Interactions Between the Vegetative States of Phages λ and T1

Bacteria containing phage lambda in the vegetative state were produced either by induction of λ lysogens or by infection of sensitive cells with λ. These cells were superinfected with T1, and assayed for the production of λ, T1, or both. Although most of the cells produced only λ or T1, approximately 10% of the infectious centers were dual yielders. Examination of the progeny phage produced by the population of mixedly-infected cells showed that there was little, if any, phenotypic mixing, as determined by adsorption phenotype. T1am mutants in a variety of T1 genes were tested for their ability to exclude λ, but none were defective in this ability. One gene of T1, gene 4, can be complemented by λ.     

Connexin-43 Interactions with ZO-1 and α- and β-tubulin

Gap junctions are composed of connexins that form transmembrane channels between adjacent cells. The C-terminal tail of connexin-43 (Cx43), the most widely expressed connexin member, has been implicated in the regulation of Cx43 channel gating. Interestingly, channel-independent processes regulated by Cx43 have also been postulated. In our studies to elucidate the mechanism of Cx43 channel gating by growth factors and to explore additional functions of gap junctions, we have identified three interacting partners of the C-terminal tail of Cx43 (Cx43CT). (i) the c-Src tyrosine kinase, which phosphorylates Cx43CT and is involved in G protein-mediated inhibition of Cx43 gap junctional communication, (ii) the ZO-1 ‘scaffold’ protein, which might recruit signaling proteins into Cx43-based gap junctions. (iii) microtubules (consisting of α/β-tubulin dimers), which extend with their distal ends to Cx43-based gap junctions, suggesting that Cx43 gap junctions may play a novel role in regulating microtubule stability in contacted cells. Here we show that Cx43 binds α-tubulin equally well as β-tubulin. In addition, we show that the second, but not the first, PDZ domain of ZO-1 binds directly to Cx43, and we confirm that the very C-terminal isoleucine residue of Cx43 is critical for ZO-1 binding.     

A Specific and Essential Role for Na,K-ATPase α3 in Neurons Co-expressing α1 and α3

Most neurons co-express two catalytic isoforms of Na,K-ATPase, the ubiquitous α1, and the more selectively expressed α3. Although neurological syndromes are associated with α3 mutations, the specific role of this isoform is not completely understood. Here, we used electrophysiological and Na⁺ imaging techniques to study the role of α3 in central nervous system neurons expressing both isoforms. Under basal conditions, selective inhibition of α3 using a low concentration of the cardiac glycoside, ouabain, resulted in a modest increase in intracellular Na⁺ concentration ([Na⁺]_i) accompanied by membrane potential depolarization. When neurons were challenged with a large rapid increase in [Na⁺]_i, similar to what could be expected following suprathreshold neuronal activity, selective inhibition of α3 almost completely abolished the capacity to restore [Na⁺]_i in soma and dendrite. Recordings of Na,K-ATPase specific current supported the notion that when [Na⁺]_i is elevated in the neuron, α3 is the predominant isoform responsible for rapid extrusion of Na⁺. Low concentrations of ouabain were also found to disrupt cortical network oscillations, providing further support for the importance of α3 function in the central nervous system. The α isoforms express a well conserved protein kinase A consensus site, which is structurally associated with an Na⁺ binding site. Following activation of protein kinase A, both the α3-dependent current and restoration of dendritic [Na⁺]_i were significantly attenuated, indicating that α3 is a target for phosphorylation and may participate in short term regulation of neuronal function.     

Binding Interactions Between α-glucans from Lactobacillus reuteri and Milk Proteins Characterised by Surface Plasmon Resonance

Interactions between milk proteins and ??-glucans at pH 4.0?C5.5 were investigated by use of surface plasmon resonance. The ??-glucans were synthesised with glucansucrase enzymes from Lactobacillus reuteri strains ATCC-55730, 180, ML1 and 121. Variations in the molecular characteristics of the ??-glucans, such as molecular weight, linkage type and degree of branching, influenced the interactions with native and denatured ??-lactoglobulin and ??-casein. The highest overall binding levels were reached with ??-(1,4) compared to ??-(1,3) linked glucans. Glucans with many ??-(1,6) linkages demonstrated the highest binding levels to ??-casein, whereas the interaction with native ??-lactoglobulin was suppressed by ??-(1,6) linkages. Glucans with a higher degree of branching generally displayed lower protein binding levels whereas a higher molecular weight resulted in increased binding to ??-casein. The interactions with ??-casein were not pH dependent, whereas binding to denatured ??-lactoglobulin was highest at pH 4.0 and binding to native ??-lactoglobulin was optimal at pH 4.5?C5.0. This study shows that molecular weight, linkage type and degree of branching of ??-glucans highly influence the binding interactions with milk proteins.     

Cardiac α1-Adrenoceptors that regulate contractile function: Subtypes and subcellular signal transduction mechanismsthat regulate contractile function: Subtypes and subcellular signal transduction mechanisms

Activation of α₁-adrenoceptors as well as endothelin (ET) and angiotensin II (Ang II) receptors in cardiac muscle is coupled to acceleration of the hydrolysis of phosphoinositide (PI), with resultant production of inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol. There is an excellent correlation between the extent of acceleration of the PI hydrolysis and the positive inotropic effect (PIE) under most experimental conditions after the administration of α-adrenoceptor agonists, ET and Ang II in the rabbit ventricular muscle. The PIE of the α-adrenoceptor agonists, ET and Ang II is associated with a negative lusitropic effect and an increase in the sensitivity of myofilaments to Ca²⁺ ions. The PIE can be selectively inhibited by inhibitors of protein kinase C (PKC) such as staurosporine, NA 0345 and H-7, with little effect on the PI hydrolysis and the PIE of isoproterenol and Bay k 8644. Surprisingly, an activator of PKC, phorbol 12,13-dibutyrate (PDBu), selectively and more completely inhibited the PIE and acceleration of PI hydrolysis induced by the α-adrenoceptor agonists as well as by ET and Ang II in the rabbit. These receptor agonists consistently cause intracellular alkalinization by activation of Na⁺−H⁺ exchange, while the effects on membrane ion channel activities are divergent. For example, α-adrenoceptor agonists cause monophasic prolongation of the action potential, the time course of which coincides well with that of the PIE, while ET and Ang II produce a biphasic change in action potential duration, i.e., the long-lasting prolongation preceded by a transient abbreviation. α-Adrenoceptor agonists scarcely affect I_Ca, whereas ET elicits a biphasic alteration of the current. In addition, the potassium current, I_Kl, is markedly suppressed by α-adrenoceptor agonists, but this effect is not revealed with Ang II under the same experimental condition. These results indicate that the effects of α₁-adrenoceptors stimulation are partially shared by those of ET and Ang II receptor activation in the heart. Approximately 60% of the total population of α₁-adrenoceptors in the rabbit ventricle are composed of α_1B subtype, which is susceptible to chlorethylclonidine (CEC) and is predominantly responsible for the α₁-mediated PIE and PI hydrolysis. The remaining fraction that belongs to α_1A-adrenoceptors subtype is further subclassified into the WB 4101-sensitive (partly coupled to PI hydrolysis) and the niguldipinesensitive (PI hydrolysis-unrelated) subtypes. Special issue dedicated to Dr. Kinya Kuriyama.     

Glycosylation Modulates Melanoma Cell α2β1 and α3β1 Integrin Interactions with Type IV Collagen

Although type IV collagen is heavily glycosylated, the influence of this post-translational modification on integrin binding has not been investigated. In the present study, galactosylated and nongalactosylated triple-helical peptides have been constructed containing the α1(IV)382–393 and α1(IV)531–543 sequences, which are binding sites for the α2β1 and α3β1 integrins, respectively. All peptides had triple-helical stabilities of 37 °C or greater. The galactosylation of Hyl³⁹³ in α1(IV)382–393 and Hyl⁵⁴⁰ and Hyl⁵⁴³ in α1(IV)531–543 had a dose-dependent influence on melanoma cell adhesion that was much more pronounced in the case of α3β1 integrin binding. Molecular modeling indicated that galactosylation occurred on the periphery of α2β1 integrin interaction with α1(IV)382–393 but right in the middle of α3β1 integrin interaction with α1(IV)531–543. The possibility of extracellular deglycosylation of type IV collagen was investigated, but no β-galactosidase-like activity capable of collagen modification was found. Thus, glycosylation of collagen can modulate integrin binding, and levels of glycosylation could be altered by reduction in expression of glycosylation enzymes but most likely not by extracellular deglycosylation activity.     

Epithelial Polarity: Interactions Between Junctions and Apical–Basal Machinery

Epithelial polarity is established and maintained by competition between determinants that define the apical and basolateral domains. Cell–cell adhesion complexes, or adherens junctions, form at the interface of these regions. Mutations in adhesion components as well as apical determinants normally lead to an expansion of the basolateral domain. Here we investigate the genetic relationship between the polarity determinants and adhesion and show that the levels of the adhesion protein Armadillo affect competition. We find that in arm mutants, even a modest reduction in the basolateral component lgl leads to a full apical domain expansion or lgl phenotype. By using an allelic series of Armadillo mutations, we show that there is a threshold at which basolateral expansion can be reversed. Further, in embryos lacking the Wingless signaling component zw3, the same full apical expansion occurs again with only a reduction in lgl. We propose a model where zw3 regulates protein levels of apical and adhesion components and suggest that a reciprocal interaction between junctions and polarity modules functions to maintain stable apical and basolateral domains.A major reason that epithelial cells require apical–basal polarity is to differentiate between the interior of the organism and the external environment. To accomplish this, epithelial cells generate molecularly distinct domains along their plasma membranes: an apical domain that is exposed to the outside, a basolateral domain that contacts the interior, and, in between, an adhesion complex that holds the cell sheet together. In Drosophila embryos, at least three polarity complexes are used to establish and maintain this subcellular organization commonly known as apical–basal cell polarity. On the apical side, the Crumbs (Crb) and Stardust (Std, Pals) proteins form one complex (Jurgens et al. 1984; Tepass et al. 1990; Tepass and Knust 1993; Wodarz et al. 1995; Muller and Wieschaus 1996). The second one is composed of Bazooka (Baz, Par-3), Par-6, and atypical protein kinase C (aPKC) (Wieschaus et al. 1984; Muller and Wieschaus 1996; Wodarz et al. 2000; Hutterer et al. 2004). On the opposite or basolateral side of the cell, Lethal giant larvae (Lgl), Discs large (Dlg), and Scribble (Scrib) determine the basolateral domain of the plasma membrane (Gateff and Schneiderman 1974; Mechler et al. 1985; Woods and Bryant 1989; Bilder and Perrimon 2000). In between the complexes lie the adherens junctions (AJ) composed of E-cadherin, Armadillo (Arm, β-catenin), and α-catenin (Oda et al. 1993, 1994; Peifer et al. 1993; Tepass et al. 1996).In Drosophila embryos, mutations that affect apical components often lead to the crumbs phenotype, where ectodermal cells lose integrity and many die through apoptosis. The surviving cells secrete cuticle in a discontinuous fashion, leaving pieces apparently floating within the eggshell (Tepass et al. 1990; Tanentzapf and Tepass 2003). This phenotype is also seen in embryos deficient for AJ proteins (Oda et al. 1993; Cox et al. 1996; Magie et al. 2002). On the other hand, mutations that affect the basolateral genes display a very different phenotype. Zygotic only (Z) mutants for scrib, lgl, and dlg have a significant maternal mRNA contribution that allows normal embryonic development to proceed. Phenotypes are observed only in larvae, which die with significantly overgrown imaginal discs (Gateff 1978; Bilder and Perrimon 2000). Removal of the maternal mRNA complement, as well as the zygotic contribution (M/Z) through the induction of germline clones, leads to a poorly differentiated and convoluted cuticle with a bubbly appearance (Figure 1) (Bilder et al. 2003; Tanentzapf and Tepass 2003).Open in a separate windowFigure 1.—Schema and cuticles representing wild-type vs. the opposing phenotypes of apical and basolateral expansion. (A) A wild-type cuticle shows rows of denticles separated by naked regions in a highly organized or patterned fashion. The apical determinants localize to the apical surface of cells, establishing the apical domain (green), the basolateral determinants localize to the basolateral surface of cells, establishing the basolateral domain (blue), and the adherens junctions (red) form at the interface between these two opposing regions. (B) The crumbs phenotype is observed when an apical determinant is mutated, causing an expansion of the basolateral domain. (C) The lgl phenotype, or bubble phenotype, is observed when a basolateral determinant is mutated, causing an expansion of the apical domain.These studies led to a comprehensive competition model where apical and basal components opposed each other (Figure 1, schema); however, a strangely neglected topic was the interaction of junctions and the apical and basal determinants. Therefore, we used a genetic approach to investigate the interaction of apical–basal polarity proteins and adherens junctions.     

Relationship Between β Adrenoceptor Occupancy and Receptor Down-Regulation Induced β Antagonists with Intrinsic Sympathomimetic Activity

Abstract

The relationship between the Ki of the β₂ adrenoceptor and EC50 values characterizing receptor down-regulation induced by isoproterenol and six β antagonists classified as having weak to strong intrinsic sympathomimetic activity (ISA) was determined using L6 myoblasts. It was hypothesized that if receptor loss induced by β antagonists with ISA was mediated through cAMP, EC50 = Ki. EC50/Ki ratios for (-)isoproterenol, (-) and (+) celiprolol were 0.006, 0.01 and 0.08, respectively (p<0.05); ratios for (-)pindolol and dilevalol were 19 and 9.5, respectively (p<0.05). EC50/Ki ratios for acebutalol and (-)alprenolol were not significantly different from 1.0. Isoproterenol and dilevalol maximally down-regulated receptor density 89 and 83%, respectively, followed by (+)celiprolol, 54%; (-)celiprolol, 53%; acebutalol, 41%; (-)pindolol, 36% and (-)alprenolol, 31%. Receptor loss was blocked in each case by ICI118,551 or sotalol. A sensitive radioimmunoassay failed to detect increased cAMP accumulation following pretreatment with concentrations of acebutalol, (-)alprenolol, celiprolol and (-)pindolol 100 times their respective Ki values. Isoproterenol and dilevalol stimulated cAMP accumulation 100-and 2-fold over basal, respectively. We conclude that receptor loss induced by & antagonists with ISA is mediated through the β₂ adrenoceptor and in at least some cases is cAMP-independent.     

High Vascular Tone of Mouse Femoral Arteries In Vivo Is Determined by Sympathetic Nerve Activity Via α1A- and α1D-Adrenoceptor Subtypes

Background and purpose

Determining the role of vascular receptors in vivo is difficult and not readily accomplished by systemic application of antagonists or genetic manipulations. Here we used intravital microscopy to measure the contributions of sympathetic receptors, particularly α₁-adrenoceptor subtypes, to contractile activation of femoral artery in vivo.

Experimental approach

Diameter and intracellular calcium ([Ca²⁺]_i) in femoral arteries were determined by intravital fluorescence microscopy in mice expressing a Myosin Light Chain Kinase (MLCK) based calcium-calmodulin biosensor. Pharmacological agents were applied locally to the femoral artery to determine the contributions of vascular receptors to tonic contraction and [Ca²⁺]_i,.

Key results

In the anesthetized animal, femoral arteries were constricted to a diameter equal to 54% of their passive diameter (i.e. tone = 46%). Of this total basal tone, 16% was blocked by RS79948 (0.1 µM) and thus attributable to α₂-adrenoceptors. A further 46% was blocked by prazosin (0.1 µM) and thus attributable to α₁-adrenoceptors. Blockade of P_2X and NPY₁ receptors with suramin (0.5 mM) and BIBP3226 (1.0 µM) respectively, reduced tone by a further 22%, leaving 16% of basal tone unaffected at these concentrations of antagonists. Application of RS100329 (α_1A-selective antagonist) and BMY7378 (α_1D-selective) decreased tone by 29% and 26%, respectively, and reduced [Ca²⁺]_i. Chloroethylclonidine (1 µM preferential for α_1B-) had no effect. Abolition of sympathetic nerve activity (hexamethonium, i.p.) reduced basal tone by 90%.

Conclusion and Implications

Tone of mouse femoral arteries in vivo is almost entirely sympathetic in origin. Activation of α_1A- and α_1D-adrenoceptors elevates [Ca²⁺]_i and accounts for at least 55% of the tone.     

Combinatorial Pharmacogenetic Interactions of Bucindolol and β1, α2C Adrenergic Receptor Polymorphisms

Background

Pharmacogenetics involves complex interactions of gene products affecting pharmacodynamics and pharmacokinetics, but there is little information on the interaction of multiple genetic modifiers of drug response. Bucindolol is a β-blocker/sympatholytic agent whose efficacy is modulated by polymorphisms in the primary target (β₁ adrenergic receptor [AR] Arg389 Gly on cardiac myocytes) and a secondary target modifier (α_2C AR Ins [wild-type (Wt)] 322–325 deletion [Del] on cardiac adrenergic neurons). The major allele homozygotes and minor allele carriers of each polymorphism are respectively associated with efficacy enhancement and loss, creating the possibility for genotype combination interactions that can be measured by clinical trial methodology.

Methodology

In a 1,040 patient substudy of a bucindolol vs. placebo heart failure clinical trial, we tested the hypothesis that combinations of β₁389 and α_2C322–325 polymorphisms are additive for both efficacy enhancement and loss. Additionally, norepinephrine (NE) affinity for β₁389 AR variants was measured in human explanted left ventricles.

Principal Findings

The combination of β₁389 Arg+α_2C322–325 Wt major allele homozygotes (47% of the trial population) was non-additive for efficacy enhancement across six clinical endpoints, with an average efficacy increase of 1.70-fold vs. 2.32-fold in β₁389 Arg homozygotes+α_2C322–325 Del minor allele carriers. In contrast, the minor allele carrier combination (13% subset) exhibited additive efficacy loss. These disparate effects are likely due to the higher proportion (42% vs. 8.7%, P = 0.009) of high-affinity NE binding sites in β₁389 Arg vs. Gly ARs, which converts α_2CDel minor allele-associated NE lowering from a therapeutic liability to a benefit.

Conclusions

On combination, the two sets of AR polymorphisms 1) influenced bucindolol efficacy seemingly unpredictably but consistent with their pharmacologic interactions, and 2) identified subpopulations with enhanced (β₁389 Arg homozygotes), intermediate (β₁389 Gly carriers+α_2C322–325 Wt homozygotes), and no (β₁389 Gly carriers+α_2C322–325 Del carriers) efficacy.     

Hyaluronan-binding protein 1 (HABP1/p32/gC1qR) induces melanoma cell migration and tumor growth by NF-kappa B dependent MMP-2 activation through integrin α(v)β(3) interaction

Cell migration is the hallmark of cancer regulating anchorage independent growth and invasiveness of tumor cells. Hyaluronan (HA), an ECM polysaccharide is shown to regulate this process. In the present report, we demonstrated, supplementation of purified recombinant hyaluronan binding protein 1(HABP1/p32/gC1qR) from human fibroblast cDNA enhanced migration potential of highly invasive melanoma (B16F10) cells. Exogenous HABP1 adhered to the cell surface transiently and was shown to interact and colocalize with α_vβ₃ integrin, a regulatory molecule of cell migration. In HABP1 treated cells, the phosphorylation of nuclear factor inducing kinase (NIK) and IκBα was observed, followed by nuclear translocation of p65 subunit of NFκB, along with its DNA-binding and transactivation, resulting in upregulation of MT1-MMP expression and finally MMP-2 activation. To substantiate our findings, prior to HABP1 treatment, the expression of NIK was reduced by small interfering RNA mediated knockdown and confirmed the inhibition of nuclear translocation of p65 subunit of NFκB and upregulation of MT1-MMP expression. In addition, the use of curcumin, an anti-cancer drug, or GRGDSP, the blocking peptide along with exogenous HABP1, inhibited such NFκB-dependent pathway, confirming that HABP1-induced cell migration is α_vβ₃ integrin-mediated and downstream signaling by NFκB. Finally, we translated the in vitro data in mice model and observed enhanced tumor growth with higher MT1-MMP expression and MMP-2 activation in the tumors upon injection of HABP1 treated melanoma cells. The treatment of curcumin, the anticancer drug along with HABP1, inhibited the migration, expression of MT1-MMP and activation of MMP-2 and finally tumor growth supports the involvement of HABP1 in tumor formation.     

Effect of Time‐of‐Day‐Specific Strength Training on Serum Hormone Concentrations and Isometric Strength in Men

A time‐of‐day influence on the neuromuscular response to strength training has been previously reported. However, no scientific study has examined the influence of the time of day when strength training is performed on hormonal adaptations. Therefore, the primary purpose of this study was to examine the effects of time‐of‐day‐specific strength training on resting serum concentrations and diurnal patterns of testosterone (T) and cortisol (CORT) as well as maximum isometric strength of knee extensors. Thirty eight diurnally active healthy, previously untrained men (age 20–45 yrs) underwent a ten‐week preparatory strength training period when sessions were conducted between 17:00–19:00 h. Thereafter, these subjects were randomized into either a morning (n=20, training times 07:00–09:00 h) or afternoon (n=18, 7:00–19:00 h) training group for another ten‐week period of time‐of‐day‐specific training (TST). Isometric unilateral knee extension peak torque (MVC) was measured at 07:00, 12:00, 17:00, and 20:30 h over two consecutive days (Day 1 & Day 2) before and after TST. Blood samples were obtained before each clock‐time measurement to assess resting serum T and CORT concentrations. A matched control group (n=11) did not train but participated in the tests. Serum T and CORT concentrations significantly declined from 07:00 to 20:30 h on all test days (Time effect, p<.001). Serum CORT at 07:00 h was significantly higher on Day 1 than Day 2 in the control and afternoon group, both in Pre and Post conditions (Day×Time interaction, p<.01). In the morning group, a similar day‐to‐day difference was present in the Pre but not Post conditions (Time×Group interaction, p<.05). MVC significantly increased after TST in both the morning and afternoon groups (Pre to Post effect, p<.001). In both groups, a typical diurnal variation in MVC (Time effect, p<.001) was found, especially on Day 2 in the Pre condition, and this feature persisted from Pre to Post in the afternoon group. In the morning group, however, diurnal variation was reduced after TST on both Day 1 and Day 2 (Pre to Post×Day×Time×Group interaction, p<.05). In conclusion, 10 weeks of morning time‐of‐day‐specific strength training resulted in reduced morning resting CORT concentrations, presumably as a result of decreased masking effects of anticipatory psychological stress prior to the morning testing. The typical diurnal pattern of maximum isometric strength was blunted by the TST period in the morning but not the afternoon group. However, the TST period had no significant effect on the resting total T concentration and its diurnal pattern and on the absolute increase in maximum strength.     

Cation—π and Amino-Acceptor Interactions Between Hydrated Metal Cations and DNA Bases. A Quantum-Chemical View

Abstract

Cation—π interactions between cytosine and hexahydrated cations have been characterized using ab initio method with inclusion of electron correlation effects, assuming idealized and crystal geometries of the interacting species. Hydrated metal cations can interact with nucleobases in a cation—π manner. The stabilization energy of such complexes would be large and comparable to the one for cation—π complex with benzene. Further, polarized water molecules belonging to the hydration shell of the cation are capable to form a strong hydrogen bond interaction with the nitrogen lone electron pair of the amino groups of bases and enforce a pronounced sp³ pyramidalization of the nucleobase amino groups. However, in contrast to the benzene—cation complexes, the cation—π configurations are highly unstable for a nucleobase since the conventional in plane binding of hydrated cations to the acceptor sites on the nucleobase is strongly preferred. Thus, a cation—π interaction with a nucle-obase can occur only if the position of the cation is locked above the nucleobase plane by another strong interaction. This indeed can occur in biopolymers and may have an effect on the local DNA architecture. Nevertheless, nucleobases have no intrinsic propensity to form cation—π interactions.     

Properties and Specific Functional Features of Wheat Grain α-Amylase/Subtilisin Inhibitor

A protein bifunctional inhibitor of endogenous α-amylase and subtilisin has been isolated from wheat grain and purified. The inhibitor specifically inactivates α-amylase isozymes with high isoelectric point values (group α-AMY1) and has almost no effect on the α-AMY2 isozymes with low isoelectric point values. This enzyme does not belong to glycoproteins and has a molecular weight of 21 kDa and an isoelectric point of 7.2. The protein displays a relatively high thermostability and pH optimum of 8.0; its inhibitory activity requires the presence of Ca²⁺ cations. The inhibition of excess α-amylase in wheat grain with a low falling number by the purified protein is studied.     

Temporal Variation in Drug Interaction Between Lithium and Morphine‐Induced Analgesia

The administration‐time‐dependent aspects of the drug interaction between lithium and morphine‐induced analgesia were studied using the mouse hot‐plate test at six different times of day, each scheduled at 4 h intervals. Lithium treatment alone, in doses of 1 to 10 mmol/kg administered intraperitoneally (i.p.) did not significantly alter test latencies compared to the corresponding clock‐time in saline‐injected controls. Basal pain sensitivity and morphine‐induced antinociceptive activity displayed significant circadian rhythms as assessed by the hot‐plate response latencies, with higher values occurring during the nocturnal activity than during the daytime rest span. Acute administration of lithium, in a dose of 3 mmol/kg, 30 min prior to morphine dosing did not influence morphine‐induced analgesia compared to all the clock‐time test‐matched morphine groups, except the 9 HALO (Hours After Lights On) one. There was a prominent potentiation of the morphine‐induced antinociception at this biological time during combined drug treatment. The latter finding demonstrates that administration‐time‐dependent differences in drug‐drug interactions need to be considered in both experimental designs and clinical settings.     

Quantitative mRNA Analysis of Eight Bovine 5‐HT Receptor Subtypes in Brain,Abomasum, and Intestine by Real‐Time RT‐PCR

Abstract

Serotoninergic pathways are involved in economically important bovine gastrointestinal (GI) motility disorders such as displaced abomasum and cecal dilatation/dislocation. The existing research tools to investigate the role of serotoninergic pathways in such disorders in ruminants comprise functional pharmacological methods, e.g., in vitro contractility studies in tissue baths, and electromyographical recordings in vivo. However, no tools for quantification of bovine serotonin receptor [5‐hydroxytryptamine receptor (5‐HTR)] expression were available so far. This study aimed to develop real‐time RT‐PCR assays for quantitative mRNA analysis of bovine 5‐HTR subtypes. Because the bovine 5‐HTR coding sequences (CDSs) were completely unknown, multiple species (human, mouse, and rat) alignment of complete CDS was used for primer design in highly homologous regions. LightCycler real‐time RT‐PCR assays (partial CDS) for the following bovine 5‐HTR subtypes were developed and validated: 5‐HTR_1A, 5‐HTR_1B, 5‐HTR_1D, 5‐HTR_1F, 5‐HTR_2A, 5‐HTR_2B, 5‐HTR_2C, and 5‐HTR₄. Intra‐ and inter‐assay coefficients of variation (CV) for the eight established assays were small, ranging from 0.49% to 2.46%. As a first physiological application, 5‐HTR mRNA expression levels were measured in brain, abomasum, and intestine of 10 healthy, lactating dairy cows. The 5‐HTR expression was quantified by normalization to the housekeeping gene glyceraldehyde‐phosphate‐dehydrogenase (GAPDH). The 5‐HTR subtype expression levels ranged from 0.001% (5‐HTR_2C in intestine) to 1% 5‐HTR/GAPDH (5‐HTR_1B and 5‐HTR₄ in intestine). There were high variations of 5‐HTR subtype mRNA expression within tissues across receptor subtypes and within receptor subtypes across tissues. In conclusion, accurate real‐time RT‐PCR assays for quantitative analysis of bovine 5‐HTR subtype gene expression were developed and validated.     

Conformational Similarity Indices Between Different Residues in Proteins and α-Helix Propensities

Abstract

Various amino acid similarity matrices have been derived using data on physicochemical properties and molecular evolution. Conformational similarity indices, CS_XX′, between different residues are computed here using the distribution of the main-chain and side-chain torsion angles and the values have been used to cluster amino acids in proteins. A subset of these parameters, CS_AX′ indicates the extent of similarity in the main-chain and side-chain conformations (φ ψ and χ₁) of different residues (X) with Ala (A) and is found to have strong correlation with α-helix propensities. However, no subset of CS_XX′ provides any linear relationship with β-sheet propensities, suggesting that the conformational feature favouring the location of a residue in an a-helix is different from the one favouring the β-sheet. Conformationally similar residues (close CS_AX values) have similar steric framework of the side-chain (linear/branched, aliphatic/aromatic), irrespective of the polarity or hydrophobicity. Cooperative nucleation of helix may be facile for a contiguous stretch of residues with high overall CS_AX values.     

NMR Structure of Integrin α4 Cytosolic Tail and Its Interactions with Paxillin

Background

Integrins are a group of transmembrane signaling proteins that are important in biological processes such as cell adhesion, proliferation and migration. Integrins are α/β hetero-dimers and there are 24 different integrins formed by specific combinations of 18 α and 8 β subunits in humans. Generally, each of these subunits has a large extracellular domain, a single pass transmembrane segment and a cytosolic tail (CT). CTs of integrins are important in bidirectional signal transduction and they associate with a large number of intracellular proteins.

Principal Findings

Using NMR spectroscopy, we determined the 3-D structure of the full-length α4 CT (Lys968-Asp999) and characterize its interactions with the adaptor protein paxillin. The α4 CT assumes an overall helical structure with a kink in its membrane proximal region. Residues Gln981-Asn997 formed a continuous helical conformation that may be sustained by potential ionic and/or hydrogen bond interactions and packing of aromatic-aliphatic side-chains. ¹⁵N-¹H HSQC NMR experiments reveal interactions of the α4 CT C-terminal region with a fragment of paxillin (residues G139-K277) that encompassed LD2-LD4 repeats. Residues of these LD repeats including their adjoining linkers showed α4 CT binding-induced chemical shift changes. Furthermore, NMR studies using LD-containing peptides showed predominant interactions between LD3 and LD4 of paxillin and α4 CT. Docked structures of the α4 CT with these LD repeats suggest possible polar and/or salt-bridge and non-polar packing interactions.

Significance

The current study provides molecular insights into the structural diversity of α CTs of integrins and interactions of integrin α4 CT with the adaptor protein paxillin.