3D Structure Prediction of Human β1-Adrenergic Receptor via Threading-Based Homology Modeling for Implications in Structure-Based Drug Designing

Dilated cardiomyopathy is a disease of left ventricular dysfunction accompanied by impairment of the β₁-adrenergic receptor (β₁-AR) signal cascade. The disturbed β₁-AR function may be based on an elevated sympathetic tone observed in patients with heart failure. Prolonged adrenergic stimulation may induce metabolic and electrophysiological disturbances in the myocardium, resulting in tachyarrhythmia that leads to the development of heart failure in human and sudden death. Hence, β₁-AR is considered as a promising drug target but attempts to develop effective and specific drug against this tempting pharmaceutical target is slowed down due to the lack of 3D structure of Homo sapiens β₁-AR (hsβADR1). This study encompasses elucidation of 3D structural and physicochemical properties of hsβADR1 via threading-based homology modeling. Furthermore, the docking performance of several docking programs including Surflex-Dock, FRED, and GOLD were validated by re-docking and cross-docking experiments. GOLD and Surflex-Dock performed best in re-docking and cross docking experiments, respectively. Consequently, Surflex-Dock was used to predict the binding modes of four hsβADR1 agonists. This study provides clear understanding of hsβADR1 structure and its binding mechanism, thus help in providing the remedial solutions of cardiovascular, effective treatment of asthma and other diseases caused by malfunctioning of the target protein.     

Tau is Endogenously Nitrated in Mouse Brain: Identification of a Tyrosine Residue Modified In vivo by NO

Nitration of tau protein is normally linked to neurodegeneration but, until now, no comprehensive information is available regarding tau nitration in healthy subjects. It has been previously reported that in differentiated PC12 cells, tau co-immunoprecipitated with alpha-tubulin is nitrated at tyrosine residues and that this post-translation modification doesn’t impair the association of tau with the cytoskeleton. The present paper is focused on the identification of tyrosine residues endogenously modified in tau from PC12 cells and reports for the first time that tau is also nitrated in vivo in normal mouse brain and that one tyrosine is endogenously modified.     

Stochastic Modeling of Gene Regulation by Noncoding Small RNAs in the Strong Interaction Limit

Noncoding small RNAs (sRNAs) are known to play a key role in regulating diverse cellular processes, and their dysregulation is linked to various diseases such as cancer. Such diseases are also marked by phenotypic heterogeneity, which is often driven by the intrinsic stochasticity of gene expression. Correspondingly, there is significant interest in developing quantitative models focusing on the interplay between stochastic gene expression and regulation by sRNAs. We consider the canonical model of regulation of stochastic gene expression by sRNAs, wherein interaction between constitutively expressed sRNAs and mRNAs leads to stoichiometric mutual degradation. The exact solution of this model is analytically intractable given the nonlinear interaction term between sRNAs and mRNAs, and theoretical approaches typically invoke the mean-field approximation. However, mean-field results are inaccurate in the limit of strong interactions and low abundances; thus, alternative theoretical approaches are needed. In this work, we obtain analytical results for the canonical model of regulation of stochastic gene expression by sRNAs in the strong interaction limit. We derive analytical results for the steady-state generating function of the joint distribution of mRNAs and sRNAs in the limit of strong interactions and use the results derived to obtain analytical expressions characterizing the corresponding protein steady-state distribution. The results obtained can serve as building blocks for the analysis of genetic circuits involving sRNAs and provide new insights into the role of sRNAs in regulating stochastic gene expression in the limit of strong interactions.     

Prediction of Protein–Protein Interaction Sites in Sequences and 3D Structures by Random Forests

Identifying interaction sites in proteins provides important clues to the function of a protein and is becoming increasingly relevant in topics such as systems biology and drug discovery. Although there are numerous papers on the prediction of interaction sites using information derived from structure, there are only a few case reports on the prediction of interaction residues based solely on protein sequence. Here, a sliding window approach is combined with the Random Forests method to predict protein interaction sites using (i) a combination of sequence- and structure-derived parameters and (ii) sequence information alone. For sequence-based prediction we achieved a precision of 84% with a 26% recall and an F-measure of 40%. When combined with structural information, the prediction performance increases to a precision of 76% and a recall of 38% with an F-measure of 51%. We also present an attempt to rationalize the sliding window size and demonstrate that a nine-residue window is the most suitable for predictor construction. Finally, we demonstrate the applicability of our prediction methods by modeling the Ras–Raf complex using predicted interaction sites as target binding interfaces. Our results suggest that it is possible to predict protein interaction sites with quite a high accuracy using only sequence information.     

Impairment of Spermatogenesis in Rats by Mercuric Chloride: Involvement of Low 17β-Estradiol Level in Induction of Acute Oxidative Stress

Mercuric chloride (HgCl₂) has been shown to affect the male reproductive organs, and oxidative stress has been linked with hypospermatogenesis and with male infertility. However, the specific mode of impairment of spermatogenesis during HgCl₂ exposure has not yet been clarified fully. Because of the involvement of 17β-estradiol (E2) in the male reproductive tract and its putative role on spermatogenesis, the present study aimed to investigate the possibility that HgCl₂-induced oxidative stress-mediated modulation of the E2 level exerts adverse effects on testicular steroidogenic and gametogenic activities. HgCl₂ treatment at 50 and 100 ppm for 90 days by continuous oral administration in the drink water resulted in significant dose-dependent fashion decrease in serum and testicular E₂ levels and an increase in testicular testosterone levels in dose-dependent manner, without statistical alteration in serum testosterone level among HgCl₂ exposed groups compared to the control. Cauda epididymal sperm count and motility were decreased significantly (p < 0.01), in a dose-dependent manner, in the HgCl₂-treated groups, and qualitative examination revealed inhibition of spermatogenesis and the preferential loss of maturing and elongated spermatids. The seminiferous tubules were dilated in treated animals. When compared to the control, increase in lipid peroxidation due to toxic effects of HgCl2 was accompanied by significant reduction (p < 0.01) in antioxidant enzymes activities, superoxide dismutase, catalase, and glutathione peroxidase of testes, implicating the presence of oxidative tissue damage. Furthermore, these tissue injuries caused functional impairment as evidenced with testicular elevated activity of lactate dehydrogenase. Unless oxidative stress can lead to cancer development, testis’ tumor markers as beta human chorionic gonadotropin and alpha-fetoprotein levels have shown no significant differences in the HgCl₂-exposed group compared with respect to the control. Large quantities of metal accumulated in the testis tissue are in agreement with the testis-activity failure verified in this tissue. These findings suggest that a decrease in E2 level after mercury exposure may render testis more susceptible to oxidative damage leading to its functional inactivation, thus providing new dimension to mechanisms underlying heavy metal-induced male infertility.     

An Origin of Cooperative Oxygen Binding of Human Adult Hemoglobin: Different Roles of the α and β Subunits in the α2β2 Tetramer

Human hemoglobin (Hb), which is an α₂β₂ tetramer and binds four O₂ molecules, changes its O₂-affinity from low to high as an increase of bound O₂, that is characterized by ‘cooperativity’. This property is indispensable for its function of O₂ transfer from a lung to tissues and is accounted for in terms of T/R quaternary structure change, assuming the presence of a strain on the Fe-histidine (His) bond in the T state caused by the formation of hydrogen bonds at the subunit interfaces. However, the difference between the α and β subunits has been neglected. To investigate the different roles of the Fe-His(F8) bonds in the α and β subunits, we investigated cavity mutant Hbs in which the Fe-His(F8) in either α or β subunits was replaced by Fe-imidazole and F8-glycine. Thus, in cavity mutant Hbs, the movement of Fe upon O₂-binding is detached from the movement of the F-helix, which is supposed to play a role of communication. Recombinant Hb (rHb)(αH87G), in which only the Fe-His in the α subunits is replaced by Fe-imidazole, showed a biphasic O₂-binding with no cooperativity, indicating the coexistence of two independent hemes with different O₂-affinities. In contrast, rHb(βH92G), in which only the Fe-His in the β subunits is replaced by Fe-imidazole, gave a simple high-affinity O₂-binding curve with no cooperativity. Resonance Raman, ¹H NMR, and near-UV circular dichroism measurements revealed that the quaternary structure change did not occur upon O₂-binding to rHb(αH87G), but it did partially occur with O₂-binding to rHb(βH92G). The quaternary structure of rHb(αH87G) appears to be frozen in T while its tertiary structure is changeable. Thus, the absence of the Fe-His bond in the α subunit inhibits the T to R quaternary structure change upon O₂-binding, but its absence in the β subunit simply enhances the O₂-affinity of α subunit.     

Plant—Phytoseiid Interactions Mediated by Herbivore-Induced Plant Volatiles: Variation in Production of Cues and in Responses of Predatory Mites

Phytoseiid mites use herbivore-induced plant volatiles in long-range prey-habitat location and are arrested by these volatiles in a prey patch. The responses of predatory mites to these volatiles are considered to be an important factor in the local extermination of prey populations by phytoseiids such as Phytoseiulus persimilis. Prey-induced plant volatiles are highly detectable and can be reliable indicators of prey presence and prey identity. The composition of herbivore-induced plant volatiles depends on plant species and plant cultivar. Moreover, the composition may also vary with the herbivore species that infests a plant. The responses of phytoseiids to prey-induced plant volatiles from a specific plant-herbivore combination are highly variable. Causal factors include starvation, specific hunger, experience, pathogen infestation and the presence of competitors. Investigating variation in the phytoseiid's behavioural response in relation to these factors is important for understanding how and why behavioural strategies maximize phytoseiid fitness.     

Localization of lipocaline-type prostaglandin D synthase in rat brain: immunoelectron microscopic study

The distribution of lipocaline-type prostaglandin D synthase (L-PGDS) in rat brain was investigated by immunoelectron microscopy using a protein A-gold technique. In perivascular cells adjacent to the basement membrane of arterioles in the pia-arachnoid and of blood vessels in the subpial cortex, gold labeling was confined to the lumen of the dilated rough endoplasmic reticulum, and not found in the few lysosomes present in the cytoplasm. The results suggest that the perivascular cells secrete L-PGDS and seem not to degrade lipophilic molecules carried by L-PGDS. Moreover, gold particles representing the antigenic sites of L-PGDS were found in the Golgi apparatus, rough endoplasmic reticulum, vesicles, and nuclear envelope of arachnoid trabecular cells, arachnoid barrier cells, and arachnoid pia mater cells. The labeling was less detectable in the same organelles of choroid plexus epithelial cells, compared with leptomeningeal cells. In meningeal macrophages and parenchymal microglia, L-PGDS was detected in lysosomes, multivesicular bodies, and endocytic vesicles. The production of L-PGDS in perivascular cells is important to the various functions of this enzyme in brain parenchyma.     

Studies of α-Helicity and Intersegmental Interactions in Voltage-Gated Na+ Channels: S2D4

Much data, including crystallographic, support structural models of sodium and potassium channels consisting of S1–S4 transmembrane segments (the “voltage-sensing domain”) clustered around a central pore-forming region (S5–S6 segments and the intervening loop). Voltage gated sodium channels have four non-identical domains which differentiates them from the homotetrameric potassium channels that form the basis for current structural models. Since potassium and sodium channels also exhibit many different functional characteristics and the fourth domain (D4) of sodium channels differs in function from other domains (D1–D3), we have explored its structure in order to determine whether segments in D4 of sodium channels differ significantly from that determined for potassium channels. We have probed the secondary and tertiary structure and the role of the individual amino acid residues of the S2D4) of Na_v1.4 by employing cysteine-scanning mutagenesis (with tryptophan and glutamine substituted for native cysteine). A Fourier transform power spectrum of perturbations in free energy of steady-state inactivation gating (using midpoint potentials and slopes of Boltzmann equation fits of channel availability, h_∞-V plots) indicates a substantial amount of α-helical structure in S2D4 (peak at 106°, α-Periodicity Index (α-PI) of 3.10), This conclusion is supported by α-PI values of 3.28 and 2.84 for the perturbations in rate constants of entry into (β) and exit from (α) fast inactivation at 0 mV for mutant channels relative to WT channels assuming a simple two-state model for transition from the open to inactivated state. The results of cysteine substitution at the two most sensitive sites of the S2D4 α-helix (N1382 and E1392C) support the existence of electrostatic network interactions between S2 and other transmembrane segments within Na_v1.4D4 similar to but not identical to those proposed for K⁺ channels.     

Incidence of mosaic cell lines in vivo and malsegregation of chromosome 21 in lymphocytes in vitro of trisomy 21 patients: detection by fluorescence in situ hybridization on binucleated lymphocytes

In order to detect aneuploidy in interphase human lymphocytes, both in vivo and in vitro, fluorescence in situ hybridization (FISH) was carried out on binucleated cells cytokinesis-blocked by cytochalasin B at the first mitosis after phytohemagglutinin stimulation. A pericentric chromosome-21-specific DNA probe prepared from yeast artificial chromosome clone 881D2 by the polymerase chain reaction was employed. One thousand binucleated cells per individual were scored from cultures from twelve trisomy 21 patients aged 0.01-8.9 years (mean 4.3 years) and 20 normal children of similar age. Of trisomy 21 patients, increased frequencies of disomic cells in vivo (1.690+/-1.070%) and cells containing six signals with nondisjunction (0.822+/-0.554%) were found, compared with those of monosomic 21 cells in vivo (0.265+/-0.130%) and cells containing four signals with nondisjunction in normal children (0.369+/-0.250%; P=0.000 and P=0.000, respectively). These results show that malsegregation of chromosome 21 occurs more often in trisomic 21 cells than in disomic cells from normal children. The frequency of nondisjunction was significantly higher than the loss of chromosome 21 in both cultured trisomic (0.822+/-0.554% vs 0.043+/-0.049%, P=0.000) and disomic (0.369+/-0.250% vs 0.010+/-0.30%, P=0.000) cells. Comparisons of in vivo and in vitro data on aneuploidy indicate that a cell selection mechanism may exist in vivo. All these results show that FISH, with a chromosome-specific probe, on binucleated lymphocytes is a powerful tool for simultaneously detecting mosaic cell lines in vivo and malsegregation (loss and nondisjunction) of a corresponding chromosome in vitro in the same cell population.     

Prediction of charge-induced molecular alignment: residual dipolar couplings at pH 3 and alignment in surfactant liquid crystalline phases

Recently we reported that the alignment tensor of a biological macromolecule, which was dissolved in a dilute suspension of highly negatively charged filamentous phage at close to neutral pH, can be predicted from the molecule’s 3D charge distribution and shape (Zweckstetter et al. 2004). Here it is demonstrated that this approach is also applicable to alignment of proteins in liquid crystalline phases formed by filamentous phage at low pH. Residual dipolar couplings (RDCs) predicted by our simple electrostatic model for the B1 domain of protein G in fd phage at pH 3 fit very well with the experimental values. The sign of charge–shape predicted one-bond ¹H–¹⁵N dipolar couplings for the B1 domain of protein G (GB1) was inverted at pH 3 compared to neutral pH, in agreement with experimental observations. Our predictions indicate that this is a feature specific for GB1. In addition, it is shown that RDCs induced in the protein ubiquitin by the presence of a positively charged surfactant system comprising cetylpyridinium bromide/hexanol/sodium bromide can be predicted accurately by a simple electrostatic alignment model. This shows that steric and electrostatic interactions dominate weak alignment of biomolecules for a wide range of pH values both in filamentous phage and in surfactant liquid crystalline phases.     

Regulation of apoptosis by Caspases under oxidative stress conditions in mice testicular cells: in vitro molecular mechanism

Exposure to various toxicants is known to cause apoptosis in various cell types. The spermatogenic cells are particularly sensitive to various deleterious conditions including toxicant exposure. The affected cells might undergo apoptosis; however, the mechanisms may be different for different kinds of insults to the cells. In the present study, we looked into the mechanisms involved in apoptosis after exposure of testicular cells from mice to two different chemicals, diethyl maleate (DEM) and tert-butyl hydroperoxide (TBHP). For the study, cells were maintained for 4 h under various treatments: control (media only), 0.25 mM DEM, 0.5 mM DEM, 0.25 mM TBHP, and 0.5 mM TBHP. The treated cells were then harvested for various estimations, viz. viability, reduced and oxidized glutathione, redox ratio, free radical generation, and ethidium bromide/acridine orange co-staining. mRNA was extracted for RT-PCR analysis of Caspase 3, Caspase 8, Caspase 9, p53, p21, Bax, and Bcl-2. It was observed that both the treatments resulted in decreased levels of reduced glutathione and a concomitant increase in the oxidized form and ROS levels in a dose-dependent manner. The apoptotic cell death was evident from ethidium bromide/acridine orange staining. The mRNA expression pattern of various Caspases showed progressive increase in Caspase 3 and Caspase 9 mRNA in both the treatments in a dose-dependent manner, whereas there was no change in Caspase 8 mRNA expression. p53, p21, and Bax also showed increased expression, whereas Bcl-2 expression remained unchanged in DEM treatments and increased significantly in both TBHP treatments. Hence, the present study indicates the involvement and activation of various apoptotic factors, particularly Caspase 3 and 9 along with p53, in response to exposure of testicular cells to DEM and TBHP.     

Resilient Plant–Bird Interactions in a Volcanic Island Ecosystem: Pollination of Japanese Camellia Mediated by the Japanese White-Eye

Observations of interspecies interactions during volcanic activity provide important opportunities to study how organisms respond to environmental devastation. Japanese camellia (Camellia japonica L.) and its main avian pollinator, the Japanese White-eye (Zosterops japonica), offer an excellent example of such an interaction as key members of the biotic community on Miyake-jima, which erupted in 2000 and continues to emit volcanic gases. Both species exhibit higher resistance to volcanic damage than other species. We examined the effects of volcanic activity on this plant–pollinator system by estimating pollen flow and the genetic diversity of the next generation. The results showed that despite a decrease in Camellia flowers, the partitioning of allelic richness among mother-tree pollen pools and seeds decreased while the migration rate of pollen from outside the study plot and the pollen donor diversity within a fruit increased as the index of volcanic damage increased. In areas with low food (flower) density due to volcanic damage, Z. japonica ranged over larger areas to satisfy its energy needs rather than moving to areas with higher food density. Consequently, the genetic diversity of the seeds (the next plant generation) increased with the index of volcanic damage. The results were consistent with previously published data on the movement of Z. japonica based on radio tracking and the genetic diversity of Camellia pollen adhering to pollinators. Overall, our results indicated that compensation mechanisms ensured better pollination after volcanic disturbance.     

Subunit Interacting Surfaces of Human Hemoglobin in Solution: Localization of the α–β Subunit Interacting Surfaces on the α-Chain by a Comprehensive Synthetic Strategy

By using synthetic overlapping peptides encompassing the entire -chain of adult human hemoglobin (HbA), we have mapped on the -chain the regions responsible for its binding to the -chain in solution. These binding surfaces were, in general, in good agreement with those expected from the crystal structure (peptides 81–95, 101–115, 111–125, and 131–141). However, we observed some significant differences in the levels of binding found here in solution and those expected from the crystal structure. Peptide 31–45, which in the crystal had the highest number of contact residues of all the -chain peptides, did not bind the -chain in solution. Similarly, peptide 91–105, with seven contact residues in the crystal, showed low binding with the -chain in solution. On the other hand, peptides 41–55 and 121–135 possessed much higher binding activity in solution than would be expected from their contribution to subunit association in the crystal. In fact, peptide 121–135 had the highest binding activity of the -chain peptides. These studies and our previous findings, which localized on the -chain the regions that bind to the -chain in solution, have shown that the regions of subunit association in solution are close to, but not identical with, those in the crystal. The approach should be quite useful for mapping subunit association in oligomeric proteins and could even be applied to proteins that are isolated only in traces or whose three-dimensional structure is not yet known.     

Attempt at Affinity Labeling of α- and β- Amylases by α- and β-d-Glucopyranosides and α- and β-Maltooligosaccharides with 2,3-Epoxypropyl Residue as Aglycone: Specific Inactivation of β-Amylases

Among 2,3-epoxypropyl α-d-glucopyranoside and 2,3-epoxypropyl α-maltooligosaccharides and the β-anomers, 2,3-epoxypropyl α-d-glucopyranoside (α-EPG) strongly inactivated the β-amylases [EC 3.2.1.2] of sweet potato, barley, and Bacillus, cereus, in addition to soybean β amylase [J. Biochem., 99, 1631 (1986)]. However, none of the compounds used inactivated any α-amylases [EC 3.2.1.1] of porcine pancreas, Aspergillus oryzae, or Bacillus amyloliquefaciens. Irreversible incorporation of ¹⁴C-labeled α-EPG into β-amylases was stoichiometric, i.e., one α-EPG per active site of the enzyme was bound, and the inactivations were almost complete. The results suggest that α-EPG is an affinity labeling reagent selective for β-amylase. Slow inactivations by the other compounds were also observed, depending on the difference of source of β amylase.     

Plant–Microbe Interactions: Wetting of Ivy (Hedera helix L.) Leaf Surfaces in Relation to Colonization by Epiphytic Microorganisms

Abstract Leaf wettability, cuticular wax composition, and microbial colonization of upper and lower leaf surfaces of ivy (Hedera helix L.) was investigated for young and old leaves sampled in June and September. Contact angles of aqueous buffered solutions measured on young leaf surfaces ranged between 76° and 86° and were not dependent on the pH value of the applied droplets. Contact angles measured on old leaf surfaces were up to 32°, significantly lower than on young leaf surfaces. Furthermore, contact angles were significantly lower using aqueous solutions of pH 9.0 compared to pH 3.0, indicating the influence of ionizable functional groups on leaf surface wetting properties. Observed changes in leaf wetting properties did not correlate with different levels of alkanoic acids in cuticular waxes. However, microscopic examination of the leaf surfaces indicated the influence of epiphytic microorganisms on wetting properties of old leaves, since their surfaces were always colonized by epiphytic microorganisms (filamentous fungi, yeasts, and bacteria), whereas surfaces of young leaves were basically clean. In order to analyze the effect of epiphytic microorganisms on leaf surface wetting, surfaces of young and clean ivy leaves were artificially colonized with Pseudomonas fluorescens. This resulted in a significant increase and a pH dependence of leaf surface wetting in the same way as it was observed on old ivy leaf surfaces. From these results it can be deduced that the native wetting properties of leaf surfaces can be significantly masked by the presence of epiphytic microorganisms. The ecological implications of altered wetting properties for microorganisms using the leaf/atmosphere interface as habitat are discussed. Received: 20 March 1999; Accepted: 5 July 1999; Online Publication: 18 July 2000