma membrane was unaffected by co-expression of human kanadaptin. Moreover, we found that no interaction between human kanadaptin and kAE1 or eAE1 could be detected in co-transfected cells either by co-immunoprecipitation or by histidine6-tagged co-purification. Taken together, we found that human kanadaptin did not interact with kAE1 and had no effect on trafficking of kAE1 to the plasma membrane in transfected cells. Kanadaptin may not be involved in the biosynthesis and targeting of kAE1. As such, defects in kanadaptin and its interaction with kAE1 are unlikely to be involved in the pathogenesis of the inherited kidney disease, distal renal tubular acidosis (dRTA).     

Derivatives (halogen,nitro and amino) of 8-hydroxyquinoline with highly potent antimicrobial and antioxidant activities

8-Hydroxyquinoline (8HQ) compounds have been reported to possess diverse bioactivities. In recent years, drug repositioning has gained considerable attention in drug discovery and development. Herein, 8HQ (1) and its derivatives (2–9) bearing various substituents (amino, nitro, cyano and halogen) were investigated for their antimicrobial against 27 microorganisms (agar dilution method) and antioxidant (DPPH method) activities. The parent 8HQ (1) exerted a highly potent antimicrobial activity against Gram-positive bacteria including diploid fungi and yeast with MIC values in the range of 3.44–13.78 μM. Moreover, the halogenated 8HQ, especially 7-bromo-8HQ (4) and clioquinol (6), displayed a high antigrowth activity against Gram-negative bacteria compared with the parent compound (1). Apparently, the derivatives with a relatively high safely index, e.g., nitroxoline (2), exhibited strong antibacterial activity against Aeromonas hydrophila (MIC=5.26 μM) and selectively inhibited the growth of P. aeruginosa with the MIC value of 84.14 μM; cloxyquin (3) showed a strong activity against Listseria monocytogenes and Plesiomonas shigelloides with MIC values of 5.57 and 11.14 μM, respectively. Most compounds displayed an antioxidant activity. Specifically, 5-amino-8HQ (8) was shown to be the most potent antioxidant (IC₅₀=8.70 μM) compared with the positive control (α-tocopherol) with IC₅₀ of 13.47 μM. The findings reveal that 8HQ derivatives are potential candidates to be further developed as antimicrobial and antioxidant agents.     

The current status of opisthorchiasis and clonorchiasis in the Mekong Basin

This review highlights the current status and control of liver fluke infections in the Mekong Basin countries where Opisthorchis and Clonorchis are highly endemic. Updated data on prevalence and distribution have been summarized from presentations in the “96 Years of Opisthorchiasis. International Congress of Liver Flukes”. It is disturbing that despite treatment and control programs have been in place for decades, all countries of the Lower Mekong Basin are still highly endemic with O. viverrini and/or C. sinensis as well as alarmingly high levels of CCA incidence. A common pattern that is emerging in each country is the difference in transmission of O. viverrini between lowlands which have high prevalence versus highlands which have low prevalence. This seems to be associated with wetlands, flooding patterns and human movement and settlement. A more concerted effort from all community, educational, public health and government sectors is necessary to successfully combat this fatal liver disease of the poor.     

CaMKII inhibition in heart failure, beneficial, harmful, or both

Calmodulin-dependent protein kinase II (CaMKII) has been proposed to be a therapeutic target for heart failure (HF). However, the cardiac effect of chronic CaMKII inhibition in HF has not been well understood. We have tested alterations of Ca(2+) handling, excitation-contraction coupling, and in vivo β-adrenergic regulation in pressure-overload HF mice with CaMKIIδ knockout (KO). HF was produced in wild-type (WT) and KO mice 1 wk after severe thoracic aortic banding (sTAB) with a continuous left ventricle (LV) dilation and reduction of ejection fraction for up to 3 wk postbanding. Cardiac hypertrophy was similar between WT HF and KO HF mice. However, KO HF mice manifested exacerbation of diastolic function and reduction in cardiac reserve to β-adrenergic stimulation. Compared with WT HF, L-type calcium channel current (I(Ca)) density in KO HF LV was decreased without changes in I(Ca) activation and inactivation kinetics, whereas I(Ca) recovery from inactivation was accelerated and Ca(2+)-dependent I(Ca) facilitation, a positive staircase blunted in WT HF, was recovered. However, I(Ca) response to isoproterenol was reduced. KO HF myocytes manifested dramatic decrease in sarcoplasmic reticulum (SR) Ca(2+) leak and slowed cytostolic Ca(2+) concentration decline. Sarcomere shortening was increased, but relaxation was slowed. In addition, an increase in myofilament sensitivity to Ca(2+) and the slow skeletal muscle troponin I-to-cardiac troponin I ratio and interstitial fibrosis and a decrease in Na/Ca exchange function and myocyte apoptosis were observed in KO HF LV. CaMKIIδ KO cannot suppress severe pressure-overload-induced HF. Although cellular contractility is improved, it reduces in vivo cardiac reserve to β-adrenergic regulation and deteriorates diastolic function. Our findings challenge the strategy of CaMKII inhibition in HF.     

Genetic history of Southeast Asian populations as revealed by ancient and modern human mitochondrial DNA analysis

The 360 base-pair fragment in HVS-1 of the mitochondrial genome were determined from ancient human remains excavated at Noen U-loke and Ban Lum-Khao, two Bronze and Iron Age archaeological sites in Northeastern Thailand, radio-carbon dated to circa 3,500-1,500 years BP and 3,200-2,400 years BP, respectively. These two neighboring populations were parts of early agricultural communities prevailing in northeastern Thailand from the fourth millennium BP onwards. The nucleotide sequences of these ancient samples were compared with the sequences of modern samples from various ethnic populations of East and Southeast Asia, encompassing four major linguistic affiliations (Altaic, Sino-Tibetan, Tai-Kadai, and Austroasiatic), to investigate the genetic relationships and history among them. The two ancient samples were most closely related to each other, and next most closely related to the Chao-Bon, an Austroasiatic-speaking group living near the archaeological sites, suggesting that the genetic continuum may have persisted since prehistoric times in situ among the native, perhaps Austroasiatic-speaking population. Tai-Kadai groups formed close affinities among themselves, with a tendency to be more closely related to other Southeast Asian populations than to populations from further north. The Tai-Kadai groups were relatively distant from all groups that have presumably been in Southeast Asia for longer-that is, the two ancient groups and the Austroasiatic-speaking groups, with the exception of the Khmer group. This finding is compatible with the known history of the Thais: their late arrival in Southeast Asia from southern China after the 10th-11th century AD, followed by a period of subjugation under the Khmers.     

Human kanadaptin and kidney anion exchanger 1 (kAE1) do not interact in transfected HEK 293 cells

Kanadaptin (k¯idney anion exchanger adaptor protein) is a widely expressed protein, shown previously to interact with the cytosolic domain of mouse Cl^?/HCO₃^? anion exchanger 1 (kAE1) but not erythroid AE1 (eAE1) by a yeast-two hybrid assay. Kanadaptin was co-localized with kAE1 in intracellular membranes but not at the plasma membrane in α-intercalated cells of rabbit kidney. It was suggested that kanadaptin is an adaptor protein or chaperone involved in targeting kAE1 to the plasma membrane. To test this hypothesis, the interaction of human kanadaptin with human kAE1 was studied in co-transfected HEK293 cells. Human kanadaptin contains 796 amino acids and was immuno-detected as a 90 kDa protein in transfected cells. Pulse-chase experiments showed that it has a half-life (t_1/2) of 7 h. Human kanadaptin was localized predominantly to the nucleus, whereas kAE1 was present intracellularly and at the plasma membrane. Trafficking of kAE1 from its site of synthesis in the endoplasmic reticulum to the plasma membrane was unaffected by co-expression of human kanadaptin. Moreover, we found that no interaction between human kanadaptin and kAE1 or eAE1 could be detected in co-transfected cells either by co-immunoprecipitation or by histidine6-tagged co-purification. Taken together, we found that human kanadaptin did not interact with kAE1 and had no effect on trafficking of kAE1 to the plasma membrane in transfected cells. Kanadaptin may not be involved in the biosynthesis and targeting of kAE1. As such, defects in kanadaptin and its interaction with kAE1 are unlikely to be involved in the pathogenesis of the inherited kidney disease, distal renal tubular acidosis (dRTA).     

Antimicrobial resistance markers as a monitoring index of gonorrhoea in Thailand

Multiplex PCR was applied to explore the antimicrobial-resistance profiles of 145 gonococci isolated from Bangrak Hospital, Thailand in 2007. All isolates were clearly identified for the plasmid-mediated resistant types of penicillin (Asia, Africa and Toronto) and tetracycline (American and Dutch). This method can also predict the decreased susceptibility to ciprofloxacin by detection of Ser-91 mutation. Prevalence rates of penicillinase-producing Neisseria gonorrhoeae (PPNG) and high-level tetracycline-resistance N. gonorrhoeae (TRNG) were shown to be high as 82.1% and 84.1%, respectively. Most PPNG carried the Africa-type (78.2%) while the American-type (61.8%) was harboured in most TRNG. Mono- and triple-resistance patterns were presented in 2.6% and 79.5% of male, 20.7% and 62.1% of men who have sex with men (MSM), 0% and 75.0% of female, and 10% and 70% of female sex workers (FSW). Additionally, the rate of the Dutch type was high in patients among the age of 35-44 years (57.1%) and female patients (43.8%). The changing types of plasmids have been noticed during the time period of study. The multi-resistance patterns of the gonococcal isolates can be used as an epidemiological index of gonorrhoea and human sexual behaviours. This information will support the management of individual patients as well as the public health surveillance.