Effects of α-Tubulin K40 Acetylation and Detyrosination on Kinesin-1 Motility in a Purified System

Long-range transport in cells is achieved primarily through motor-based transport along a network of microtubule tracks. Targeted transport by kinesin motors can be correlated with posttranslational modifications (PTMs) of the tubulin subunits in specific microtubules. To directly examine the influence of specific PTMs on kinesin-1 motility, we generated tubulin subunits that were either enriched in or lacking acetylation of α-tubulin lysine 40 (K40) or detyrosination of the α-tubulin C-terminal tail. We show that K40 acetylation does not result in significant changes in kinesin-1’s landing rate or motility parameters (velocity and run length) across experimental conditions. In contrast, detyrosination causes a moderate increase in kinesin-1’s landing rate. The fact that the effects of detyrosination are dampened by prior K40 acetylation indicates that the combination of PTMs may be an important aspect of the functional output of microtubule heterogeneity. Importantly, our results indicate that the moderate influences that single PTMs have on kinesin-1 in vitro do not explain the strong correlation between specific PTMs and kinesin-1 transport in cells. Thus, additional mechanisms for regulating kinesin-1 transport in cells must be explored in future work.     

Effects of body size and environment on diet-tissue δN fractionation in fishes

Nitrogen stable isotope natural abundance data are often used in trophodynamic research. The assumed nitrogen diet-tissue fractionation (Δδ¹⁵N) determines conclusions about trophic level, potential food sources and ontogenetic diet shifts. Δδ¹⁵N is usually assumed to be 3.0-3.4‰ per trophic level and unaffected by the size or age of animals or their environment. To assess the effects of body size, experimental duration and environmental conditions on fish tissue Δδ¹⁵N, two populations of European sea bass (Dicentrarchus labrax) were reared on constant diets of dab (Limanda limanda) muscle or sandeel (Ammodytes marinus) for 2 years under natural light and temperature regimes. Bass were sampled at approximately monthly intervals to determine Δδ¹⁵N for muscle, heart and liver tissue. Mean values of Δδ¹⁵N were 3.83‰, 3.54‰, 2.05‰ (sandeel diet) and 3.98‰, 3.32‰, 1.95‰ (dab diet) for muscle, heart and liver tissue respectively. The assumption that fractionation was independent of body mass was upheld for muscle and heart tissue, but not for liver. Time effects on muscle Δδ¹⁵N were explainable by a sinusoidal function with a period of 1 year and wave height ∼ 0.3‰. Time resulted in increases in heart δ¹⁵N and decreases in liver δ¹⁵N which were small compared to background variation, equating to 1/6 of a trophic level over 2 years, and unlikely to have great significance in ecological studies. Heart and liver δ¹⁵N were also affected by temperature probably reflecting the metabolic functions of these tissues and their associated rates of turnover. However in heart the explanatory power of temperature appeared tied to that of time. Although the Δδ¹⁵N for bass muscle on both diets approached 4‰, the Δδ¹⁵N values from this study, when combined with those from the literature, suggest that where fish species specific data are not available, a mean Δδ¹⁵N for fish muscle of 3.2‰ should be applied (mean white muscle Δδ¹⁵N = 3.15). The literature based mean Δδ¹⁵N for whole fish was lower than that of white muscle suggesting that a separate Δδ¹⁵N (2.9‰) should be applied when sampling whole fish.     

Effects of body size and environment on diet-tissue δC fractionation in fishes

δ¹³C data are often used in trophodynamic research where diet-tissue fractionation (Δδ¹³C) is assumed to be 0-1‰ per trophic level and unaffected by the size of animals or their environment. Variation in Δδ¹³C will influence conclusions about food sources, energy pathways and trophic level. To assess the effects of body size, age and environmental conditions on Δδ¹³C, European sea bass (Dicentrarchus labrax) were reared on constant diets of dab (Limanda limanda) or (Ammodytes marinus) for 2years under natural environmental regimes. Bass were sampled approximately monthly to determine Δδ¹³C for muscle, heart and liver tissue and were 1.66‰, − 0.18‰, − 1.77‰ (sandeel diet) and 1.34‰, − 1.18‰, − 1.75‰ (dab diet) respectively. Arithmetic lipid correction increased Δδ¹³C to > 2‰ for muscle and liver. Δδ¹³C was dependent on body mass and experimental duration (age) and generally declined with weight or time even after correction for lipid content. For liver, increasing temperature increased Δδ¹³C. The Δδ¹³C estimates from this study were compared with all available published Δδ¹³C estimates for fish. Bass muscle Δδ¹³C was similar to previous estimates for fish white muscle Δδ¹³C (1.56 ± 1.10‰) and whole body Δδ¹³C (1.52 ± 1.13‰). Fractionations derived in this study, combined with those from the literature, support the use of diet-tissue fractionation values of between 1‰-2‰ for δ¹³C, rather than the commonly used 0‰ − 1‰. For muscle Δδ¹³C, 1.5‰ is appropriate.     

Conformational heterogeneity of α-synuclein in membrane

α-Synuclein (αS) is a natively disordered protein in solution, thought to be involved in the fusion of neurotransmitter vesicles to cellular membranes during neurotransmission. Monomeric αS has been previously characterized in two distinct membrane-associated conformations: a broken-helix structure, and an extended helix. By employing atomistic molecular dynamics and a novel membrane representation with significantly enhanced lipid mobility (HMMM), we investigate the process of spontaneous membrane binding of αS and the conformational dynamics of monomeric αS in its membrane-bound form.     

Acetylation of α-Tubulin on Lys40 Is a Widespread Post-Translational Modification in Angiosperms

Acetylation of α-tubulin on Lys⁴⁰ is thought to be a modification that regulates the dynamic instability of microtubules, but little is known about the occurrence of α-tubulin acetylation in plants. Here we report on a growth stage-dependent change in levels of α-tubulin acetylation and the organ distribution of acetylated α-tubulin in Arabidopsis thaliana plants. Widespread occurrence of α-tubulin acetylation in the leaves of 15 species (20 cultivars) of angiosperms was also confirmed. Our data indicate that acetylated α-tubulin is widespread in many angiosperms, but levels can differ, sometimes considerably, among different organs and developmental stages.     

Effects of β-Cyclodextrin on the Structure of Sphingomyelin/Cholesterol Model Membranes

The interaction of β-cyclodextrin (β-CD) with mixed bilayers composed of sphingomylein and cholesterol (Chol) above and below the accepted stable complexation ratio (67:33) was investigated. Membranes with the same (symmetric) and different (asymmetric) compositions in their inner and outer leaflets were deposited at surface pressures of 20, 30, and 40 mN/m at the solid-liquid interface. Using neutron reflectometry, membranes of various global molar ratios (defined as the sum of the molar ratios of the inner and outer leaflets), were characterized before and after β-CD was added to the subphase. The structure of bilayers with global molar ratios at or above the stable complexation ratio was unchanged by β-CD, indicating that β-CD is unable to remove sphingomyelin or complexed Chol. However, β-CD removed all uncomplexed Chol from bilayers composed of global molar ratios below the stable complexation ratio. The removal of Chol by β-CD was independent of the initial structure of the membranes as deposited, suggesting that asymmetric membranes homogenize by the exchange of molecules between leaflets. The interaction of β-CD with the aforementioned membranes was independent of the deposition surface pressure except for a symmetric 50:50 membrane deposited at 40 mN/m. The scattering from 50:50 bilayers with higher packing densities (deposited at 40 mN/m) was unaffected by β-CD, suggesting that the removal of Chol can depend on both the composition and packing density of the membrane.     

A 3D Structure Model of Integrin α4β1 Complex: I. Construction of a Homology Model of β1 and Ligand Binding Analysis

It is well established that integrin α4β1 binds to the vascular cell adhesion molecule (VCAM) and fibronectin and plays an important role in signal transduction. Blocking the binding of VCAM to α4β1 is thought to be a way of controlling a number of disease processes. To better understand how various inhibitors might block the interaction of VCAM and fibronectin with α4β1, we began constructing a structure model for the integrin α4β1 complex. As the first step, we have built a homology model of the β1 subunit based on the I domain of the integrin CD11B subunit. The model, including a bound Mg²⁺ ion, was optimized through a specially designed relaxation scheme involving restrained minimization and dynamics steps. The native ligand VCAM and two highly active small molecules (TBC772 and TBC3486) shown to inhibit binding of CS-1 and VCAM to α4β1 were docked into the active site of the refined model. Results from the binding analysis fit well with a pharmacophore model that was independently derived from active analog studies. A critical examination of residues in the binding site and analysis of docked ligands that are both potent and selective led to the proposal of a mechanism for β1/β7 ligand binding selectivity.     

Effects of Detergent β-Octylglucoside and Phosphate Salt Solutions on Phase Behavior of Monoolein Mesophases

Using small-angle x-ray scattering (SAXS), we investigated the phase behavior of mesophases of monoolein (MO) mixed with additives commonly used for the crystallization of membrane proteins from lipidic mesophases. In particular, we examined the effect of sodium and potassium phosphate salts and the detergent β-octylglucoside (βOG) over a wide range of compositions relevant for the crystallization of membrane proteins in lipidic mesophases. We studied two types of systems: 1), ternary mixtures of MO with salt solutions above the hydration boundary; and 2), quaternary mixtures of MO with βOG and salt solutions over a wide range of hydration conditions. All quaternary mixtures showed highly regular lyotropic phase behavior with the same sequence of phases (Lα, Ia3d, and Pn3m) as MO/water mixtures at similar temperatures. The effects of additives in quaternary systems agreed qualitatively with those found in ternary mixtures in which only one additive is present. However, quantitative differences in the effects of additives on the lattice parameters of fully hydrated mesophases were found between ternary and quaternary mixtures. We discuss the implications of these findings for mechanistic investigations of membrane protein crystallization in lipidic mesophases and for studies of the suitability of precipitants for mesophase-based crystallization methods.     

Acetylation of αA-crystallin in the human lens: Effects on structure and chaperone function

α-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including αA-crystallin are acetylated in vivo. We found that K70 and K99 in αA-crystallin and, K92 and K166 in αB-crystallin are acetylated in the human lens. To determine the effect of acetylation on the chaperone function and structural changes, αA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a N^ε-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated αA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in αA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against γ-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of αA-crystallin occurs in the human lens and that it affects the chaperone function of the protein.     

Crystal structure of α/β-galactoside α2,3-sialyltransferase from a luminous marine bacterium, Photobacterium phosphoreum

α/β-Galactoside α2,3-sialyltransferase produced by Photobacterium phosphoreum JT-ISH-467 is a unique enzyme that catalyzes the transfer of N-acetylneuraminic acid residue from cytidine monophosphate N-acetylneuraminic acid to acceptor carbohydrate groups. The enzyme recognizes both mono- and di-saccharides as acceptor substrates, and can transfer Neu5Ac to both α-galactoside and β-galactoside, efficiently. To elucidate the structural basis for the broad acceptor substrate specificity, we determined the crystal structure of the α2,3-sialyltransferase in complex with CMP. The overall structure belongs to the glycosyltransferase-B structural group. We could model a reasonable active conformation structure based on the crystal structure. The predicted structure suggested that the broad substrate specificity could be attributed to the wider entrance of the acceptor substrate binding site.     

Effect of 6α,7β-dihydroxyvouacapan-17β-oic acid and its lactone derivatives on the growth of human cancer cells

The furanditerpene 6α,7β-dihydroxyvouacapan-17β-oic acid (1) is a natural product biosynthesized by some species from the genus Pterodon (Leguminosae). This secondary metabolite has multiple biological activities that include anti-inflammatory, analgesic, plant growth regulatory, anti-edematogenic, photosystem II inhibitory and photosynthesis uncoupler, and antifungal properties. However, few studies on the antiproliferative profile of compound 1 and/or its derivatives have been reported up to date. Here, we describe the isolation of compound 1 from hexane extract of P. polygalaeflorus fruits as well as the semisynthesis of three lactone derivatives: 6α-hydroxyvouacapan-7β,17β-lactone (2), 6α-acetoxyvouacapan-7β,17β-lactone (3), and 6-oxovouacapan-7β,17β-lactone (4). Additionally, antiproliferative activity of these compounds against nine human cancer cell lines was investigated. Our results revealed that 6α-hydroxyvouacapan-7β,17β-lactone (2) was the most potent furanditerpene against all cancer cell lines studied. The presence of non-substituted hydroxyl group at C-6 and the presence of 7β,17β-lactone ring are important for the antiproliferative activity of these compounds.     

The effect of α1-acid glycoprotein (orosomucoid) on triglyceride metabolism in the nephrotic syndrome

We have identified α₁-acid glycoprotein as a new co-factor in the lipoprotein lipase reaction. We isolated an active form of the compound from nephrotic urine that is effective both $\text{in vitro}$ and $\text{in vivo}$. α₁-acid glycoprotein increased lipolysis 100% in the presence of C-II apolipoprotein in a lipoprotein lipase assay system. Rats with induced nephrotic syndrome showed a decrease in triglyceride clearance. $\text{T}\text{1}\text{2}$ was increased from 14 min to 43 min. The injection of α₁-acid glycoprotein restored the lipid clearance to normal. These findings suggest that elevated plasma triglycerides in human nephrotic patients is the direct result of excessive loss of α₁-acid glycoprotein from plasma into urine. We propose that replacement therapy may be possible.     

δN and δC analysis of a Posidonia sinuosa seagrass bed

Potential food sources and dominant invertebrates and fishes were collected for the examination of variability in ¹³C/¹²C and ¹⁵N/¹⁴N to determine the sources of carbon available to consumers within a Western Australian Posidonia sinuosa-dominated seagrass bed. Autotrophs showed a wide distribution of δ¹³C values, with P. sinuosa at −11.3 ± 0.8‰ and macroalgae ranging from −16.6 to −31.7‰. This variation allowed us to successfully identify macroalgae as the main contributor of carbon to the trophic structure, although no distinction could be made between epiphytic macroalgae on seagrass, or allochthonous macroalgal sources. The range in δ¹⁵N ratios among potential food items at the trophic base was too small to make it useful as tracer of nitrogen flow pathways, but it consistently increased from macrophytes and detritus (4.1–6.8‰), to invertebrates (5.7–7.4‰) located near the middle of the food web, to fishes (8.3–11.9‰), with piscivorous species such as Leviprora inops generally having a higher ¹⁵N. δ¹³C of seston (−12.8‰) and sedimentary organic matter (−8.7‰) indicate that seagrass material is the main contributor to these two carbon pools, and that very little of it contributes to animal biomass.     

Aminoterminal acetylation of synthetic Nα-desacetyl thymosin α1

A transacetylase associated with the ribosome fraction from wheat germ catalyzes the transfer of acetyl groups from acetyl CoA to synthetic N^α-desacetyl thymosin α₁. The product was identified by high-performance liquid chromatography and by the isolation of a tryptic peptide containing the acetylated NH₂-terminus. In 20 min, with 13 μg of enzyme protein, 15% of the desacetyl thymosin α₁ added was converted to the acetylated form. Under the conditions employed only the α-NH₂ group was acetylated.     

Regio- and stereospecific synthesis of rac-carbasugar-based cyclohexane pentols; Investigations of their α- and β-glucosidase inhibitions

In the present study, (3aR,7aS)-1,3,3a,4,7,7a-hexahydroisobenzofuran was submitted to photooxygenation and two isomeric hydroperoxides were successfully obtained. Without any further purification, reduction of the hydroperoxides with titanium tetraisopropoxide catalyzed by dimethyl sulfide gave two alcohol isomers in high yields. After acetylation of alcohol with Ac₂O in pyridine, epoxidation reaction of formed monoacetates with m-CPBA, then chromatographed and followed by hydrolysis of the acetate groups with NH₃ in CH₃OH resulted in the formation of epoxy alcohol isomers respectively. These epoxy alcohol isomers were subjected to trans-dihydroxylation reaction with acid (H₂SO₄) in the presence of water to afford triols. Acetylation of the free hydroxyl groups produced benzofuran triacetates in high yields. Ring-opening reaction of furan triacetates with sulfamic acid catalyzed in the presence of acetic acid/acetic anhydrate and subsequently hydrolysis of the acetate groups with ammonia gave the targeted cyclohexane carbasugar-based pentols. All products were separated and purified by chromatographic and crystallographic methods. Structural analyses of all compounds were conducted by spectral techniques including NMR and X-ray analyses. The biological inhibition activity of the target compounds was tested against glycosidase enzymes, α- and β-glucosidase.     

Effects of repeated daily injections of prostaglandin F2α on ovaries in mares

In experiment 1, seven groups of pony mares (2 or 3/group) were given either no injections (controls), or 5(5X) or 10(10X) daily subcutaneous (SC) injections of 1.25 mg PGF_2α beginning on days 1, 7 or 13 post-ovulation. Compared to controls (24.5 days), the interovulatory interval was longer (P<.05) for day 7, 10X (33.5 days) and day 13, 10X mares (49.0 days) but was not different for the remaining groups. In experiment 2, nine groups of pony mares (4/group) were given either no injections (controls) or 1(1X) or 10(10X) daily SC injections of 1.25 mg PGF_2α beginning on day 2 of estrus or on days 1, 7 or 13 post-ovulation. Compared to controls (25.0 days), the interovulatory interval was longer (P<.05) for day 13 post-ovulation, 10X mares (40.0 days) and shorter (P<.05) for day 1 post-ovulation, 10X mares (14.5 days). The interovulatory interval for the remaining groups was not different (P>.05) from that for controls. In day 13 post-ovulation, 10X mares, the longer interovulatory interval did not appear to be related to a depression in either peripheral LH concentration (no effect of treatment on LH) or on follicular development (no effect of treatment on diameter of largest follicle). This suggests that circulating levels of gonadotropins were adequate for ovarian follicular development and ovulation and the effect of repeated daily injections of PGF_2α in preventing ovulation was likely exerted at the ovarian level directly on the follicle.     

Effects of point mutations in pVHL on the binding of HIF-1α

The von Hippel-Lindau tumor suppressor protein (pVHL) has an essential role in the regulation of the hypoxia response pathway in animal cells. Under normoxic conditions, the hypoxia-inducible factor (HIF) undergoes trans-4-prolyl hydroxylation and is subsequently recognised by the β-domain of pVHL, leading to the ubiquitination and degradation of HIF. Mutations of pVHL alter the binding of HIF. A subset of relevant clinically observed mutations to pVHL are thought to cause weaker binding of HIF-1α and are associated with cancer and cardiovascular diseases. Here, we present computational studies analyzing the interaction of HIF with mutant forms of pVHL, describing at atomic detail the local structural reorganization caused by substitution of certain residues of pVHL. The results reveal that the canonical configuration in the wild-type system is vital for the efficient functioning of the complex and that mutation of any of the residues implicated in the h-bond network in the binding site disrupts HIF binding. Although the experimentally observed ordering of binding energies for mutants of Tyr98 is reproduced, our examination of a broader range of mutations does not support the hypothesis of a correlation between the degree of disruption of the pVHL/HIF-1α interaction caused by a mutation and the phenotype with which the mutation is associated. We suggest that disruption of the binding interaction is one of many factors behind the manifestation of VHL disease.     

Differences in β-strand Populations of Monomeric Aβ40 and Aβ42

Using homonuclear ¹H NOESY spectra, with chemical shifts, ³J_H^N_H^α scalar couplings, residual dipolar couplings, and ¹H-¹⁵N NOEs, we have optimized and validated the conformational ensembles of the amyloid-β 1–40 (Aβ40) and amyloid-β 1–42 (Aβ42) peptides generated by molecular dynamics simulations. We find that both peptides have a diverse set of secondary structure elements including turns, helices, and antiparallel and parallel β-strands. The most significant difference in the structural ensembles of the two peptides is the type of β-hairpins and β-strands they populate. We find that Aβ42 forms a major antiparallel β-hairpin involving the central hydrophobic cluster residues (16–21) with residues 29–36, compatible with known amyloid fibril forming regions, whereas Aβ40 forms an alternative but less populated antiparallel β-hairpin between the central hydrophobic cluster and residues 9–13, that sometimes forms a β-sheet by association with residues 35–37. Furthermore, we show that the two additional C-terminal residues of Aβ42, in particular Ile-41, directly control the differences in the β-strand content found between the Aβ40 and Aβ42 structural ensembles. Integrating the experimental and theoretical evidence accumulated over the last decade, it is now possible to present monomeric structural ensembles of Aβ40 and Aβ42 consistent with available information that produce a plausible molecular basis for why Aβ42 exhibits greater fibrillization rates than Aβ40.     

The use of multivariable standard curves in the radioimmunoassay of testosterone and 5α-dihydrotestosterone

Multivariable calibration curves have been used to enable testosterone and 5α-dihydrotestosterone to be assayed directly in plasma extracts without further pre-purification of the sample. Two antisera were used, both with relatively high, but different affinities for the substances measured, and with relatively low affinity towards all other substances tested. The antisera were obtained from rabbits immunized against testosterone-3-BSA and 5α-dihydrotestosterone-3-BSA. The technique was of adequate precision, accuracy and specificity. The last was examined by comparison of values obtained by the present method and those obtained following prepurification by thin layer chromatography.