Base stacking in the dinucleoside phosphate rapa.

The pH-induced unstacking of rRpA has been investigated by batch calorimetry and uv spectroscopy. Equilibrium uv melting curves confirmed that the adenine bases in rApA are stacked at pH7 but unstacked at pH 1.5. The enthalpy change accompanying this pH-induced unstacking is +2.65 kcal (mole of A-A stack)-1 as measured by batch calorimetry. This represents the first direct determination of this important parameter for a dinucleoside phosphate. It is noted that the calorimetrically determined value reported here is considerably lower than published van't Hoff enthalpies but is consistent with values that can be derived from calorimetric data on polymers.     

The interaction of β-cyclodextrin with dinucleoside phosphates

Circular dichroism studies of the interaction of β-cyclodextrin with a series of eight dinucleoside phosphates, and with 3′-AMP are reported. From results with ApU and ApA, it is shown that β-cyclodextrin binding is sensitive to dinucleoside stacking in approximately the same way as optical measures of stacking. Some qualitative uses of β-cyclodextrin binding are suggested, based on the fact that the change in the CD spectrum caused by cyclodextrin binding is unique to each of the dinucleosides studied. Hoffman and Bock have previously suggested the use of β-cyclodextrin as a probe of nucleic acid structure. Their work indicated that only binding to adenosine and inosine would have to be considered. The present paper shows that binding to other bases cannot be neglected, and will impose serious restrictions on the use of β-cyclodextrin as a structural probe.     

The β‐sheet breakers and π‐stacking

Fibrillation of β‐amyloid is recognized as a key process leading to the development of Alzheimer's disease. Small peptides called β‐sheet breakers were found to inhibit the process of β‐amyloid fibrillation and to dissolve amyloid fibrils in vitro, in vivo, and in cell culture studies [1,2]. The mechanism by which peptide inhibition takes place remains elusive and a detailed model needs to be established. Here, we present new insights into the possible role of consecutive Phe residues, present in the structure of β‐sheet breakers, supported by the results obtained by means of MD simulations. We performed a 30‐ns MD of two β‐sheet breakers: iAβ5 (LPFFD) and iAβ6 (LPFFFD) which have two and three consecutive Phe residues, respectively. We have found that Phe rings in these peptides tend to form stacked conformations. For one of the peptides – iAβ6 – the calculated electrostatic contribution to free energy of one of the conformers with three rings stacked (c2) is significantly lower than that corresponding to the unstacked one (c1), two rings stacked (c0) and second conformer with three rings stacked (c3). This may favor the interaction of the c2 conformer with the target on amyloid fibril. We hypothesize that the mechanism of inhibition of amyloidogenesis by β‐sheet breaker involves competition among π‐stacked Phe residues of the inhibitor and π‐stacking within the β‐amyloid fibril. iAβ6 may be a promising candidate for a lead compound of amyloidogenesis inhibitors. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Studies on the 5α-androstane-3β, 17β-diol-6α-hydroxylase and on the 5α-androstane-3β, 17β-diol-7α-hydroxylase in anterior hypophysis of male rats.

In anterior pituitaries from male rats, it appeared that 5α-androstane-3β, 17β-diol was quickly metabolized into 5α-androstane-3β,6α-17β-triol and 5α-androstane-3β,7α, 17β-triol by action of 6α- and 7α-hydroxylases. Hydroxysteroid hydroxylases were located in endoplasmic reticulum and were dependent on NADPH⁺. Their optimum pH was 8.0, optima temperature, 37°C, and their apparent Km was 2.7 μM. Hydroxylative reactions were not reversible and not modified by gonadectomy. Hydroxylation seemed an efficient control of the pituitary level of 5α-andros-tane-3β, 17β-diol.     

A study of adenosine 3′–5′ cyclic monophosphate binding sites of human erythrocyte membranes using 8-azidoadenosine 3′–5′ cyclic monophosphate,a photoaffinity probe

An earlier report (1a) has shown the utility of 8-N₃cAMP (8-azidoadenosine-3′, 5′-cyclic monophosphate) as a photoaffinity probe for cAMP binding sites in human erythrocyte membranes. The increased resolution obtained using a linear-gradient SDS polyacrylamide gel system now shows that: (1) both cAMP and 8-N₃cAMP stimulate the phosphorylation by [γ-³²P]-ATP of the same red cell membrane proteins; (2) the protein of approximately 48,000 molecular weight whose phosphorylation by [γ-³²P]-ATP is stimulated by cAMP and 8-N₃cAMP migrates at a solwer rate than the protein in the same molecular weight range which is heavily photolabeled with [³²P]-8-N₃cAMP; (3) other cyclic nucleotide binding sites exist besides those initailly reported; (4) the variation in the ratio of incorporation of ³²P-8-N₃cAMP into the two highest affinity binding sites appears to be the result of a specific proteolysis of the larger protein.     

5α,6α-Epoxy-cholestan-3β-ol (cholesterol α-oxide): A specific substrate for rat liver glutathione transferase B

A semi-micro assay was developed for the conjugation of 5α,6α-epoxy-cholestan-3β-ol (cholesterol α-oxide) with glutathione. The soluble supernatant of rat liver homogenate catalysed the reaction at a rate of 0.2–0.5 pmol.min⁻¹ .mg protein⁻¹ with 4μM cholesterol α-oxide, while the reaction in the presence of GSH alone was barely detectable. Enzymic activity in the soluble supernatant was due equally to the two forms of glutathione transferase B (100 pmol.min⁻.mg protein⁻¹), glutathione transferases AA, A, C and E being unreactive. The activity of purified glutathione transferase B was about 5-times that expected from the activity of the soluble supernatant. Complex enzyme kinetics were obtained suggestive of substrate inhibition.     

Human α‐amino‐β‐carboxymuconate‐ε‐semialdehyde decarboxylase (ACMSD): A structural and mechanistic unveiling

Human α‐amino‐β‐carboxymuconate‐ε‐semialdehyde decarboxylase determines the fate of tryptophan metabolites in the kynurenine pathway by controlling the quinolinate levels for de novo nicotinamide adenine dinucleotide biosynthesis. The unstable nature of its substrate has made gaining insight into its reaction mechanism difficult. Our electron paramagnetic resonance (EPR) spectroscopic study on the Cu‐substituted human enzyme suggests that the native substrate does not directly ligate to the metal ion. Substrate binding did not result in a change of either the hyperfine structure or the super‐hyperfine structure of the EPR spectrum. We also determined the crystal structure of the human enzyme in its native catalytically active state (at 1.99 Å resolution), a substrate analogue‐bound form (2.50 Å resolution), and a selected active site mutant form with one of the putative substrate binding residues altered (2.32 Å resolution). These structures illustrate that each asymmetric unit contains three pairs of dimers. Consistent with the EPR findings, the ligand‐bound complex structure shows that the substrate analogue does not directly coordinate to the metal ion but is bound to the active site by two arginine residues through noncovalent interactions. Proteins 2015; 83:178–187. © 2014 Wiley Periodicals, Inc.     

Engineered cystine knot peptides that bind αvβ3, αvβ5, and α5β1 integrins with low‐nanomolar affinity

There is a critical need for compounds that target cell surface integrin receptors for applications in cancer therapy and diagnosis. We used directed evolution to engineer the Ecballium elaterium trypsin inhibitor (EETI‐II), a knottin peptide from the squash family of protease inhibitors, as a new class of integrin‐binding agents. We generated yeast‐displayed libraries of EETI‐II by substituting its 6‐amino acid trypsin binding loop with 11‐amino acid loops containing the Arg‐Gly‐Asp integrin binding motif and randomized flanking residues. These libraries were screened in a high‐throughput manner by fluorescence‐activated cell sorting to identify mutants that bound to α_vβ₃ integrin. Select peptides were synthesized and were shown to compete for natural ligand binding to integrin receptors expressed on the surface of U87MG glioblastoma cells with half‐maximal inhibitory concentration values of 10–30 nM. Receptor specificity assays demonstrated that engineered knottin peptides bind to both α_vβ₃ and α_vβ₅ integrins with high affinity. Interestingly, we also discovered a peptide that binds with high affinity to α_vβ₃, α_vβ₅, and α₅β₁ integrins. This finding has important clinical implications because all three of these receptors can be coexpressed on tumors. In addition, we showed that engineered knottin peptides inhibit tumor cell adhesion to the extracellular matrix protein vitronectin, and in some cases fibronectin, depending on their integrin binding specificity. Collectively, these data validate EETI‐II as a scaffold for protein engineering, and highlight the development of unique integrin‐binding peptides with potential for translational applications in cancer. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

A Potential Role for 5‐Androstene‐3β,7β,17β‐triol in Obesity and Metabolic Syndrome

Metabolic syndrome is marked by perturbed glucocorticoid (GC) signaling, systemic inflammation, and altered immune status. Dehydroepiandrosterone (DHEA), a major circulating adrenal steroid and dietary supplement, demonstrates antiobesity, anti‐inflammatory, GC‐opposing and immune‐modulating activity when administered to rodents. However, plasma DHEA levels failed to correlate with metabolic syndrome and oral replacement therapy provided only mild benefits to patients. Androstene‐3β,7β,17β‐triol (β‐AET) an anti‐inflammatory metabolite of DHEA, also exhibits GC‐opposing and immune‐modulating activity when administered to rodents. We hypothesized a role for β‐AET in obesity. We now report that plasma levels of β‐AET positively correlate with BMI in healthy men and women. Together with previous studies, the observations reported here may suggest a compensatory role for β‐AET in preventing the development of metabolic syndrome. The β‐AET structural core may provide the basis for novel pharmaceuticals to treat this disease.     

Structural classification of αββ and ββα supersecondary structure units in proteins

We present a fully automatic structural classification of supersecondary structure units, consisting of two hydrogen-bonded β strands, preceded or followed by an α helix. The classification is performed on the spatial arrangement of the secondary structure elements, irrespective of the length and conformation of the intervening loops. The similarity of the arrangements is estimated by a structure alignment procedure that uses as similarity measure the root mean square deviation of superimposed backbone atoms. Applied to a set of 141 well-resolved nonhomologous protein structures, the classification yields 11 families of recurrent arrangements. In addition, fragments that are structurally intermediate between the families are found; they reveal the continuity of the classification. The analysis of the families shows that the α helix and β hairpin axes can adopt virtually all relative orientations, with, however, some preferable orientations; moreover, according to the orientation, preferences in the left/right handedness of the α–β connection are observed. These preferences can be explained by favorable side by side packing of the α helix and the β hairpin, local interactions in the region of the α–β connection or stabilizing environments in the parent protein. Furthermore, fold recognition procedures and structure prediction algorithms coupled to database-derived potentials suggest that the preferable nature of these arrangements does not imply their intrinsic stability. They usually accommodate a large number of sequences, of which only a subset is predicted to stabilize the motif. The motifs predicted as stable could correspond to nuclei formed at the very beginning of the folding process. Proteins 30:193–212, 1998. © 1998 Wiley-Liss, Inc.     

Stacking-unstacking of the dinucleoside monophosphate guanylyl-3'',5''-uridine studied with molecular dynamics.

Molecular dynamics simulations were carried out on two conformations of the dinucleoside monophosphate guanylyl-3',5'-uridine (GpU) in aqueous solution with one sodium counterion. One stacked conformation and one with the C3'-O3'-P-O5' backbone torsion angle twisted 180 degrees to create an unstacked conformation. We observed a relatively stable behavior of the stacked conformation, which remained stacked throughout the simulation, whereas the unstacked conformation showed major changes in the backbone torsion and glycosidic angles. During the simulation the unstacked conformation transformed into a more stacked form and then back again to an unstacked one. The calculated correlation times for rotational diffusion from the molecular dynamics simulations are in agreement with fluorescence anisotropy and nuclear magnetic resonance data. As expected, the correlation times for rotational diffusion of the unstacked conformation were observed to be longer than for the stacked conformation. The 2'OH group may contribute in stabilizing the stacked conformation, where the O2'-H...O4' hydrogen bond occurred in 82.7% of the simulation.     

A chemiluminescent immunoassay for the direct measurement of urinary 5α-androstane-3α, 17β-diol-glucuronide in urine

We described a chemiluminescent immunoassay (CIA) for 5α-androstane-3α, 17β-diol-glucuronide (3α-diol-G) in human diluted urine. This method allowed the direct measurement in 1μl of urine avoiding the hydrolysis and extraction steps for sample pretreatment commonly used in routine methods. The hapten 3α-diol-G was synthesized by a Koenigs–Knorr reaction. The immunogenic complex, 3α-diol-G conjugated to bovine serum albumin (BSA), was employed to induce the formation of specific antibodies in New Zealand rabbits. In addition, the required chemiluminescent (CL) tracer was prepared. The characteristics of the antibody was determined as regard to specificity and sensitivity and the precision of the assay methods established. In 22 hirsute women affected by policystic ovarian syndrome we found 3α-diol-G values significantly (p < 0.01) higher (146.28 ± 73.77μg/g of creatinine; mean ± SD) than those observed in normal women (72.1 ± 32.58 μg/g of creatinine; mean ± SD).     

Ecosystem stability in space: α, β and γ variability

The past two decades have seen great progress in understanding the mechanisms of ecosystem stability in local ecological systems. There is, however, an urgent need to extend existing knowledge to larger spatial scales to match the scale of management and conservation. Here, we develop a general theoretical framework to study the stability and variability of ecosystems at multiple scales. Analogously to the partitioning of biodiversity, we propose the concepts of alpha, beta and gamma variability. Gamma variability at regional (metacommunity) scale can be partitioned into local alpha variability and spatial beta variability, either multiplicatively or additively. On average, variability decreases from local to regional scales, which creates a negative variability–area relationship. Our partitioning framework suggests that mechanisms of regional ecosystem stability can be understood by investigating the influence of ecological factors on alpha and beta variability. Diversity can provide insurance effects at the various levels of variability, thus generating alpha, beta and gamma diversity–stability relationships. As a consequence, the loss of biodiversity and habitat impairs ecosystem stability at the regional scale. Overall, our framework enables a synthetic understanding of ecosystem stability at multiple scales and has practical implications for landscape management.     

Involvement of integrins α3β1 and α5β1 and glycoprotein IIb in megakaryocyte‐induced osteoblast proliferation

As the prevalence of osteoporosis is expected to increase over the next few decades, the development of novel therapeutic strategies to combat this disorder becomes clinically imperative. These efforts draw extensively from an expanding body of knowledge pertaining to the physiologic mechanisms of skeletal homeostasis. To this body of knowledge, we contribute that cells of hematopoietic lineage may play a crucial role in balancing osteoblastic bone formation against osteoclastic resorption. Specifically, our laboratory has previously demonstrated that megakaryocytes (MKs) can induce osteoblast (OB) proliferation in vitro, but do so only when direct cell‐to‐cell contact is permitted. To further investigate the nature of this interaction, we have effectively neutralized several adhesion molecules known to function in the analogous interaction of MKs with another cell type of mesenchymal origin—the fibroblast (FB). Our findings implicate the involvement of fibronectin/RGD‐binding integrins including α3β1 (VLA‐3) and α5β1 (VLA‐5) as well as glycoprotein (gp) IIb (CD41), all of which are known to be expressed on MK membranes. Furthermore, we demonstrate that interleukin (IL)‐3 can enhance MK‐induced OB activation in vitro, as demonstrated in the MK–FB model system. Taken together, these results suggest that although their physiologic and clinical implications are very different, these two models of hematopoietic–mesenchymal cell activation are mechanistically analogous in several ways. J. Cell. Biochem. 109: 927–932, 2010. © 2010 Wiley‐Liss, Inc.     

5α‐Androst‐16‐en‐3α‐ol β‐D‐Glucuronide,Precursor of 5α‐Androst‐16‐en‐3α‐ol in Human Sweat

5α‐Androst‐16‐en‐3α‐ol (α‐androstenol) is an important contributor to human axilla sweat odor. It is assumed that α‐andostenol is excreted from the apocrine glands via a H₂O‐soluble conjugate, and this precursor was formally characterized in this study for the first time in human sweat. The possible H₂O‐soluble precursors, sulfate and glucuronide derivatives, were synthesized as analytical standards, i.e., α‐androstenol, β‐androstenol sulfates, 5α‐androsta‐5,16‐dien‐3β‐ol (β‐androstadienol) sulfate, α‐androstenol β‐glucuronide, α‐androstenol α‐glucuronide, β‐androstadienol β‐glucuronide, and α‐androstenol β‐glucuronide furanose. The occurrence of α‐androstenol β‐glucuronide was established by ultra performance liquid chromatography (UPLC)/MS (heated electrospray ionization (HESI)) in negative‐ion mode in pooled human sweat, containing eccrine and apocrine secretions and collected from 25 female and 24 male underarms. Its concentration was of 79 ng/ml in female secretions and 241 ng/ml in male secretions. The release of α‐androstenol was observed after incubation of the sterile human sweat or α‐androstenol β‐glucuronide with a commercial glucuronidase enzyme, the urine‐isolated bacteria Streptococcus agalactiae, and the skin bacteria Staphylococcus warneri DSM 20316, Staphylococcus haemolyticus DSM 20263, and Propionibacterium acnes ATCC 6919, reported to have β‐glucuronidase activities. We demonstrated that if α‐ and β‐androstenols and androstadienol sulfates were present in human sweat, their concentrations would be too low to be considered as potential precursors of malodors; therefore, the H₂O‐soluble precursor of α‐androstenol in apocrine secretion should be a β‐glucuronide.     

The Muscle-Specific Laminin Receptor α7β1 Integrin Negatively Regulates α5β1 Fibronectin Receptor Function

α7β1 is the major integrin complex expressed in differentiated muscle cells where it functions as a laminin receptor. In this work we have expressed the α7 integrin subunit in CHO cells to investigate the functional properties of this receptor. After transfection with α7 CHO cells acquired the ability to adhere and spread on laminin 1 consistent with the laminin receptor activity of the α7β1. α7 transfectants, however, showed a 70% reduction in the ability to adhere to fibronectin and were unable to assemble a fibronectin matrix. The degree of reduction was inversely related to the level of α7 expression. To define the mechanisms underlying this adhesive defect we analyzed surface expression and functional properties of the α5β1 fibronectin receptor. Although cell surface expression of α5β1 was reduced by a factor of 20–25% in α7 transfectants compared to control untransfected cells, this slight reduction was not sufficient to explain the dramatic reduction in cell adhesion (70%) and matrix assembly (close to 100%). Binding studies showed that the affinity of¹²⁵I-fibronectin for its surface receptor was decreased by 50% in α7 transfectants, indicating that the α5β1 integrin is partially inactivated in these cells. Inactivation can be reversed by Mn²⁺, a cation known to increase integrin affinity for their ligands. In fact, incubation of cells with Mn²⁺restored fibronectin binding affinity, adhesion to fibronectin, and assembly of fibronectin matrix in α7 transfectants. These data indicate that α7 expression leads to the functional down regulation of α5β1 integrin by decreasing ligand binding affinity and surface expression. In conclusion, the data reported establish the existence of anegative cooperativitybetween α7 and α5 integrins that may be important in determining functional regulation of integrins during myogenic differentiation.     

Modulation of α5β1 and αVβ3 integrins on the cell surface during mitosis

One of the hallmarks of cells undergoing mitotic division is their rounded morphology and reduced adhesion to the substratum. We have studied and compared the attachment of interphase and mitotic cells to substrata coated with fibronectin and vitronectin. We have found that adhesion of mitotic cells, as compared to interphase cells, is significantly reduced to fibronectin, but is higher to vitronectin. These results correlate well with the expression of α5β1 and αVβ3 integrins, the respective receptors for fibronectin and vitronectin, on the cell surface. Mitotic cells show higher levels of αVβ3 and very low levels of α5β1 proteins on the cell surface as compared to interphase cells. This difference in the levels of these integrins also reflects in the total amounts of fibronectin and vitronectin present on the cell surface of these cells. We have further shown, by flow cytometry, that binding of vitronectin, or the synthetic peptide-GRGDSP-, causes an increase in the intracellular levels of Ca²⁻ in mitotic cells, but no change is seen in the interphase cells. Binding of fibronectin to either of these cells fails to elicit any response. One interesting feature of our results is that the levels of total, i.e., cytoplasmic plus membrane bound, α5β1 and αVβ3 integrins of mitotic and interphase cells remain the same, thus implying an alteration in the distribution of integrin chains between the plasma membrane and the cytoplasm during the conversion of interphase cells into the mitotic phase. © 1996 Wiley-Liss, Inc.     

The cystathionine‐β‐synthase domains on the guanosine 5′’‐monophosphate reductase and inosine 5′‐monophosphate dehydrogenase enzymes from Leishmania regulate enzymatic activity in response to guanylate and adenylate nucleotide levels

The Leishmania guanosine 5′‐monophosphate reductase (GMPR) and inosine 5′‐monophosphate dehydrogenase (IMPDH) are purine metabolic enzymes that function maintaining the cellular adenylate and guanylate nucleotide. Interestingly, both enzymes contain a cystathionine‐β‐synthase domain (CBS). To investigate this metabolic regulation, the Leishmania GMPR was cloned and shown to be sufficient to complement the guaC (GMPR), but not the guaB (IMPDH), mutation in Escherichia coli. Kinetic studies confirmed that the Leishmania GMPR catalyzed a strict NADPH‐dependent reductive deamination of GMP to produce IMP. Addition of GTP or high levels of GMP induced a marked increase in activity without altering the K_m values for the substrates. In contrast, the binding of ATP decreased the GMPR activity and increased the GMP K_m value 10‐fold. These kinetic changes were correlated with changes in the GMPR quaternary structure, induced by the binding of GMP, GTP, or ATP to the GMPR CBS domain. The capacity of these CBS domains to mediate the catalytic activity of the IMPDH and GMPR provides a regulatory mechanism for balancing the intracellular adenylate and guanylate pools.     

A fluorescent probe for biothiols based on the conjugate addition of thiols to α,β‐unsaturated ester

A sensitive fluorogenic probe 1 for biothiols was developed based on the Michael addition reaction. The probe 1 was readily synthesized via the reaction of 2‐(4′‐hydroxyphenyl) benzimidazole (HPBI) with acryloyl chloride and shows weak fluorescence emission. Upon mixing with biothiols, the fluorescence of 1 is significantly enhanced due to the conjugate addition of thiols to the α,β‐unsaturated carbonyl moiety, thus eliminating the photoinduced electron transfer (PET) quenching of the fluorophore by the intramolecular carbon–carbon double bond. Cysteine (Cys) was selected as the representative thiol in the spectral experiment. A good linear relationship was obtained from 1.0 to 30.0 µmol L^?1 for Cys and the detection limit was 0.17 µmol L^?1. Furthermore, probe 1 was highly selective for biothiols without the interference of some biologically relevant analytes and has been applied to detecting biothiols in human urine samples. Copyright © 2010 John Wiley & Sons, Ltd.