Traité de l'autorité des deux puissances.

Manuscript notes on front fly-leaf of vol. I.

Julii Bernhards von Rohr Einleitung zu der allgemeinen Land- und Feld-Wirthschaffts-Kunst derer Teutschen : darinnen die allgemeinen Regeln und Anmerckungen die so wohl bey der Land und Feld-Oeconomie überhaupt als insonderheit [b]ey dem Ackerbau, der Viehzucht, Gärtne[re]y ... und Fischereyen fast in allen Provinzien Teutschlandes in acht zu nehmen [i]n einer guten Ordnung ohne Einmischung unnöthiger Sachen vorgetragen werden.

Goldsmiths'-Kress no. 05628.10, suppl.

Poems.

[Bookplate of Anatole de Montaiglon and his monogram on the binding.]

Flavii Aviani fabulae ad ms. cd. collatae.

Parchment binding.

Manual for the use of the Legislature of the State of New York.

Part of the illustrative material is folded.

Phosphate forms an unusual tripodal complex with the Fe-Mn center of sweet potato purple acid phosphatase

Purple acid phosphatases (PAPs) are a family of binuclear metalloenzymes that catalyze the hydrolysis of phosphoric acid esters and anhydrides. A PAP in sweet potato has a unique, strongly antiferromagnetically coupled Fe(III)-Mn(II) center and is distinguished from other PAPs by its increased catalytic efficiency for a range of activated and unactivated phosphate esters, its strict requirement for Mn(II), and the presence of a mu-oxo bridge at pH 4.90. This enzyme displays maximum catalytic efficiency (k(cat)/K-m) at pH 4.5, whereas its catalytic rate constant (k(cat)) is maximal at near-neutral pH, and, in contrast to other PAPs, its catalytic parameters are not dependent on the pK(a) of the leaving group. The crystal structure of the phosphate-bound Fe(III)-Mn(II) PAP has been determined to 2.5-Angstrom resolution (final R-free value of 0.256). Structural comparisons of the active site of sweet potato, red kidney bean, and mammalian PAPs show several amino acid substitutions in the sweet potato enzyme that can account for its increased catalytic efficiency. The phosphate molecule binds in an unusual tripodal mode to the two metal ions, with two of the phosphate oxygen atoms binding to Fe(III) and Mn(II), a third oxygen atom bridging the two metal ions, and the fourth oxygen pointing toward the substrate binding pocket. This binding mode is unique among the known structures in this family but is reminiscent of phosphate binding to urease and of sulfate binding to A protein phosphatase. The structure and kinetics support the hypothesis that the bridging oxygen atom initiates hydrolysis.

New insights into the interactions of serum proteins with bis(maltolato)oxovanadium(IV): Transport and biotransformation of insulin-enhancing vanadium pharmaceuticals

Significant new insights into the interactions of the potent insulin-enhancing compound bis(maltolato)oxovanadium(IV) (BMOV) with the serum proteins, apo-transferrin and albumin, are presented. Identical reaction products are observed by electron paramagnetic resonance (EPR) with either BMOV or vanadyl sulfate (VOSO4) in solutions of human serum apo-transferrin. Further detailed study rules out the presence of a ternary ligand-vanadyl-transferrin complex proposed previously. By contrast, differences in reaction products are observed for the interactions of BMOV and VOSO4 with human serum albumin (HSA), wherein adduct formation between albumin and BMOV is detected. In BMOV-albumin solutions, vanadyl ions are bound in a unique manner not observed in comparable solutions Of VOSO4 and albumin. Presentation of chelated vanadyl ions precludes binding at the numerous nonspecific sites and produces a unique EPR spectrum which is assigned to a BMOV-HSA adduct. The adduct species cannot be produced, however, from a solution Of VOSO4 and HSA titrated with maltol. Addition of maltol to a VOSO4-HSA solution instead results in formation of a different end product which has been assigned as a ternary complex, VO(ma)(HSA). Furthermore, analysis of solution equilibria using a model system of BMOV with 1-methylimidazole (formation constant log K = 4.5(1), by difference electronic absorption spectroscopy) lends support to an adduct binding mode (VO(ma)(2)-HSA) proposed herein for BMOV and HSA. This detailed report of an in vitro reactivity difference between VOSO4 and BMOV may have bearing on the form of active vanadium metabolites delivered to target tissues. Albumin binding of vanadium chelates is seen to have a potentially dramatic effect on pharmacokinetics, transport, and efficacy of these antidiabetic chelates.

Molecular dissection of the Munc18c/syntaxin4 interaction: Implications for regulation of membrane trafficking

Sec1p/Munc18 (SM) proteins are believed to play an integral role in vesicle transport through their interaction with SNAREs. Different SM proteins have been shown to interact with SNAREs via different mechanisms, leading to the conclusion that their function has diverged. To further explore this notion, in this study, we have examined the molecular interactions between Munc18c and its cognate SNAREs as these molecules are ubiquitously expressed in mammals and likely regulate a universal plasma membrane trafficking step. Thus, Munc18c binds to monomeric syntaxin4 and the N-terminal 29 amino acids of syntaxin4 are necessary for this interaction. We identified key residues in Munc18c and syntaxin4 that determine the N-terminal interaction and that are consistent with the N-terminal binding mode of yeast proteins Sly1p and Sed5p. In addition, Munc18c binds to the syntaxin4/SNAP23/VAMP2 SNARE complex. Pre-assembly of the syntaxin4/Munc18c dimer accelerates the formation of SNARE complex compared to assembly with syntaxin4 alone. These data suggest that Munc18c interacts with its cognate SNAREs in a manner that resembles the yeast proteins Sly1p and Sed5p rather than the mammalian neuronal proteins Munc18a and syntaxin1a. The Munc18c-SNARE interactions described here imply that Munc18c could play a positive regulatory role in SNARE assembly.

Single-molecule fluorescence resonance energy transfer study of SNARE-mediated membrane fusion

This is a comprehensive study of protein-mediated membrane fusion through single-molecule fluorescence resonance energy transfer (smFRET). Membrane fusion is one of the important cellular processes by which two initially distinct lipid bilayers merge their hydrophobic cores, resulting in one interconnected structure. For example, exocytosis, fertilization of an egg by a sperm and communication between neurons are a few among many processes that rely on some form of fusion. Proteins called soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) play a central role in fusion processes which is also regulated by many accessory proteins, such as synaptotagmin, complexin and Munc18. By a new lipid mixing method at the single-vesicle level, we are able to accurately detect different stages of SNARE-mediated membrane fusion including docking, hemi and full fusion via FRET value of single donor/acceptor vesicle pair. Through this single-vesicle lipid mixing assay, we discovered the vesicle aggregation induced by C2AB/Ca2+, the dual function of complexin, and the fusion promotion role of Munc18/SNARE-core binding mode. While this new method provides the information regarding the extent of the ensemble lipid mixing, the fusion pore opening between two vesicular cavities and the interaction between proteins cannot be detected. In order to overcome these limitations, we then developed a single-vesicle content mixing method to reveal the key factor of pore expansion by detecting the FRET change of dual-labeled DNA probes encapsulated in vesicles. Through our single-vesicle content mixing assay, we found the fusion pore expansion role of yeast SNAREs as well as neuronal SNAREs plus synaptotagmin 1.

New cholinesterase inhibitors for Alzheimer's disease: Structure Activity Studies (SARs) and molecular docking of isoquinolone and azepanone derivatives

A library of isoquinolinone and azepanone derivatives were screened for both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activity. The strategy adopted included (a) in vitro biological assays, against eel AChE (EeAChE) and equine serum BuChE (EqBuChE) in order to determine the compounds IC50 and their dose-response activity, consolidated by (b) molecular docking studies to evaluate the docking poses and interatomic interactions in the case of the hit compounds, validated by STD-NMR studies. Compound (1f) was identified as one of these hits with an IC50 of 89.5 mu M for EeAChE and 153.8 mu M for EqBuChE, (2a) was identified as a second hit with an IC50 of 108.4 mu M (EeAChE) and 277.8 mu M (EqBuChE). In order to gain insights into the binding mode and principle active site interactions of these molecules, (R)-(1f) along with 3 other analogues (also as the R-enantiomer) were docked into both RhAChE and hBuChE models. Galantamine was used as the benchmark. The docking study was validated by performing an STD-NMR study of (1f) with EeAChE using galantamine as the benchmark.

Functional dynamics of PDZ binding domains: a normal-mode analysis.

Postsynaptic density-95/disks large/zonula occludens-1 (PDZ) domains are relatively small (80-120 residues) protein binding modules central in the organization of receptor clusters and in the association of cellular proteins. Their main function is to bind C-terminals of selected proteins that are recognized through specific amino acids in their carboxyl end. Binding is associated with a deformation of the PDZ native structure and is responsible for dynamical changes in regions not in direct contact with the target. We investigate how this deformation is related to the harmonic dynamics of the PDZ structure and show that one low-frequency collective normal mode, characterized by the concerted movements of different secondary structures, is involved in the binding process. Our results suggest that even minimal structural changes are responsible for communication between distant regions of the protein, in agreement with recent NMR experiments. Thus, PDZ domains are a very clear example of how collective normal modes are able to characterize the relation between function and dynamics of proteins, and to provide indications on the precursors of binding/unbinding events.

Structural Insight into the Mode of Action of a Direct Inhibitor of Coregulator Binding to the Thyroid Hormone Receptor.

The development of nuclear hormone receptor antagonists that directly inhibit the association of the receptor with its essential coactivators would allow useful manipulation of nuclear hormone receptor signaling. We previously identified 3-(dibutylamino)-1-(4-hexylphenyl)-propan-1-one (DHPPA), an aromatic β-amino ketone that inhibits coactivator recruitment to thyroid hormone receptor β (TRβ), in a high-throughput screen. Initial evidence suggested that the aromatic β-enone 1-(4-hexylphenyl)-prop-2-en-1-one (HPPE), which alkylates a specific cysteine residue on the TRβ surface, is liberated from DHPPA. Nevertheless, aspects of the mechanism and specificity of action of DHPPA remained unclear. Here, we report an x-ray structure of TRβ with the inhibitor HPPE at 2.3-Å resolution. Unreacted HPPE is located at the interface that normally mediates binding between TRβ and its coactivator. Several lines of evidence, including experiments with TRβ mutants and mass spectroscopic analysis, showed that HPPE specifically alkylates cysteine residue 298 of TRβ, which is located near the activation function-2 pocket. We propose that this covalent adduct formation proceeds through a two-step mechanism: 1) β-elimination to form HPPE; and 2) a covalent bond slowly forms between HPPE and TRβ. DHPPA represents a novel class of potent TRβ antagonist, and its crystal structure suggests new ways to design antagonists that target the assembly of nuclear hormone receptor gene-regulatory complexes and block transcription.

Structural Insight into the Mode of Action of a Direct Inhibitor of Coregulator Binding to the Thyroid Hormone Receptor.

The development of nuclear hormone receptor antagonists that directly inhibit the association of the receptor with its essential coactivators would allow useful manipulation of nuclear hormone receptor signaling. We previously identified 3-(dibutylamino)-1-(4-hexylphenyl)-propan-1-one (DHPPA), an aromatic β-amino ketone that inhibits coactivator recruitment to thyroid hormone receptor β (TRβ), in a high-throughput screen. Initial evidence suggested that the aromatic β-enone 1-(4-hexylphenyl)-prop-2-en-1-one (HPPE), which alkylates a specific cysteine residue on the TRβ surface, is liberated from DHPPA. Nevertheless, aspects of the mechanism and specificity of action of DHPPA remained unclear. Here, we report an x-ray structure of TRβ with the inhibitor HPPE at 2.3-Å resolution. Unreacted HPPE is located at the interface that normally mediates binding between TRβ and its coactivator. Several lines of evidence, including experiments with TRβ mutants and mass spectroscopic analysis, showed that HPPE specifically alkylates cysteine residue 298 of TRβ, which is located near the activation function-2 pocket. We propose that this covalent adduct formation proceeds through a two-step mechanism: 1) β-elimination to form HPPE; and 2) a covalent bond slowly forms between HPPE and TRβ. DHPPA represents a novel class of potent TRβ antagonist, and its crystal structure suggests new ways to design antagonists that target the assembly of nuclear hormone receptor gene-regulatory complexes and block transcription.

Do N-arachidonyl-glycine (NA-glycine) and 2-arachidonoyl glycerol (2-AG) share mode of action and the binding site on the β 2 subunit of GABA A receptors?

NA-glycine is an endogenous lipid molecule with analgesic properties, which is structurally similar to the endocannabinoids 2-AG and anandamide but does not interact with cannabinoid receptors. NA-glycine has been suggested to act at the G-protein coupled receptors GPR18 and GPR92. Recently, we have described that NA-glycine can also modulate recombinant α1β2γ2 GABAA receptors. Here we characterize in more detail this modulation and investigate the relationship of its binding site with that of the endocannabinoid 2-AG.