Kinetics and Equilibrium Studies of Tet Repressor–Operator Interaction

Binding of a Tet repressor mutant containing a single Trp43 residue in the tet operator recognition α-helix leads to the quenching of the protein fluorescence down to about 23% in the case of the tet O₁ operator and to 40% in the case of the tet O₂ operator. We have used fluorescence detection to describe the binding equilibrium and kinetics of the Tet repressor interaction with the 20-bp DNA operators tet O₁ and tet O₂. Stopped-flow measurements in an excess of the tet operators performed in 5 mM NaCl or 150 mM NaCl indicate that the reaction can be described by at least three exponentials characterized by different relaxation times. The mechanism of interaction for both operators as well as for two salt concentrations used can be described as TetR + Operator ? Complex 1 ? Complex 2 ? Complex 3. Only the much faster process can be described as a second-order reaction characterized by a bimolecular rate constant equal to 2.8 × 10⁶ M^?1 sec^?1 for both operators. The medium and slow processes may be described by relaxational times ranging from 50 msec to seconds. The results of the binding equilibrium measurements extrapolated to 1 M NaCl concentration, which reflects the specific nonionic interaction between TetR and tet operators, indicate K _as equal to 3.2 × 10⁴ and 4.0 × 10⁵ M^?1 for tet O₁ and tet O₂, respectively. The number of monovalent ions replaced upon binding can be calculated as about 5 and 3 for tet O₁ and tet O₂, respectively. The binding of Tet repressor to the operators leads to changes in the circular dichroism spectra of the DNA which could indicate transitions of B-DNA into A-like DNA structure.     

An affinity-column procedure for the purification of veratrate O-demethylase from fungi.

An affinity column procedure is reported for purifying veratrate O-demethylase from higher fungi. The procedure is based on the affinity of the fungal demethylases for veratrate, which was coupled to AH-Sepharose 4B. An over 300-fold purification of the enzyme from an Ascomycete (Chaetomium piluliferum), and a lower degree of purification (20-fold) from a Basidiomycete (Xerocomus badius), were obtained. The O-demethylases from higher fungi require NADH and oxygen. The enzyme activity is sensitive to exposure to oxygen. The pH optima are 5 for enzyme from Chaetomium, and 7 for demethylase from Xerocomus, respectively. The enzymes are not specific for veratrate. They also demethylate p- and m-anisate and 3,4-dimethoxycinnamate, but to a lower degree.     

Fluorescence and Phosphorescence Study of Tet Repressor–Operator Interaction

Fluorescence and phosphorescence measurements have been carried out on single-p tryptophan (Trp 43 or Trp 75)-containing mutants of Tet repressor (Tet R). Tet R containing Trp 43, the residue localized in the DNA recognition helix of the repressor, has been used to observe the binding of Tet R to two 20-bp DNA sequences of tet O₁ and tet O₂ operators. Binding of Tet R to tet O₁ operator leads to a 78% decrease of the repressor fluorescence intensity, with an accompanying 20-nm blue shift of its fluorescence emission maximum to 330 nm. Upon binding of Tet R to tet O₂ operator, the Trp 43 fluorescence intensity is quenched by 60%, and a 10-nm shift of its emission maximum to 340 nm occurs. Solute fluorescence quenching studies, using acrylamide, performed at low ionic strength indicate that in both the complex of Tet R with the O₁ and that with the O₂ operator, Trp 43 is moderately buried, as indicated by a bimolecular rate quenching constant of about 1.8 × 10⁹ M^–1 sec^–1. In contrast to the Tet R–tet O₂ complex, the Stern–Volmer acrylamide quenching constant K _sv of the complex with tet O₁ operator changes from 7.5 M^–1 at 5 mM NaCl to 22 M^–1 at 200 mM NaCl, indicating different exposures of Trp 43 in the two complexes in solutions of higher ionic strength. Phosphorescence studies showed a 0–0 vibronic transition at 408 and 403 nm for Trp 43 and Trp 75, respectively. Upon binding of Tet R to the tet operators, we observed red shifts of 0–0 vibronic bands of Trp 43 to 413 and 412 nm for tet O₁ and tet O₂ operator, respectively, and the phosphorescence triplet lifetime of Trp 43 at 75 K was quenched from 6.0–5.5 to 3.5–3.3 sec. The thermal phosphorescence quenching profile ranged from –200°C to –20°C, and differed drastically for the two complexes, suggesting different dynamics of the microenvironment of the Trp 43 residue. The luminescence data for Trp 43 of Tet R suggest that the recognition helix of Tet R interacts in different fashions with the tet O₁ and tet O₂ operators.     

Alpha-synuclein-induced aggregation of cytoplasmic vesicles in Saccharomyces cerevisiae

Aggregated alpha-synuclein (alpha-syn) fibrils form Lewy bodies (LBs), the signature lesions of Parkinson's disease (PD) and related synucleinopathies, but the pathogenesis and neurodegenerative effects of LBs remain enigmatic. Recent studies have shown that when overexpressed in Saccharomyces cerevisiae, alpha-syn localizes to plasma membranes and forms cytoplasmic accumulations similar to human alpha-syn inclusions. However, the exact nature, composition, temporal evolution, and underlying mechanisms of yeast alpha-syn accumulations and their relevance to human synucleinopathies are unknown. Here we provide ultrastructural evidence that alpha-syn accumulations are not comprised of LB-like fibrils, but are associated with clusters of vesicles. Live-cell imaging showed alpha-syn initially localized to the plasma membrane and subsequently formed accumulations in association with vesicles. Imaging of truncated and mutant forms of alpha-syn revealed the molecular determinants and vesicular trafficking pathways underlying this pathological process. Because vesicular clustering is also found in LB-containing neurons of PD brains, alpha-syn-mediated vesicular accumulation in yeast represents a model system to study specific aspects of neurodegeneration in PD and related synucleinopathies.     

TDP-43 proteinopathies: neurodegenerative protein misfolding diseases without amyloidosis

In this review, we summarize recent advances in understanding frontotemporal lobar degeneration (FTLD), amyotrophic lateral sclerosis (ALS) and related neurodegenerative disorders that are collectively known as TDP-43 proteinopathies, since transactive response DNA-binding protein 43 (TDP-43) was recently shown to be the major component of the ubiquitinated inclusions that are their pathological hallmarks. TDP-43 proteinopathies are distinct from most other neurodegenerative disorders because TDP-43 inclusions are not amyloid deposits. Besides TDP-43-positive inclusions, both sporadic and familial forms of FTLD and ALS have the pathologic TDP-43 signature of abnormal hyperphosphorylation, ubiquitination and C-terminal fragments in affected brain and spinal cord, suggesting that they share a common mechanism of pathogenesis. Thus, these findings support the concept that FTLD and ALS represent a clinicopathologic spectrum of one disease, that is, TDP-43 proteinopathy.     

Structural and magnetic characteristics of tetramethylammonium tetrahalogenoferrates(III)

A series of tetramethylammonium tetrahalogenoferrates(III), [FeBr_4−nCl_n]⁻ (n = 0, 1, 3, 4), of general formula [(CH₃)₄N][FeBr_4−nCl_n], have been synthesized. The crystal and molecular structures of [(CH₃)₄N][FeCl₄] were determined. The compound is isostructural with its [FeBr_4−nCl_n]⁻ (n = 0, 1, 3, 4) analogues. Magnetic measurements of the powdered samples of [(CH₃)₄N][FeBr_4−nCl_n] gave negative values of the Weiss constant, which suggest antiferromagnetic coupling. The strength of the antiferromagnetic interactions strongly depends on the kind of halide ligands in the coordination sphere of iron(III) and increases with an increasing number of the bromide anions.     

Atrial natriuretic factor receptor guanylate cyclase signaling: new ATP-regulated transduction motif

ANF-RGC membrane guanylate cyclase is the receptor for the hypotensive peptide hormones, atrial natriuretic factor (ANF) and type B natriuretic peptide (BNP). It is a single transmembrane spanning protein. Binding the hormone to the extracellular domain activates its intracellular catalytic domain. This results in accelerated production of cyclic GMP, a second messenger in controlling blood pressure, cardiac vasculature, and fluid secretion. ATP is the obligatory transducer of the ANF signal. It works through its ATP regulated module, ARM, which is juxtaposed to the C-terminal side of the transmembrane domain. Upon interaction, ATP induces a cascade of temporal and spatial changes in the ARM, which, finally, result in activation of the catalytic module. Although the exact nature and the details of these changes are not known, some of these have been stereographed in the simulated three-dimensional model of the ARM and validated biochemically. Through comprehensive techniques of steady state, time-resolved tryptophan fluorescence and Forster Resonance Energy Transfer (FRET), site-directed and deletion-mutagenesis, and reconstitution, the present study validates and explains the mechanism of the model-based predicted transduction role of the ARM’s structural motif, ⁶⁶⁹WTAPELL⁶⁷⁵. This motif is critical in the ATP-dependent ANF signaling. Molecular modeling shows that ATP binding exposes the ⁶⁶⁹WTAPELL⁶⁷⁵ motif, the exposure, in turn, facilitates its interaction and activation of the catalytic module. These principles of the model have been experimentally validated. This knowledge brings us a step closer to our understanding of the mechanism by which the ATP-dependent spatial changes within the ARM cause ANF signaling of ANF-RGC.     

Tetraiodobenzimidazoles are potent inhibitors of protein kinase CK2

A series of novel iodinated benzimidazoles have been prepared by iodination of respective benzimidazole with iodine and periodic acid in sulfuric acid solution. Additionally several 2-substituted- and N-1-carboxymethyl-substituted derivatives of 4,5,6,7-tetraiodobenzimidazole (TIBI) were obtained. For sake of comparison, some new 4,5,6,7-tetrabromobenzimidazoles were also synthesized. The ability of the new compounds to inhibit protein kinase CK2 has been evaluated. The results show that 4,5,6,7-tetraiodobenzimidazoles are more powerful inhibitors of CK2 than their tetrabrominated analogs. Molecular modeling supports the experimental data showing that tetraiodobenzimidazole moiety fills better the binding pocket than respective tetrabromo and tetrachlorocompounds. To note that 4,5,6,7-tetraiodobenzimidazole (TIBI) is one of the most efficient CK2 inhibitors (K_i = 23 nM) described to date.     

TRPV1 in Brain Is Involved in Acetaminophen-Induced Antinociception

Background

Acetaminophen, the major active metabolite of acetanilide in man, has become one of the most popular over-the-counter analgesic and antipyretic agents, consumed by millions of people daily. However, its mechanism of action is still a matter of debate. We have previously shown that acetaminophen is further metabolized to N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z -eicosatetraenamide (AM404) by fatty acid amide hydrolase (FAAH) in the rat and mouse brain and that this metabolite is a potent activator of transient receptor potential vanilloid 1 (TRPV₁) in vitro. Pharmacological activation of TRPV₁ in the midbrain periaqueductal gray elicits antinociception in rats. It is therefore possible that activation of TRPV₁ in the brain contributes to the analgesic effect of acetaminophen.

Methodology/Principal Findings

Here we show that the antinociceptive effect of acetaminophen at an oral dose lacking hypolocomotor activity is absent in FAAH and TRPV₁ knockout mice in the formalin, tail immersion and von Frey tests. This dose of acetaminophen did not affect the global brain contents of prostaglandin E₂ (PGE₂) and endocannabinoids. Intracerebroventricular injection of AM404 produced a TRPV₁-mediated antinociceptive effect in the mouse formalin test. Pharmacological inhibition of TRPV₁ in the brain by intracerebroventricular capsazepine injection abolished the antinociceptive effect of oral acetaminophen in the same test.

Conclusions

This study shows that TRPV₁ in brain is involved in the antinociceptive action of acetaminophen and provides a strategy for developing central nervous system active oral analgesics based on the coexpression of FAAH and TRPV₁ in the brain.     

Interactions between <Emphasis Type="Italic">Glu-1</Emphasis> and <Emphasis Type="Italic">Glu-3</Emphasis> loci and associations of selected molecular markers with quality traits in winter wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) DH lines

The quality of wheat depends on a large complex of genes and environmental factors. The objective of this study was to identify quantitative trait loci controlling technological quality traits and their stability across environments, and to assess the impact of interaction between alleles at loci Glu-1 and Glu-3 on grain quality. DH lines were evaluated in field experiments over a period of 4 years, and genotyped using simple sequence repeat markers. Lines were analysed for grain yield (GY), thousand grain weight (TGW), protein content (PC), starch content (SC), wet gluten content (WG), Zeleny sedimentation value (ZS), alveograph parameter W (APW), hectolitre weight (HW), and grain hardness (GH). A number of QTLs for these traits were identified in all chromosome groups. The Glu-D1 locus influenced TGW, PC, SC, WG, ZS, APW, GH, while locus Glu-B1 affected only PC, ZS, and WG. Most important marker-trait associations were found on chromosomes 1D and 5D. Significant effects of interaction between Glu-1 and Glu-3 loci on technological properties were recorded, and in all types of this interaction positive effects of Glu-D1 locus on grain quality were observed, whereas effects of Glu-B1 locus depended on alleles at Glu-3 loci. Effects of Glu-A3 and Glu-D3 loci per se were not significant, while their interaction with alleles present at other loci encoding HMW and LMW were important. These results indicate that selection of wheat genotypes with predicted good bread-making properties should be based on the allelic composition both in Glu-1 and Glu-3 loci, and confirm the predominant effect of Glu-D1d allele on technological properties of wheat grains.