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Aristolochic acids I and II (AA-I/II) are carcinogenic principles of Aristolochia plants, which have been employed in traditional medicinal practices and discovered as food contaminants. While the deleterious effects of AAs are broadly acknowledged, there is a dearth of information to define the mechanisms underlying their carcinogenicity. Following bioactivation in the liver, N-hydroxyaristolactam and N-sulfonyloxyaristolactam metabolites are transported via circulation and elicit carcinogenic effects by reacting with cellular DNA. In this study, we apply DNA adduct analysis, X-ray crystallography, isothermal titration calorimetry, and fluorescence quenching to investigate the role of human serum albumin (HSA) in modulating AA carcinogenicity. We find that HSA extends the half-life and reactivity of N-sulfonyloxyaristolactam-I with DNA, thereby protecting activated AAs from heterolysis. Applying novel pooled plasma HSA crystallization methods, we report high-resolution structures of myristic acid-enriched HSA (HSA MYR ) and its AA complexes (HSA MYR /AA-I and HSA MYR /AA-II) at 1.9 Å resolution. While AA-I is located within HSA subdomain IB, AA-II occupies subdomains IIA and IB. ITC binding profiles reveal two distinct AA sites in both complexes with association constants of 1.5 and 0.5 · 10 6  M -1 for HSA/AA-I versus 8.4 and 9.0 · 10 5  M -1 for HSA/AA-II. Fluorescence quenching of the HSA Trp 214 suggests variable impacts of fatty acids on ligand binding affinities. Collectively, our structural and thermodynamic characterizations yield significant insights into AA binding, transport, toxicity, and potential allostery, critical determinants for elucidating the mechanistic roles of HSA in modulating AA carcinogenicity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Influenza remains one of the most widespread infections, causing an annual illness in adults and children. Therefore, the search for new antiviral drugs is one of the priorities of practical health care. Eight isorhamnetin glycosides were purified from Persicaria species, characterized by nuclear magnetic resonance spectroscopy and mass spectrometry and then evaluated as potential agents against influenza virus. A comprehensive in vitro and in vivo assessment of the compounds revealed that compound 5 displayed the most potent inhibitory activity with an EC 50 value of 1.2-1.3 μM, better than standard drugs (isorhamnetin 28.0-56.0 μM and oseltamivir 1.3-9.1 μM). Molecular docking results also revealed that compound 5 has the lowest binding energy (-10.7 kcal/mol) among the tested compounds and isorhamnetin (-8.1 kcal/mol). The ability of the isorhamnetin glycosides to suppress the reproduction of the influenza virus was studied on a model of a cell culture and chicken embryos. The ability of active compounds to influence the structure of the virion, as well as the activity of hemagglutinin and neuraminidase, has been demonstrated. Compound 1, 5, and 6 demonstrated the most effective inhibition of virus replication for all tested viruses. Molecular dynamics simulation techniques were run for 100 ns for compound 5 with two protein receptors Hem (1RUY) and Neu (3BEQ). These results revealed that the Hem-complex system acquired a relatively more stable conformation and even better descriptors than the other Neu-complex studied systems, suggesting that it can be an effective inhibiting drug toward hemagglutinin than neuraminidase inhibition. Based on the reported results, compound 5 can be a good candidate to be evaluated for effectiveness in preclinical testing., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Aristolochic acids are potent human carcinogens; the role of phase II metabolism in their bioactivation is unclear. Accordingly, we tested the ability of the partially reduced metabolites, N-hydroxyaristolactams (AL-NOHs), and their N-O-sulfonated and N-O-acetylated derivatives to react with DNA to form aristolactam-DNA adducts. AL-NOHs displayed little or no activity in this regard while the sulfo- and acetyl compounds readily form DNA adducts, as detected by (32)P-post-labeling analysis. Mouse hepatic and renal cytosols stimulated binding of AL-NOHs to DNA in the presence of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) but not of acetyl-CoA. Using Time of Flight liquid chromatography/mass spectrometry, N-hydroxyaristolactam I formed the sulfated compound in the presence of PAPS and certain human sulfotransferases, SULT1B1 >>> SULT1A2 > SULT1A1 >>> SULT1A3. The same pattern of SULT reactivity was observed when N-hydroxyaristolactam I was incubated with these enzymes and PAPS and the reaction was monitored by formation of aristolactam-DNA adducts. In the presence of human, Nad(p)h: quinone oxidoreductase, the ability of aristolochic acid I to bind DNA covalently was increased significantly by addition of PAPS and SULT1B1. We conclude from these studies that AL-NOHs, formed following partial nitroreduction of aristolochic acids, serve as substrates for SULT1B1, producing N-sulfated esters, which, in turn, are converted to highly active species that react with DNA and, potentially, cellular proteins, resulting in the genotoxicity and nephrotoxicity associated with ingestion of aristolochic acids by humans., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Although the important role of the non-structural (NS1 and NEP) gene of influenza A in virulence of the virus is well established, our knowledge about the extent of variation in the NS gene pool of influenza A viruses in their natural reservoirs in Kazakhstan is incomplete. 17 influenza A viruses of different subtypes were studied in this paper. Seven types of haemagglutinin and five different neuraminidase subtypes in eight combinations were found among the isolated viruses. A comparison of nucleotide sequences of isolated viruses revealed a substantial number of silent mutations, which results in high degree of homology in amino acid sequences. By phylogenetic analysis it was shown that two distinct gene pools, corresponding to both NS allele A with 5 Clades and B, were present at the same time in Kazakhstan. The degree of variation within the alleles was very low. In our study allele A viruses had a maximum of 5% amino acid divergence in Clade while allele B viruses had only 4% amino acid divergence.

The juxtamembrane domain of vesicle-associated membrane protein (VAMP) 2 (also known as synaptobrevin2) contains a conserved cluster of basic/hydrophobic residues that may play an important role in membrane fusion. Our measurements on peptides corresponding to this domain determine the electrostatic and hydrophobic energies by which this domain of VAMP2 could bind to the adjacent lipid bilayer in an insulin granule or other transport vesicle. Mutation of residues within the juxtamembrane domain that reduce the VAMP2 net positive charge, and thus its interaction with membranes, inhibits secretion of insulin granules in beta cells. Increasing salt concentration in permeabilized cells, which reduces electrostatic interactions, also results in an inhibition of insulin secretion. Similarly, amphipathic weak bases (e.g., sphingosine) that reverse the negative electrostatic surface potential of a bilayer reverse membrane binding of the positively charged juxtamembrane domain of a reconstituted VAMP2 protein and inhibit membrane fusion. We propose a model in which the positively charged VAMP and syntaxin juxtamembrane regions facilitate fusion by bridging the negatively charged vesicle and plasma membrane leaflets.

Calcium/calmodulin (Ca/CaM) binds to the intracellular juxtamembrane domain (JMD) of the epidermal growth factor receptor (EGFR). The basic JMD also binds to acidic lipids in the inner leaflet of the plasma membrane, and this interaction may contribute an extra level of autoinhibition to the receptor. Binding of a ligand to the EGFR produces a rapid increase in intracellular calcium, [Ca2+]i, and thus Ca/CaM. How does Ca/CaM compete with the plasma membrane for the JMD? Does Ca/CaM directly pull the JMD off the membrane or does Ca/CaM only bind to the JMD after it has dissociated spontaneously from the bilayer? To answer this question, we studied the effect of Ca/CaM on the rate of dissociation of fluorescent JMD peptides from phospholipid vesicles by making kinetic stop-flow measurements. Ca/CaM increases the rate of dissociation: an analysis of the differential equations that describe the dissociation shows that Ca/CaM must directly pull the basic JMD peptide off the membrane surface. These measurements lead to a detailed atomic-level mechanism for EGFR activation that reconciles the existence of preformed EGFR dimers/oligomers with the Kuriyan allosteric model for activation of the EGFR kinase domains.

Phosphatidylinositol 4,5-bisphosphate (PIP(2)) controls a surprisingly large number of processes in cells. Thus, many investigators have suggested that there might be different pools of PIP(2) on the inner leaflet of the plasma membrane. If a significant fraction of PIP(2) is bound electrostatically to unstructured clusters of basic residues on membrane proteins, the PIP(2) diffusion constant, D, should be reduced. We microinjected micelles of Bodipy TMR-PIP(2) into cells, and we measured D on the inner leaflet of fibroblasts and epithelial cells by using fluorescence correlation spectroscopy. The average +/- SD value from all cell types was D = 0.8 +/- 0.2 microm(2)/s (n = 218; 25 degrees C). This is threefold lower than the D in blebs formed on Rat1 cells, D = 2.5 +/- 0.8 microm(2)/s (n = 26). It is also significantly lower than the D in the outer leaflet or in giant unilamellar vesicles and the diffusion coefficient for other lipids on the inner leaflet of these cell membranes. The simplest interpretation is that approximately two thirds of the PIP(2) on inner leaflet of these plasma membranes is bound reversibly.

Phospholipase C-zeta (PLC-zeta) is a sperm-specific enzyme that initiates the Ca2+ oscillations in mammalian eggs that activate embryo development. It shares considerable sequence homology with PLC-delta1, but lacks the PH domain that anchors PLC-delta1 to phosphatidylinositol 4,5-bisphosphate, PIP2. Thus it is unclear how PLC-zeta interacts with membranes. The linker region between the X and Y catalytic domains of PLC-zeta, however, contains a cluster of basic residues not present in PLC-delta1. Application of electrostatic theory to a homology model of PLC-zeta suggests this basic cluster could interact with acidic lipids. We measured the binding of catalytically competent mouse PLC-zeta to phospholipid vesicles: for 2:1 phosphatidylcholine/phosphatidylserine (PC/PS) vesicles, the molar partition coefficient, K, is too weak to be of physiological significance. Incorporating 1% PIP2 into the 2:1 PC/PS vesicles increases K about 10-fold, to 5x10(3) M-1, a biologically relevant value. Expressed fragments corresponding to the PLC-zeta X-Y linker region also bind with higher affinity to polyvalent than monovalent phosphoinositides on nitrocellulose filters. A peptide corresponding to the basic cluster (charge=+7) within the linker region, PLC-zeta-(374-385), binds to PC/PS vesicles with higher affinity than PLC-zeta, but its binding is less sensitive to incorporating PIP2. The acidic residues flanking this basic cluster in PLC-zeta may account for both these phenomena. FRET experiments suggest the basic cluster could not only anchor the protein to the membrane, but also enhance the local concentration of PIP2 adjacent to the catalytic domain.

Membrane-permeable calmodulin inhibitors, such as the napthalenesulfonamide derivatives W-7/W-13, trifluoperazine, and calmidazolium, are used widely to investigate the role of calcium/calmodulin (Ca2+/CaM) in living cells. If two chemically different inhibitors (e.g. W-7 and trifluoperazine) produce similar effects, investigators often assume the effects are due to CaM inhibition. Zeta potential measurements, however, show that these amphipathic weak bases bind to phospholipid vesicles at the same concentrations as they inhibit Ca2+/CaM; this suggests that they also bind to the inner leaflet of the plasma membrane, reducing its negative electrostatic surface potential. This change will cause electrostatically bound clusters of basic residues on peripheral (e.g. Src and K-Ras4B) and integral (e.g. epidermal growth factor receptor (EGFR)) proteins to translocate from the membrane to the cytoplasm. We measured inhibitor-mediated translocation of a simple basic peptide corresponding to the calmodulin-binding juxtamembrane region of the EGFR on model membranes; W-7/W-13 causes translocation of this peptide from membrane to solution, suggesting that caution must be exercised when interpreting the results obtained with these inhibitors in living cells. We present evidence that they exert dual effects on autophosphorylation of EGFR; W-13 inhibits epidermal growth factor-dependent EGFR autophosphorylation under different experimental conditions, but in the absence of epidermal growth factor, W-13 stimulates autophosphorylation of the receptor in four different cell types. Our interpretation is that the former effect is due to W-13 inhibition of Ca2+/CaM, but the latter results could be due to binding of W-13 to the plasma membrane.

Several biologically important peripheral (e.g., myristoylated alanine-rich C kinase substrate) and integral (e.g., the epidermal growth factor receptor) membrane proteins contain clusters of basic residues that interact with acidic lipids in the plasma membrane. Previous measurements demonstrate that the polyvalent acidic lipid phosphatidylinositol 4,5-bisphosphate is bound electrostatically (i.e., sequestered) by membrane-adsorbed basic peptides corresponding to these clusters. We report here three experimental observations that suggest monovalent acidic lipids are not sequestered by membrane-bound basic peptides. 1), Binding of basic peptides to vesicles does not decrease when the temperature is lowered below the fluid-to-gel phase transition. 2), The binding energy of Lys-13 to lipid vesicles increases linearly with the fraction of monovalent acidic lipids. 3), Binding of basic peptides to vesicles produces no self-quenching of fluorescent monovalent acidic lipids. One potential explanation for these results is that membrane-bound basic peptides diffuse too rapidly for the monovalent lipids to be sequestered. Indeed, our fluorescence correlation spectroscopy measurements show basic peptides bound to phosphatidylcholine/phosphatidylserine membranes have a diffusion coefficient approximately twofold higher than that of lipids, and those bound to phosphatidylcholine/phosphatidylinositol 4,5-bisphosphate membranes have a diffusion coefficient comparable to that of lipids.

Newcastle disease virus (NDV) infects domesticated and wild birds throughout the world and has the possibility to cause outbreaks in chicken flocks in future. To assess the evolutionary characteristics of 10 NDV strains isolated from chickens in Kazakhstan during 1998 we investigated the phylogenetic relationships among these viruses and viruses described previously. For genotyping, fusion (F) gene phylogenetic analysis (nucleotide number 47-421) was performed using sequences of Kazakhstanian isolates as compared to sequences of selected NDV strains from GenBank. Phylogenetic analysis showed that all newly characterized strains belonged to the genetic group designated as VIIb. All strains possessed a virulent fusion cleavage site (RRQRR/F) belonging to velogenic or mesogenic pathotypes with intracerebral pathogenicity indexes (ICPI) varying from 1.05 to 1.87.

The inner leaflet of a typical mammalian plasma membrane contains 20-30% univalent PS (phosphatidylserine) and 1% multivalent PtdIns(4,5)P(2). Numerous proteins have clusters of basic (or basic/hydrophobic) residues that bind to these acidic lipids. The intracellular effector CaM (calmodulin) can reverse this binding on a wide variety of proteins, including MARCKS (myristoylated alanine-rich C kinase substrate), GAP43 (growth-associated protein 43, also known as neuromodulin), gravin, GRK5 (G-protein-coupled receptor kinase 5), the NMDA (N-methyl-D-aspartate) receptor and the ErbB family. We used the first principles of physics, incorporating atomic models and the Poisson-Boltzmann equation, to describe how the basic effector domain of MARCKS binds electrostatically to acidic lipids on the plasma membrane. The theoretical calculations show the basic cluster produces a local positive electrostatic potential that should laterally sequester PtdIns(4,5)P(2), even when univalent acidic lipids are present at a physiologically relevant 100-fold excess; four independent experimental measurements confirm this prediction. Ca(2+)/CaM binds with high affinity (K(d) approximately 10nM) to this domain and releases the PtdIns(4,5)P(2). MARCKS, a major PKC (protein kinase C) substrate, is present at concentrations comparable with those of PtdIns(4,5)P(2) (approx. 10 microM) in many cell types. Thus MARCKS can act as a reversible PtdIns(4,5)P(2) buffer, binding PtdIns(4,5)P(2) in a quiescent cell, and releasing it locally when the intracellular Ca(2+) concentration increases. This reversible sequestration is important because PtdIns(4,5)P(2) plays many roles in cell biology. Less is known about the role of CaM-mediated reversible membrane binding of basic/hydrophobic clusters for the other proteins.

The basic effector domain of myristoylated alanine-rich C kinase substrate (MARCKS), a major protein kinase C substrate, binds electrostatically to acidic lipids on the inner leaflet of the plasma membrane; interaction with Ca2+/calmodulin or protein kinase C phosphorylation reverses this binding. Our working hypothesis is that the effector domain of MARCKS reversibly sequesters a significant fraction of the L-alpha-phosphatidyl-D-myo-inositol 4,5-bisphosphate (PIP2) on the plasma membrane. To test this, we utilize three techniques that measure the ability of a peptide corresponding to its effector domain, MARCKS(151-175), to sequester PIP2 in model membranes containing physiologically relevant fractions (15-30%) of the monovalent acidic lipid phosphatidylserine. First, we measure fluorescence resonance energy transfer from Bodipy-TMR-PIP2 to Texas Red MARCKS(151-175) adsorbed to large unilamellar vesicles. Second, we detect quenching of Bodipy-TMR-PIP2 in large unilamellar vesicles when unlabeled MARCKS(151-175) binds to vesicles. Third, we identify line broadening in the electron paramagnetic resonance spectra of spin-labeled PIP2 as unlabeled MARCKS(151-175) adsorbs to vesicles. Theoretical calculations (applying the Poisson-Boltzmann relation to atomic models of the peptide and bilayer) and experimental results (fluorescence resonance energy transfer and quenching at different salt concentrations) suggest that nonspecific electrostatic interactions produce this sequestration. Finally, we show that the PLC-delta1-catalyzed hydrolysis of PIP2, but not binding of its PH domain to PIP2, decreases markedly as MARCKS(151-175) sequesters most of the PIP2.