Electronic spectral studies of dinuclear Cobalt(II) complexes with a phenol-based dinucleating ligand containing four methoxyethyl chelating arms

The electronic spectra of dinuclear cobalt(II) complexes [Co₂(bomp)(MeCO₂)₂]BPh₄ (1) and [Co₂(bomp)(PhCO₂)₂]BPh₄ (2) were studied [H(bomp): 2,6-bis[bis(2-methoxyethyl)aminomethyl]-4-methylphenol]; the spectral components obtained by Gaussian curve analysis were well simulated based on the angular overlap model using the aomx program. The first transition band ⁴T₁ → ⁴T₂(⁴F) of an octahedral high-spin cobalt(II) complex was found to be sensitive to the distortion around the cobalt(II) ion.     

Phosphorylation and stabilization of ATP binding cassette transporter A1 by synthetic amphiphilic helical peptides

To investigate structural requirement of helical apolipoprotein to phosphorylate and stabilize ATP-binding cassette transporter A1 (ABCA1), synthetic peptides (Remaley, A. T., Thomas, F., Stonik, J. A., Demosky, S. J., Bark, S. E., Neufeld, E. B., Bocharov, A. V., Vishnyakova, T. G., Patterson, A. P., Eggerman, T. L., Santamarina-Fojo, S., and Brewer, H. B. (2003) J. Lipid Res. 44, 828-836) were examined for these activities. L37pA, an L amino acid peptide that contains two class-A amphiphilic helices, and D37pA, the same peptide with all D amino acids, both removed cholesterol and phospholipid from differentiated THP-1 cells more than apolipoproteins (apos) A-I, A-II, and E. Both peptides also mediated lipid release from human fibroblasts WI-38 similar to apoA-I. L2D37pA, an L-peptide whose valine and tyrosine were replaced with D amino acids also promoted lipid release from WI-38 but less so with THP-1, whereas L3D37pA, in which alanine, lysine, and asparatic acid were replaced with D amino acids was ineffective in lipid release for both cell lines. ABCA1 protein in THP-1 and WT-38 was stabilized against proteolytic degradation by apoA-I, apoA-II, and apoE and by all the peptides tested except for L3D37pA, and ABCA1 phosphorylation closely correlated with its stabilization. The analysis of the relationship among these parameters indicated that removal of phospholipid triggers signals for phosphorylation and stabilization of ABCA1. We thus concluded that an amphiphilic helical motif is the minimum structural requirement for a protein to stabilize ABCA1 against proteolytic degradation.     

Protein phosphatase 2B dephosphorylates twitchin, initiating the catch state of invertebrate smooth muscle

"Catch" is the state where some invertebrate muscles sustain high tension for long periods at low ATP hydrolysis rates. Physiological studies using muscle fibers have not yet fully provided the details of the initiation process of the catch state. The process was extensively studied by using an in vitro reconstitution assay with several phosphatase inhibitors. Actin filaments bound to thick filaments pretreated with the soluble protein fraction of muscle homogenate and Ca2+ (catch treatment) in the presence of MgATP at a low free Ca2+ concentration (the catch state). Catch treatment with > 50 microm okadaic acid, > 1 microm microcystin LR, 1 microm cyclosporin A, 1 microm FK506, or 0.2 mm calcineurin autoinhibitory peptide fragment produced almost no binding of the actin filaments, indicating protein phosphatase 2B (PP2B) was involved. Use of bovine calcineurin (PP2B) and its activator calmodulin instead of the soluble protein fraction initiated the catch state, indicating that only PP2B and calmodulin in the soluble protein fraction are essential for the initiation process. The initiation was reproduced with purified actin, myosin, twitchin, PP2B, and calmodulin. 32P autoradiography showed that only twitchin was dephosphorylated during the catch treatment with either the soluble protein fraction or bovine calcineurin and calmodulin. These results indicate that PP2B directly dephosphorylates twitchin and initiates the catch state and that no other component is required for the initiation process of the catch state.     

Involvement of caspase-9 in execution of the maternal program of apoptosis in Xenopus late blastulae overexpressed with S-adenosylmethionine decarboxylase

We previously demonstrated that overexpression of S-adenosylmethionine decarboxylase (SAMDC) in Xenopus early embryos induces execution of maternal program of apoptosis shortly after midblastula transition, which likely serves as a fail-safe mechanism of early development to eliminate physiologically damaged cells before they entering the gastrula stage. To determine how caspases are involved in this process, we microinjected peptide inhibitors and "dominant-negative forms" of caspase-9 and -1 into Xenopus fertilized eggs, and found that inhibitors of caspase-9, but not caspase-1, completely suppress SAMDC-induced apoptosis. The lysate of SAMDC-overexpressing late blastulae contained activity to cleave in vitro-synthesized [(35)S]procaspase-9, but not [(35)S]procaspase-1, and mRNA for caspase-9, but not caspase-1, occurred abundantly in the unfertilized egg as maternal mRNA. We also found that overexpression of caspase-9 and -1 equally executes the apoptosis, but the apoptosis executed by these mRNAs was only partially rescued by Bcl-2 and rescued embryos did not develop beyond neurula stage. These results indicate that activation of caspase-9 is a key step for execution of the maternally preset program of apoptosis in Xenopus early embryos.     

Circulation of type 1 vaccine-derived poliovirus in the Philippines in 2001

In 2001, highly evolved type 1 circulating vaccine-derived poliovirus (cVDPV) was isolated from three acute flaccid paralysis patients and one contact from three separate communities in the Philippines. Complete genomic sequencing of these four cVDPV isolates revealed that the capsid region was derived from the Sabin 1 vaccine strain but most of the noncapsid region was derived from an unidentified enterovirus unrelated to the oral poliovirus vaccine (OPV) strains. The sequences of the cVDPV isolates were closely related to each other, and the isolates had a common recombination site. Most of the genetic and biological properties of the cVDPV isolates were indistinguishable from those of wild polioviruses. However, the most recently identified cVDPV isolate from a healthy contact retained the temperature sensitivity and partial attenuation phenotypes. The sequence relationships among the isolates and Sabin 1 suggested that cVDPV originated from an OPV dose given in 1998 to 1999 and that cVDPV circulated along a narrow chain of transmission. Type 1 cVDPV was last detected in the Philippines in September 2001, and population immunity to polio was raised by extensive OPV campaigns in late 2001 and early 2002.     

Structural analysis of human hair single fibres by scanning microbeam SAXS

The origin of the curliness of human hair was revealed by scanning microbeam small angle X-ray scattering (SAXS), based on the nanostructure of keratin fibre arrangement. Scanning microbeam SAXS patterns of single hair fibres have been measured across the fibres and the differences in the patterns between the inner and the outer sides of the curvature were successfully detected. The analysis of the equatorial and azimuthal scattering intensity profiles showed that the arrangement of the intermediate filaments was different between the inner and the outer sides of the curvature. From the analogy with Merino and Romny wool, it is suggested that different types of cortices exist in human hair. It is concluded that, regardless of the ethnic origins (African, Caucasian, and Asian), the macroscopic curl shape of the hair fibre originate from the inhomogeneity of the internal nanostructure, arising from inhomogeneous distribution of two types of cortices.     

Proteomic analysis of serum marker proteins in recipient mice with liver cirrhosis after bone marrow cell transplantation

We previously found that transplantation with bone marrow cells (BMCs) improves liver function and liver fibrosis in cirrhotic mice. In the presence of liver damage induced by carbon tetrachloride (CCl4), transplanted BMC migrated into the peri-portal region and trans-differentiated into hepatocytes that produce albumin. Thus under these conditions, BMC transplantation induces liver regeneration. Detecting serum marker proteins is important to monitor the recovery of liver function of cirrhotic mice after BMC transplantation. We therefore initially resolved proteins extracted from serum samples at 48 h after BMC transplantation by 2-DE and compared spot intensity between control and BMC groups of mice. Six protein spots increased in the BMC group compared with the control group. MS revealed that these spots comprised apolipoprotein A1 (apoA1), apolipoprotein C3 (apoC3), vitamin D-binding protein, alpha-1-antitrypsin and proteasome subunit alpha type 1. We subsequently confirmed the levels of apoA1 in serum and liver samples by immunoblotting. ApoA1 increased at early stage (48 h and 1 wk) after BMC transplantation in this mouse model of liver cirrhosis. The early elevation of apoA1 might be useful to predict liver regeneration in cirrhotic mice after BMC transplantation.     

Mass spectrometric identification of N-linked glycopeptides using lectin-mediated affinity capture and glycosylation site-specific stable isotope tagging

Protein post-translational modifications (PTMs), such as glycosylation and phosphorylation, are crucial for various signaling and regulatory events, and are therefore an important objective of proteomics research. We describe here a protocol for isotope-coded glycosylation site-specific tagging (IGOT), a method for the large-scale identification of N-linked glycoproteins from complex biological samples. The steps of this approach are: (1) lectin column-mediated affinity capture of glycopeptides generated by protease digestion of protein mixtures; (2) purification of the enriched glycopeptides by hydrophilic interaction chromatography (HIC); (3) peptide-N-glycanase-mediated incorporation of a stable isotope tag, 18O18O, specifically at the N-glycosylation site; and (4) identification of 18O-tagged peptides by liquid chromatography-coupled mass spectrometry (LC/MS)-based proteomics technology. The application of this protocol to the characterization of N-linked glycoproteins from crude extracts of the nematode Caenorhabditis elegans or mouse liver provides a list of hundreds to a thousand glycoproteins and their sites of glycosylation within a week.     

Contribution of comparative fish studies to general endocrinology: structure and function of some osmoregulatory hormones

Fish endocrinologists are commonly motivated to pursue their research driven by their own interests in these aquatic animals. However, the data obtained in fish studies not only satisfy their own interests but often contribute more generally to the studies of other vertebrates, including mammals. The life of fishes is characterized by the aquatic habitat, which demands many physiological adjustments distinct from the terrestrial life. Among them, body fluid regulation is of particular importance as the body fluids are exposed to media of varying salinities only across the thin respiratory epithelia of the gills. Endocrine systems play pivotal roles in the homeostatic control of body fluid balance. Judging from the habitat-dependent control mechanisms, some osmoregulatory hormones of fish should have undergone functional and molecular evolution during the ecological transition to the terrestrial life. In fact, water-regulating hormones such as vasopressin are essential for survival on the land, whereas ion-regulating hormones such as natriuretic peptides, guanylins and adrenomedullins are diversified and exhibit more critical functions in aquatic species. In this short review, we introduce some examples illustrating how comparative fish studies contribute to general endocrinology by taking advantage of such differences between fishes and tetrapods. In a functional context, fish studies often afford a deeper understanding of the essential actions of a hormone across vertebrate taxa. Using the natriuretic peptide family as an example, we suggest that more functional studies on fishes will bring similar rewards of understanding. At the molecular level, recent establishment of genome databases in fishes and mammals brings clues to the evolutionary history of hormone molecules via a comparative genomic approach. Because of the functional and molecular diversification of ion-regulating hormones in fishes, this approach sometimes leads to the discovery of new hormones in tetrapods as exemplified by adrenomedullin 2.