Computational and Functional Analyses of a Small-Molecule Binding Site in ROMK

The renal outer medullary potassium channel (ROMK, or Kir1.1, encoded by KCNJ1) critically regulates renal tubule electrolyte and water transport and hence blood volume and pressure. The discovery of loss-of-function mutations in KCNJ1 underlying renal salt and water wasting and lower blood pressure has sparked interest in developing new classes of antihypertensive diuretics targeting ROMK. The recent development of nanomolar-affinity small-molecule inhibitors of ROMK creates opportunities for exploring the chemical and physical basis of ligand-channel interactions required for selective ROMK inhibition. We previously reported that the bis-nitro-phenyl ROMK inhibitor VU591 exhibits voltage-dependent knock-off at hyperpolarizing potentials, suggesting that the binding site is located within the ion-conduction pore. In this study, comparative molecular modeling and in silico ligand docking were used to interrogate the full-length ROMK pore for energetically favorable VU591 binding sites. Cluster analysis of 2498 low-energy poses resulting from 9900 Monte Carlo docking trajectories on each of 10 conformationally distinct ROMK comparative homology models identified two putative binding sites in the transmembrane pore that were subsequently tested for a role in VU591-dependent inhibition using site-directed mutagenesis and patch-clamp electrophysiology. Introduction of mutations into the lower site had no effect on the sensitivity of the channel to VU591. In contrast, mutations of Val¹⁶⁸ or Asn¹⁷¹ in the upper site, which are unique to ROMK within the Kir channel family, led to a dramatic reduction in VU591 sensitivity. This study highlights the utility of computational modeling for defining ligand-ROMK interactions and proposes a mechanism for inhibition of ROMK.     

Heterogeneous genetic invasions of three insecticide resistance mutations in Indo‐Pacific populations of Aedes aegypti (L.)

Nations throughout the Indo‐Pacific region use pyrethroid insecticides to control Aedes aegypti, the mosquito vector of dengue, often without knowledge of pyrethroid resistance status of the pest or origin of resistance. Two mutations (V1016G + F1534C) in the sodium channel gene (Vssc) of Ae. aegypti modify ion channel function and cause target‐site resistance to pyrethroid insecticides, with a third mutation (S989P) having a potential additive effect. Of 27 possible genotypes involving these mutations, some allelic combinations are never seen whereas others predominate. Here, five allelic combinations common in Ae. aegypti from the Indo‐Pacific region are described and their geographical distributions investigated using genome‐wide SNP markers. We tested the hypothesis that resistance allele combinations evolved de novo in populations versus the alternative that dispersal of Ae. aegypti between populations facilitated genetic invasions of allele combinations. We used latent factor mixed‐models to detect SNPs throughout the genome that showed structuring in line with resistance allele combinations and compared variation at SNPs within the Vssc gene with genome‐wide variation. Mixed‐models detected an array of SNPs linked to resistance allele combinations, all located within or in close proximity to the Vssc gene. Variation at SNPs within the Vssc gene was structured by resistance profile, whereas genome‐wide SNPs were structured by population. These results demonstrate that alleles near to resistance mutations have been transferred between populations via linked selection. This indicates that genetic invasions have contributed to the widespread occurrence of Vssc allele combinations in Ae. aegypti in the Indo‐Pacific region, pointing to undocumented mosquito invasions between countries.     

Purification and characterization of a novel enzyme, arylalkyl acylamidase, from Pseudomonas putida Sc2.

A novel enzyme, arylalkyl acylamidase, which shows a strict specificity for N-acetyl arylalkylamines, but not acetanilide derivatives, was purified from the culture broth of Pseudomonas putida Sc2. The purified enzyme appeared to be homogeneous, as judged by native and SDS/PAGE. The enzyme has a molecular mass of approximately 150 kDa and consists of four identical subunits. The purified enzyme catalyzed the hydrolysis of N-acetyl-2-phenylethylamine to 2-phenylethylamine and acetic acid at the rate of 6.25 mumol.min-1.mg-1 at 30 degrees C. It also catalyzed the hydrolysis of various N-acetyl arylalkylamines containing a benzene or indole ring, and acetic acid arylalkyl esters. The enzyme did not hydrolyze acetanilide, N-acetyl aliphatic amines, N-acetyl amino acids, N-acetyl amino sugars or acylthiocholine. The apparent Km for N-acetylbenzylamine, N-acetyl-2-phenylethylamine and N-acetyl-3-phenylpropylamine are 41 mM, 0.31 mM and 1.6 mM, respectively. The purified enzyme was sensitive to thiol reagents such as Ag2SO4, HgCl2 and p-chloromercuribenzoic acid, and its activity was enhanced by divalent metal ions such as Zn2+, Mg2+ and Mn2+.     

Rainfall, phycocyanin, and N:P ratios related to cyanobacterial blooms in a Korean large reservoir

Nutrient concentrations and other environmental factors were measured in the Daechung Reservoir for 25 weeks from spring until autumn in 1999. The high irradiance after heavy rainfall provided optimal meteorological conditions for bloom formation during summer, therefore, rain would also appear to forecast imminent bloom. The bloom formation was largely governed by cyanobacteria, in particular, Microcystis spp. and Anabaenaspp. Phycocyanin showed higher correlation with cyanobacteria (r = 0.744, P < 0.001) compared to chlorophyll-a(r = 0.599, P < 0.01). Therefore, phycocyanin was more accurate and useful than chlorophyll-a in quantitatively measuring cyanobacterial blooms. The atomic N:P ratio of the particulate form also showed a high correlation with cyanobacteria (r = 0.541, P < 0.01), increasing from 4.3 to 14.6 during bloom formation, while that of the dissolved form decreased from 25.5 to 8.7. These results indicated that the algae assimilated N significantly without comparable P uptake during the blooming season, which was in sharp contrast to the excessive storage of P during the spring.     

Production of reactive oxygen species by monocyte-derived macrophages from blood of healthy donors and patients with ischemic heart disease

Production of reactive oxygen species (ROS) by macrophages derived from blood monocytes of healthy donors (MP_N) and patients with ischemic heart disease (IHD) (MP_IHD) before, during, and after their incubation with low-density lipoprotein (LDL) isolated from blood plasma of healthy donors (LDL_N) and patients with a high cholesterol level (LDL_H) was investigated by the method of luminol-dependent (spontaneous) and stimulated chemiluminescence (CL) using opsonized zymosan (OZ) or phorbol-12-myristate-13-acetate (PMA) as the CL stimulators. It was shown that proper, luminol-dependent, and zymosan-or PMA-stimulated chemiluminescence of MP_IHD was 1.4-, 1.8-, 2.7-, and 1.6-fold higher than the same types of chemiluminescence of MP_N, respectively, (p<0.05–0.01). Although the effect of OZ on MP_N and MP_IHD was more potent than that of PMA (by 4.3- and 3.2-fold, respectively), but it appeared in 2.5–3.0 times slower than that of PMA. LDL_N and LDL_H incubated with MP_N for the first 15 and 60 min caused the 1.4- and 2.5-increase of the luminol-dependent CL of MP_N; the same treatment of MP_IHD did not influence ROS production by these cells. Repeated increase in the OZ-stimulated CL of MP_N was also observed after preincubation for 15–180 min with LDL_N and LDL_H followed by LDL removal, subsequent MP_N washing and addition of Hanks solution and OZ; the repeated increase in OZ-stimulated CL of MP_N was only observed after incubation with LDL_H than with LDL_N. No increase of CL was observed in experiments with MP_IHD. Thus, more intensive chemiluminescence of macrophages obtained from blood of patients with IHD suggests their in vivo stimulation. LDL_N and LDL_H may cause both primary and secondary (after preincubation) stimulating effect on CL of MP_N but not of MP_IHD. Thus, the analysis of macrophage chemiluminescence is a sensitive test for evaluation the degree of macrophage stimulation; it may be effectively used for monitoring of effectiveness of medical treatment of patients.     

Epigenetic inactivation of the miR-124-1 in haematological malignancies

miR-124-1 is a tumour suppressor microRNA (miR). Epigenetic deregulation of miRs is implicated in carcinogenesis. Promoter DNA methylation and histone modification of miR-124-1 was studied in 5 normal marrow controls, 4 lymphoma, 8 multiple myeloma (MM) cell lines, 230 diagnostic primary samples of acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL), MM, and non-Hodgkin's lymphoma (NHL), and 53 MM samples at stable disease or relapse. Promoter of miR-124-1 was unmethylated in normal controls but homozygously methylated in 4 of 4 lymphoma and 4 of 8 myeloma cell lines. Treatment of 5-Aza-2'-deoxycytidine led to miR-124-1 demethylation and re-expression of mature miR-124, which also associated with emergence of euchromatic trimethyl H3K4 and consequent downregulation of CDK6 in myeloma cells harboring homozygous miR-124-1 methylation. In primary samples at diagnosis, miR-124-1 methylation was absent in CML but detected in 2% each of MM at diagnosis and relapse/progression, 5% ALL, 15% AML, 14% CLL and 58.1% of NHL (p<0.001). Amongst lymphoid malignancies, miR-124-1 was preferentially methylated in NHL than MM, CLL or ALL. In primary lymphoma samples, miR-124-1 was preferentially hypermethylated in B- or NK/T-cell lymphomas and associated with reduced miR-124 expression. In conclusion, miR-124-1 was hypermethylated in a tumour-specific manner, with a heterochromatic histone configuration. Hypomethylation led to partial restoration of euchromatic histone code and miR re-expression. Infrequent miR-124-1 methylation detected in diagnostic and relapse MM samples showed an unimportant role in MM pathogenesis, despite frequent methylation found in cell lines. Amongst haematological cancers, miR-124-1 was more frequently hypermethylated in NHL, and hence warrants further study.     

Iron metabolism in diabetes-induced Alzheimer’s disease: a focus on insulin resistance in the brain

Alzheimer’s disease (AD) is characterized by an excessive accumulation of toxic amyloid beta (Aβ) plaques and memory dysfunction. The onset of AD is influenced by age, genetic background, and impaired glucose metabolism in the brain. Several studies have demonstrated that diabetes involving insulin resistance and glucose tolerance could lead to AD, ultimately resulting in cognitive dysfunction. Even though the relationship between diabetes and AD was indicated by significant evidences, the critical mechanisms and metabolic alterations in diabetes induced AD are not clear until now. Recently, iron metabolism has been shown to play multiple roles in the central nervous system (CNS). Iron deficiency and overload are associated with neurodegenerative diseases. Iron binds to Aβ and subsequently regulates Aβ toxicity in the CNS. In addition, previous studies have shown that iron is involved in the aggravation of insulin resistance. Considering these effects of iron metabolism in CNS, we expect that iron metabolism may play crucial roles in diabetic AD brain. Thus, we review the recent evidence regarding the relationship between diabetes-induced AD and iron metabolism.