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.     

Replication of single-stranded porcine circovirus DNA by DNA polymerases α and δ

Porcine circovirus is the only mammalian DNA virus so far known to contain a single-stranded circular genome (Tischer et al. (1982) Nature 295, 64–66). Replication of its small viral DNA (1.76 kb) appears to be dependent on cellular enzymes expressed during S-phase of the cell cycle (Tischer et al. (1987) Arch. Virol. 96, 39–57). In this paper we have exploited the porcine circovirus genome to probe for in vitro initiation and elongation of DNA replication by different preparations of calf thymus DNA polymerase α and δ as well as by a partially purified preparation from pig thymus. The results indicated that three different purification fractions of calf thymus DNA polymerase α and one from pig thymus initiate DNA synthesis at several sites on the porcine circovirus DNA. It appears that the sites at which DNA primase synthesizes primers are not entirely random. Subsequent DNA elongation by a highly purified DNA polymerase α holoenzyme which had been isolated by the criterion of replicating single-stranded M13 DNA (Ottiger et al. (1987) Nucleic Acids Res. 15, 4789–4807) is very efficient. Complete conversion to the double-stranded form is obtained in less than 1 min. When the DNA synthesis by DNA polymerase α is blocked with the DNA polymerase α specific monoclonal antibody SJK 132-20 after initiation by DNA primase, DNA polymerase δ can efficiently replicate from the primers. This in vitro DNA replication system may be used in analogy to the bacteriophage systems in E. coli to study initiation and elongation of DNA replication.     

Syntheses and anti‐tubercular activity of β‐substituted and α,β‐disubstituted peptidyl β‐aminoboronates and boronic acids

Tuberculosis is still affecting millions of people worldwide, and new resistant strains of Mycobacterium tuberculosis are being found. It is therefore necessary to find new compounds for treatment. In this paper, we report the synthesis and in vitro testing of peptidyl β‐aminoboronic acids and β‐aminoboronates with anti‐tubercular activity. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

Potential of the bean α‐amylase inhibitor αAI‐1 to inhibit α‐amylase activity in true bugs (Hemiptera)

True bugs (Hemiptera) are an important pest complex not controlled by Bt‐transgenic crops. An alternative source of resistance includes inhibitors of digestive enzymes, such as protease or amylase inhibitors. αAI‐1, an α‐amylase inhibitor from the common bean, inhibits gut‐associated α‐amylases of bruchid pests of grain legumes. Here we quantify the in vitro activity of α‐amylases of 12 hemipteran species from different taxonomic and functional groups and the in vitro inhibition of those α‐amylases by αAI‐1. α‐Amylase activity was detected in all species tested. However, susceptibility to αAI‐1 varied among the different groups. α‐Amylases of species in the Lygaeidae, Miridae and Nabidae were highly susceptible, whereas those in the Auchenorrhyncha (Cicadellidae, Membracidae) had a moderate susceptibility, and those in the Pentatomidae seemed to be tolerant to αAI‐1. The species with αAI‐1 susceptible α‐amylases represented families which include both important pest species but also predatory species. These findings suggest that αAI‐1‐expressing crops have potential to control true bugs in vivo.     

Conformations of peptides containing a chiral cyclic α, α‐disubstituted α‐amino acid within the sequence of Aib residues

A single chiral cyclic α,α‐disubstituted amino acid, (3S,4S)‐1‐amino‐(3,4‐dimethoxy)cyclopentanecarboxylic acid [(S,S)‐Ac₅c^dOM], was placed at the N‐terminal or C‐terminal positions of achiral α‐aminoisobutyric acid (Aib) peptide segments. The IR and ¹H NMR spectra indicated that the dominant conformations of two peptides Cbz‐[(S,S)‐Ac₅c^dOM]‐(Aib)₄‐OEt ( 1) and Cbz‐(Aib)₄‐[(S,S)‐Ac₅c^dOM]‐OMe (2) in solution were helical structures. X‐ray crystallographic analysis of 1 and 2 revealed that a left‐handed (M) 3₁₀‐helical structure was present in 1 and that a right‐handed (P) 3₁₀‐helical structure was present in 2 in their crystalline states. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis

Inflammatory cytokines are closely related to pigmentary changes. In this study, the effects of IFN‐γ on melanogenesis were investigated. IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis in B16 melanoma cells and normal human melanocytes. MITF mRNA and protein expressions were significantly inhibited in response to IFN‐γ. IFN‐γ inhibited CREB binding to the MITF promoter but did not affect CREB phosphorylation. Instead, IFN‐γ inhibited the association of CBP and CREB through the increased association between CREB binding protein (CBP) and STAT1. These findings suggest that IFN‐γ inhibits both basal and α‐MSH‐induced melanogenesis by inhibiting MITF expression. The inhibitory action of IFN‐γ in α‐MSH‐induced melanogenesis is likely to be associated with the sequestration of CBP via the association between CBP and STAT1. These data suggest that IFN‐γ plays a role in controlling inflammation‐ or UV‐induced pigmentary changes.     

Asymmetric Allylation of α‐Ketoester‐Derived N‐Benzoylhydrazones Promoted by Chiral Sulfoxides/N‐Oxides Lewis Bases: Highly Enantioselective Synthesis of Quaternary α‐Substituted α‐Allyl‐α‐Amino Acids

Chiral sulfoxides/N‐oxides (R)‐ 1 and (R,R)‐ 2 are effective chiral promoters in the enantioselective allylation of α‐keto ester N‐benzoylhydrazone derivatives 3a , 3b , 3c , 3d , 3e , 3f , 3g to generate the corresponding N‐benzoylhydrazine derivatives 4a , 4b , 4c , 4d , 4e , 4f , 4g , with enantiomeric excesses as high as 98%. Representative hydrazine derivatives 4a , 4b were subsequently treated with SmI₂, and the resulting amino esters 5a , 5b with LiOH to obtain quaternary α‐substituted α‐allyl α‐amino acids 6a , 6b , whose absolute configuration was assigned as (S), with fundament on chemical correlation and electronic circular dichroism (ECD) data. Chirality 25:529–540, 2013. © 2013 Wiley Periodicals, Inc.     

Interaction of antidiabetic α‐glucosidase inhibitors and gut bacteria α‐glucosidase

Carbohydrate hydrolyzing α‐glucosidases are commonly found in microorganisms present in the human intestine microbiome. We have previously reported crystal structures of an α‐glucosidase from the human gut bacterium Blaubia (Ruminococcus) obeum (Ro‐αG1) and its substrate preference/specificity switch. This novel member of the GH31 family is a structural homolog of human intestinal maltase‐glucoamylase (MGAM) and sucrase–isomaltase (SI) with a highly conserved active site that is predicted to be common in Ro‐αG1 homologs among other species that colonize the human gut. In this report, we present structures of Ro‐αG1 in complex with the antidiabetic α‐glucosidase inhibitors voglibose, miglitol, and acarbose and supporting binding data. The in vitro binding of these antidiabetic drugs to Ro‐αG1 suggests the potential for unintended in vivo crossreaction of the α‐glucosidase inhibitors to bacterial α‐glucosidases that are present in gut microorganism communities. Moreover, analysis of these drug‐bound enzyme structures could benefit further antidiabetic drug development.     

Cyclic oligomer design with de novo αβ‐proteins

We have previously shown that monomeric globular αβ‐proteins can be designed de novo with considerable control over topology, size, and shape. In this paper, we investigate the design of cyclic homo‐oligomers from these starting points. We experimented with both keeping the original monomer backbones fixed during the cyclic docking and design process, and allowing the backbone of the monomer to conform to that of adjacent subunits in the homo‐oligomer. The latter flexible backbone protocol generated designs with shape complementarity approaching that of native homo‐oligomers, but experimental characterization showed that the fixed backbone designs were more stable and less aggregation prone. Designed C2 oligomers with β‐strand backbone interactions were structurally confirmed through x‐ray crystallography and small‐angle X‐ray scattering (SAXS). In contrast, C3‐C5 designed homo‐oligomers with primarily nonpolar residues at interfaces all formed a range of oligomeric states. Taken together, our results suggest that for homo‐oligomers formed from globular building blocks, improved structural specificity will be better achieved using monomers with increased shape complementarity and with more polar interfaces.     

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.     

14‐3‐3γ mediates Cdc25A proteolysis to block premature mitotic entry after DNA damage

14‐3‐3 proteins control various cellular processes, including cell cycle progression and DNA damage checkpoint. At the DNA damage checkpoint, some subtypes of 14‐3‐3 (β and ζ isoforms in mammalian cells and Rad24 in fission yeast) bind to Ser345‐phosphorylated Chk1 and promote its nuclear retention. Here, we report that 14‐3‐3γ forms a complex with Chk1 phosphorylated at Ser296, but not at ATR sites (Ser317 and Ser345). Ser296 phosphorylation is catalysed by Chk1 itself after Chk1 phosphorylation by ATR, and then ATR sites are rapidly dephosphorylated on Ser296‐phosphorylated Chk1. Although Ser345 phosphorylation is observed at nuclear DNA damage foci, it occurs more diffusely in the nucleus. The replacement of endogenous Chk1 with Chk1 mutated at Ser296 to Ala induces premature mitotic entry after ultraviolet irradiation, suggesting the importance of Ser296 phosphorylation in the DNA damage response. Although Ser296 phosphorylation induces the only marginal change in Chk1 catalytic activity, 14‐3‐3γ mediates the interaction between Chk1 and Cdc25A. This ternary complex formation has an essential function in Cdc25A phosphorylation and degradation to block premature mitotic entry after DNA damage.     

Differential physiologic responses of α7 nicotinic acetylcholine receptors to β‐amyloid1–40 and β‐amyloid1–42

The β‐amyloid peptides (Aβ), Aβ_1–40 and Aβ_1–42, have been implicated in Alzheimer's disease (AD) pathology. Although Aβ_1–42 is generally considered to be the pathological peptide in AD, both Aβ_1–40 and Aβ_1–42 have been used in a variety of experimental models without discrimination. Here we show that monomeric or oligomeric forms of the two Aβ peptides, when interact with the neuronal cation channel, α7 nicotinic acetylcholine receptors (α7nAChR), would result in distinct physiologic responses as measured by acetylcholine release and calcium influx experiments. While Aβ_1–42 effectively attenuated these α7nAChR‐dependent physiology to an extent that was apparently irreversible, Aβ_1–40 showed a lower inhibitory activity that could be restored upon washings with physiologic buffers or treatment with α7nAChR antagonists. Our data suggest a clear pharmacological distinction between Aβ_1–40 and Aβ_1–42. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 25–30, 2003     

Deamidation of α‐synuclein

The rates of deamidation of α-synuclein and single Asn residues in 13 Asn-sequence mutants have been measured for 5 × 10⁻⁵M protein in both the absence and presence of 10⁻²M sodium dodecyl sulfate (SDS). In the course of these experiments, 370 quantitative protein deamidation measurements were performed and 37 deamidation rates were determined by ion cyclotron resonance Fourier transform mass spectrometry, using an improved whole protein isotopic envelope method and a mass defect method with both enzymatic and collision-induced fragmentation. The measured deamidation index of α-synuclein was found to be 0.23 for an overall deamidation half-time of 23 days, without or with SDS micelles, owing primarily to the deamidation of Asn(103) and Asn(122). Deamidation rates of 15 Asn residues in the wild-type and mutant proteins were found to be primary sequence controlled without SDS. However, the presence of SDS micelles slowed the deamidation rates of nine N-terminal region Asn residues, caused by the known three-dimensional structures induced through protein binding to SDS micelles.