A Potential Link between the C5a Receptor 1 and the β1-Adrenoreceptor in the Mouse Heart

Purpose

Inflammation may contribute to the pathogenesis of specific cardiovascular diseases, but it is uncertain if mediators released during the inflammatory process will affect the continued efficacy of drugs used to treat clinical signs of the cardiac disease. We investigated the role of the complement 5a receptor 1 (C5aR1/CD88) in the cardiac response to inflammation or atenolol, and the effect of C5aR1 deletion in control of baseline heart rate in an anesthetized mouse model.

Methods

An initial study showed that PMX53, an antagonist of C5aR1 in normal C57BL6/J (wild type, WT) mice reduced heart rate (HR) and appeared to have a protective effect on the heart following induced sepsis. C5aR1 knockout (CD88-/-) mice had a lower HR than wild type mice, even during sham surgery. A model to assess heart rate variability (HRV) in anesthetized mice was developed to assess the effects of inhibiting the β₁-adrenoreceptor (β₁-AR) in a randomized crossover study design.

Results

HR and LF Norm were constitutively lower and SDNN and HF Norm constitutively higher in the CD88-/- compared with WT mice (P< 0.001 for all outcomes). Administration of atenolol (2.5 mg/kg) reduced the HR and increased HRV (P< 0.05, respectively) in the wild type but not in the CD88-/- mice. There was no shift of the sympathovagal balance post-atenolol in either strains of mice (P> 0.05), except for the reduced LF/HF (Lower frequency/High frequency) ratio (P< 0.05) at 60 min post-atenolol, suggesting increased parasympathetic tone of the heart due to the effect of atenolol administration. The HR of the WT mice were lower post atenolol compared to the CD88-/- mice (P = 0.001) but the HRV of CD88-/- mice were significantly increased (P< 0.05), compared with WT mice.

Conclusion

Knockout of the C5aR1 attenuated the effect of β₁-AR in the heart, suggesting an association between the β₁-AR and C5aR1, although further investigation is required to determine if this is a direct or causal association.     

Expression of βA3/A1-crystallin in the developing and adult rat eye

Crystallins are very abundant structural proteins of the lens and are also expressed in other tissues. We have previously reported a spontaneous mutation in the rat βA3/A1-crystallin gene, termed Nuc1, which has a novel, complex, ocular phenotype. The current study was undertaken to compare the expression pattern of this gene during eye development in wild type and Nuc1 rats by in situ hybridization (ISH) and immunohistochemistry (IHC). βA3/A1-crystallin expression was first detected in the eyes of both wild type and Nuc1 rats at embryonic (E) day 12.5 in the posterior portion of the lens vesicle, and remained limited to the lens fibers throughout fetal life. After birth, βA3/A1-crystallin expression was also detected in the neural retina (specifically in the astrocytes and ganglion cells) and in the retinal pigmented epithelium (RPE). This suggested that βA3/A1-crystallin is not only a structural protein of the lens, but has cellular function(s) in other ocular tissues. In summary, expression of βA3/A1-crystallin is controlled differentially in various eye tissues with lens being the site of greatest expression. Similar staining patterns, detected by ISH and IHC, in wild type and Nuc1 animals suggest that functional differences in the protein, rather than changes in mRNA/protein level of expression, likely account for developmental abnormalities in Nuc1.     

A functional link between Polo-like kinase 1 and the mammalian Target-Of-Rapamycin pathway?

Polo like kinase-1 is a key effector of cell division and its over-expression in several cancers is often linked with negative prognostic. We recently described that Plk1 is over-expressed in acute myeloid leukemia, and that its inhibition selectively reduces the proliferation of leukemic cells. Here, we report that Plk1 inhibition or depletion using pharmacological and siRNA approaches decreased the phosphorylation of two mTOR substrates in AML cells. In HCT116 cells, inducible expression of a constitutively active form of Plk1 leads to activation of mTOR, as shown by increased phosphorylation of its 4E-BP1 and RPS6 down-stream targets. In addition, HCT116 cells over-expressing the active form of Plk1 were characterized by abnormal growth that could be reversed by rapamycin, a specific inhibitor of the TORC1 complex. Altogether these data suggest the existence of a molecular and functional link between the Plk1 mitotic kinase and the mTOR pathway. Given the different established functions of Plk1 and mTOR during the cell cycle, we will discuss the possible meaning of this functional relationship.     

A plea for the ‘de-migranticization' of research on migration and integration1

ABSTRACT

Migration and integration research has been institutionalized over the last few decades. However, an increasing number of voices has been calling for more reflexivity, criticizing the nation-state- and ethnicity-centred epistemology that often informs this discipline. Consistently with this line of reasoning, I argue that migration and integration research originates in a historically institutionalized nation-state migration apparatus and is thus entangled with a particular normalization discourse. Therefore, this field of study contributes to reproducing the categories of this particular migration apparatus. This entanglement poses some serious dilemmas for this research tradition, dilemmas that ask for further consideration and possible solutions. My main proposition is to ‘de-migranticize' migration and integration research. I outline possible ways of doing so and discuss the consequences of such a strategy for the future of migration and integration studies.     

Quantitative correlation between the residual activity of β-hexosaminidase A and arylsulfatase A and the severity of the resulting lysosomal storage disease

Summary A previously suggested model for the correlation between residual activity of a lysosomal enzyme and the turnover rate of its substrate(s) has been extended to a discussion of substrate accumulation rates in individual cells and whole organs. With these considerations, much of the observed variability in age of onset and clinical phenotype, as well as the phenomenon of pseudodeficiency, can be understood as the consequences of small differences in the residual activity of the affected enzyme. In order to experimentally verify the basic assumptions on which this model rests, studies were performed in cell culture. The radiolabeled substrates ganglioside G_M2 and sulfatide were added to cultures of skin fibroblasts with different activities of -hexosaminidase A or arylsulfatase A, respectively, and their uptake and turnover measured. In both series of experiments, the correlation between residual enzyme activity and the turnover rate of the substrate was essentially as predicted: degradation increased steeply with residual activity, to reach the control level at a residual activity of approximately 10–15% of normal. All cells with an activity above this critical threshold had a normal turnover. Comparison of the results of these feeding studies with the clinical status of the donor of each cell line basically confirmed our notions but also revealed the limitations of the cell culture approach.     

The β1-Subunit of the MaxiK Channel Associates with the Thromboxane A2 Receptor and Reduces Thromboxane A2 Functional Effects

The large conductance voltage- and Ca²⁺-activated K⁺ channel (MaxiK, BK_Ca, BK) is composed of four pore-forming α-subunits and can be associated with regulatory β-subunits. One of the functional roles of MaxiK is to regulate vascular tone. We recently found that the MaxiK channel from coronary smooth muscle is trans-inhibited by activation of the vasoconstricting thromboxane A₂ prostanoid receptor (TP), a mechanism supported by MaxiK α-subunit (MaxiKα)-TP physical interaction. Here, we examined the role of the MaxiK β1-subunit in TP-MaxiK association. We found that the β1-subunit can by itself interact with TP and that this association can occur independently of MaxiKα. Subcellular localization analysis revealed that β1 and TP are closely associated at the cell periphery. The molecular mechanism of β1-TP interaction involves predominantly the β1 extracellular loop. As reported previously, TP activation by the thromboxane A₂ analog U46619 caused inhibition of MaxiKα macroscopic conductance or fractional open probability (FP_o) as a function of voltage. However, the positive shift of the FP_o versus voltage curve by U46619 relative to the control was less prominent when β1 was coexpressed with TP and MaxiKα proteins (20 ± 6 mV, n = 7) than in cells expressing TP and MaxiKα alone (51 ± 7 mV, n = 7). Finally, β1 gene ablation reduced the EC₅₀ of the U46619 agonist in mediating aortic contraction from 18 ± 1 nm (n = 12) to 9 ± 1 nm (n = 12). The results indicate that the β1-subunit can form a tripartite complex with TP and MaxiKα, has the ability to associate with each protein independently, and diminishes U46619-induced MaxiK channel trans-inhibition as well as vasoconstriction.     

Defined subcomplexes of the A1 ATPase from the archaeon Methanosarcina mazei Gö1: biochemical properties and redox regulation

The potential A(1) ATPase genes ahaA, ahaB, ahaC, ahaD, ahaE, ahaF, and ahaG from the anaerobic archaeon Methanosarcina mazei G?1 were overexpressed in Escherichia coli DK8 (pTL2). An A(1) complex was purified to apparent homogeneity and shown by Western blot and N-terminal sequence analyses to contain subunits A, B, C, D, and F but to be devoid of subunits E and G. Further removal of subunit C was without effect on ATPase activity. The enzyme was most active at pH 5.2 and required bisulfite and acetate for maximal activity. Kinetic studies confirmed three new inhibitors for A(1) ATPases (diethylstilbestrol and its derivatives hexestrol and dienestrol) and identified redox modulation as a new type of regulation of archaeal A(1) ATPases.     

Crystal structure of the archaeal A1Ao ATP synthase subunit B from Methanosarcina mazei Gö1: Implications of nucleotide-binding differences in the major A1Ao subunits A and B

The A1Ao ATP synthase from archaea represents a class of chimeric ATPases/synthases, whose function and general structural design share characteristics both with vacuolar V1Vo ATPases and with F1Fo ATP synthases. The primary sequences of the two large polypeptides A and B, from the catalytic part, are closely related to the eukaryotic V1Vo ATPases. The chimeric nature of the A1Ao ATP synthase from the archaeon Methanosarcina mazei G?1 was investigated in terms of nucleotide interaction. Here, we demonstrate the ability of the overexpressed A and B subunits to bind ADP and ATP by photoaffinity labeling. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to map the peptide of subunit B involved in nucleotide interaction. Nucleotide affinities in both subunits were determined by fluorescence correlation spectroscopy, indicating a weaker binding of nucleotide analogues to subunit B than to A. In addition, the nucleotide-free crystal structure of subunit B is presented at 1.5 A resolution, providing the first view of the so-called non-catalytic subunit of the A1Ao ATP synthase. Superposition of the A-ATP synthase non-catalytic B subunit and the F-ATP synthase non-catalytic alpha subunit provides new insights into the similarities and differences of these nucleotide-binding ATPase subunits in particular, and into nucleotide binding in general. The arrangement of subunit B within the intact A1Ao ATP synthase is presented.     

1-Deaza-3′-O-methyladenosine: A Nucleoside with the Syn-conformation in the Solid State and in Solution

Abstract

The 2′-O-methyl (2) and the 3′-O-methyl (3) derivatives of 1-deazaadenosine (1) were prepared. Single crystal X-ray analysis as well as ¹H and ¹³C NMR studies were performed on the 3′-O-methyl-1-deazaadenosine 3. In the solid state, the glycosyl torsion angle (χ = 64.7°) is in the syn-range which is caused by an intramolecular (5′)CH2OH…N(3) hydrogen bond. The ribofuranose moiety adopts a ² E (C-3′-exo; S) conformation and the orientation of the exocyclic C(4′)-C(5′) bond is + sc (γ^(+)g). The conformation in solution was found to be very similar to that in solid state. Whereas the 2′-O-methyl derivative of 1 is a strong inhibitor of adenosine deaminase the 3′-O-methyl derivative is neither inhibitor nor substrate.     

Are the nail-patella syndrome and the autosomal Goltz-like syndrome the phenotypic expressions of different alleles at the COL5A1 locus?

The COL5A1 gene, which encodes the pro 1(V) chain, was recently mapped to 9q34.3 in the same region as the nail-patella locus. This was taken as an indication that the nail-patella syndrome may be an inherited connective tissue disorder. We demonstrate COL5A1 heterozygous deletion and fibroblast under-expression of 1(V) chains in a girl with an unbalanced translocation resulting in 9q32qter monosomy. The patient presents dysplastic nails, a sign typical of nail-patella syndrome, but normal patella. Moreover, she has skin and bone disorders similar to those found in the Goltz syndrome. We suggest that monosomy for the COL5A1 gene is responsible for these connective tissue disorders. Accordingly, the nail-patella syndrome could be attributable to mutations inside the COL5A1 gene rather than to a deletion of it.     

Is the functional interaction between adenosine A2A receptors and metabotropic glutamate 5 receptors a general mechanism in the brain? Differences and similarities between the striatum and the hippocampus

The aim of the present paper was to examine, in a comparative way, the occurrence and the mechanisms of the interactions between adenosine A_2A receptors (A_2ARs) and metabotropic glutamate 5 receptors (mGlu5Rs) in the hippocampus and the striatum. In rat hippocampal and corticostriatal slices, combined ineffective doses of the mGlu5R agonist 2-chloro-5-hydroxyphenylglycine (CHPG) and the A_2AR agonist CGS 21680 synergistically reduced the slope of excitatory postsynaptic field potentials (fEPSPs) recorded in CA1 and the amplitude of field potentials (FPs) recorded in the dorsomedial striatum. The cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway appeared to be involved in the effects of CGS 21680 in corticostriatal but not in hippocampal slices. In both areas, a postsynaptic locus of interaction appeared more likely. N-methyl-D-aspartate (NMDA) reduced the fEPSP slope and FP amplitude in hippocampal and corticostriatal slices, respectively. Such an effect was significantly potentiated by CHPG in both areas. Interestingly, the A_2AR antagonist ZM 241385 significantly reduced the NMDA-potentiating effect of CHPG. In primary cultures of rat hippocampal and striatal neurons (ED 17, DIV 14), CHPG significantly potentiated NMDA-induced lactate dehydrogenase (LDH) release. Again, such an effect was prevented by ZM 241385. Our results show that A_2A and mGlu5 receptors functionally interact both in the hippocampus and in the striatum, even though different mechanisms seem to be involved in the two areas. The ability of A_2ARs to control mGlu5R-dependent effects may thus be a general feature of A_2ARs in different brain regions (irrespective of their density) and may represent an additional target for the development of therapeutic strategies against neurological disorders.     

Is the association between TNF-alpha-308 A allele and DMT1 independent of HLA-DRB1, DQB1 alleles?

The aim of the study was to assess chosen factors of genetic susceptibility to DMT1: DRB1, DQB1, and TNF-alpha polymorphisms-308 (G/A) in children with DMT1 and their up-to-now healthy siblings. Then we tested whether the association between TNF-alpha genes and DMT1 is independent of HLA. 87 diabetic children, their 78 siblings, and 85 persons from healthy control group were followed up. The highest risk of DMT1 was connected with alleles: DRB1*0401 (OR = 3.39; CI: 1.55-7.41), DRB1*0301 (OR = 2.72; CI: 1.48-5.01), DQB1*0201 (OR = 4.04; CI: 2.17-7.52), DQB1*0302 (OR = 5.08; CI: 2.54-10.14), and TNF-alpha-308 A allele (OR = 2.59; CI: 1.23-5.44). Moreover linkage disequilibrium for TNF-alpha-308 A allele with DRB1*0301 and DQB1*0201 was observed in both diabetic children and their siblings. Diabetic children and their siblings present similar genetic risk factors for DMT1. The association between TNF-alpha-308 A allele and DMT1 is dependent of HLA-DRB1 and DQB1 alleles.     

MSH Receptors and the Response of Human A375 Melanoma Cells to Interleukin-1β

Abstract

α-Melanocyte-stimulating hormone (α-MSH, α-melanotropin) has been shown to be an inhibitory factor in many immunologic and inflammatory processes involving the cytokine interleukin-1 (IL-1). As the mechanism of the interaction between IL-1 and α-MSH at the receptor level is unknown, we have studied the role of MC1 melanocortin receptors in two variants of the human melanoma cell line A375 differing in their sensitivity to the cytostatic effects of IL-1β. Both IL-1 sensitive (A375r-) and resistant cells (A375r+) carry specific high affinity receptors for IL-1, albeit their concentration is 10-fold higher in A375r+ cells. In A375r- cells, MC1 receptors are absent or below the level for reliable detection in the binding assay. Conversion of A375r- to A375r+ cells by prolonged culture in medium not depleted of endotoxin led to the appearance of MC1 receptors (K_D 0.4 ± 0.123 nmol/l; 608 ± 134 receptors/cell). Stable transfection of A375r- cells with the human MC1 receptor did not, however, render them resistant to the cytostatic effect of IL-1β on concomitant treatment with α-MSH or result in the production of IL-6 on treatment with IL-1β Therefore, the presence of MC1 receptors on the surface of A375 cells or their binding to α-MSH does not seem to be a factor in cytokine resistance or IL-6 secretion. No interaction between IL-1β and α-MSH could be demonstrated at the cellular level in this melanoma cell line.     

Biochemical inhibition of the acetyltransferases ATase1 and ATase2 reduces β-secretase (BACE1) levels and Aβ generation

The cellular levels of β-site APP cleaving enzyme 1 (BACE1), the rate-limiting enzyme for the generation of the Alzheimer disease (AD) amyloid β-peptide (Aβ), are tightly regulated by two ER-based acetyl-CoA:lysine acetyltransferases, ATase1 and ATase2. Here we report that both acetyltransferases are expressed in neurons and glial cells, and are up-regulated in the brain of AD patients. We also report the identification of first and second generation compounds that inhibit ATase1/ATase2 and down-regulate the expression levels as well as activity of BACE1. The mechanism of action involves competitive and non-competitive inhibition as well as generation of unstable intermediates of the ATases that undergo degradation.     

A DFT study on the mechanisms for the cycloaddition reactions between 1−aza-2-azoniaallene cations and acetylenes

The mechanisms of cycloaddition reactions between 1-aza-2-azoniaallene cations 1 and acetylenes 2 have been investigated using the global electrophilicity and nucleophilicity of the corresponding reactants as global reactivity indexes defined within the conceptual density functional theory. The reactivity and regioselectivity of these reactions were predicted by analysis of the energies, geometries, and electronic nature of the transition state structures. The theoretical results revealed that the reaction features a tandem process: an ionic 1,3-dipolar cycloaddition to produce the cycloadducts 3?H-pyrazolium salts 3 followed by a [1,2]-shift affording the thermodynamically more stable adducts 4 or 5. The mechanism of the cycloaddition reactions can be described as an asynchronous concerted pathway with reverse electron demand. The model reaction has also been investigated at the QCISD/6-31++G(d,p) and CCSD(T)/6-31++G(d,p)//B3LYP/6-31++G(d,p) levels as well as by the DFT. The polarizable continuum model, at the B3LYP/6-31++G(d,p) level of theory, was used to study solvent effects on all the studied reactions. In solvent dichloromethane, all the initial cycloadducts 3 were obtained via direct ionic process as the result of the solvent effect. The consecutive [1,2]-shift reaction, in which intermediates 3 are rearranged to the five-membered heterocycles 4/5, is proved to be a kinetically controlled reaction, and the regioselectivity can be modulated by varying the migrant. The LOL function and RDG function based on localized electron analysis were used to analysis the covalent bond and noncovalent interactions in order to unravel the mechanism of the title reactions.     

Involvement of Cytochrome P450 1A1 and Glutathione S-Transferase P1 Polymorphisms and Promoter Hypermethylation in the Progression of Anti-Tuberculosis Drug-Induced Liver Injury: A Case–Control Study

Background

Anti-tuberculosis (anti-TB) drug-induced liver injury (ADLI) is one of the most common adverse effects associated with TB treatment. Cytochrome P450 (CYP) 1A1 and glutathione S-transferase (GST) P1 are important phase I/II metabolizing enzymes involved in drug metabolism and detoxification. Genetic polymorphism and CpG island methylation have been reported as factors influencing the expression of CYP1A1 and GSTP1.

Objective

This study aimed to determine the potential relationships of CYP1A1 and GSTP1 polymorphisms and CpG island methylation with ADLI risk.

Design

This was a population-based one-to-one matched case–control study.

Setting

The subjects were patients with TB receiving treatment in China from December 2010 to June 2013.

Patients

In total, 127 patients with TB and ADLI (case group) and 127 patients with TB but without liver injury (control group) were included in this study. Subjects were matched in terms of sex, age, and therapeutic regimen.

Methods

The general condition of each patient was assessed using questionnaires. The CYP1A1 MspI and GSTP1 Ile105Val polymorphisms as well as methylation status were detected by polymerase chain reaction (PCR)–restriction fragment length polymorphism and the methylation-specific PCR method.

Results

We found no significant difference in GSTP1 and CYP1A1 genotypes between the two groups, probably because the sample size was not large enough; however, patients with ADLI had significantly higher GSTP1 and CYP1A1 promoter methylation rates than control subjects [odds ratio (OR) = 2.467 and 2.000, respectively]. After adjusting for drinking, which significantly differed between the groups as per univariate analysis, we found that hypermethylation of GSTP1 and CYP1A1 promoters was associated with ADLI (OR = 2.645 and 2.090, respectively).

Conclusion

Hypermethylation of CpG islands of GSTP1 and CYP1A1 promoters may thus play important roles in the development of ADLI and provide evidence of being used as novel markers for ADLI risk prediction.     

The Light Chains of Microtubule-Associated Proteins MAP1A and MAP1B Interact with α1-Syntrophin in the Central and Peripheral Nervous System

Microtubule-associated proteins of the MAP1 family (MAP1A, MAP1B, and MAP1S) share, among other features, a highly conserved COOH-terminal domain approximately 125 amino acids in length. We conducted a yeast 2-hybrid screen to search for proteins interacting with this domain and identified α1-syntrophin, a member of a multigene family of adapter proteins involved in signal transduction. We further demonstrate that the interaction between the conserved COOH-terminal 125-amino acid domain (which is located in the light chains of MAP1A, MAP1B, and MAP1S) and α1-syntrophin is direct and occurs through the pleckstrin homology domain 2 (PH2) and the postsynaptic density protein 95/disk large/zonula occludens-1 protein homology domain (PDZ) of α1-syntrophin. We confirmed the interaction of MAP1B and α1-syntrophin by co-localization of the two proteins in transfected cells and by co-immunoprecipitation experiments from mouse brain. In addition, we show that MAP1B and α1-syntrophin partially co-localize in Schwann cells of the murine sciatic nerve during postnatal development and in the adult. However, intracellular localization of α1-syntrophin and other Schwann cell proteins such as ezrin and dystrophin-related protein 2 (DRP2) and the localization of the axonal node of Ranvier-associated protein Caspr1/paranodin were not affected in MAP1B null mice. Our findings add to a growing body of evidence that classical MAPs are likely to be involved in signal transduction not only by directly modulating microtubule function, but also through their interaction with signal transduction proteins.