Hydroxylysine in the N-terminal regions of the α1- and α2-chains of various collagens

The degree of hydroxylation of the lysine residue located in both alpha(1)- and alpha(2)-chains of collagen in the N-terminal, non-helical telopeptide region of the molecule has been determined in collagen from various sources after isolation of the peptides (alpha(1)- and alpha(2)-CB1) that contain the lysine residue in question and are obtained by cyanogen bromide cleavage of collagen alpha(1)- and alpha(2)-chains respectively. As with collagen from chick tibia, bone collagens from rat tibia and femur and embryonic chick frontal bone, have a high degree of hydroxylation (approx. 50% or more) of the lysine residue in both alpha(1)- and alpha(2)-CB1 peptides. This is in contrast with the lack of hydroxylation of this residue in both alpha(1)- and alpha(2)-chains of all skin collagens so far examined. The presence of hydroxylysine in alpha(1)- and alpha(2)-CB1 peptides from tendon collagen is also indicated. In rat tail tendon collagen the amount of hydroxylation is only slight but in the much less soluble tendon collagen from embryonic chick leg tendons, approximately one-third of the lysine is hydroxylated.     

Structural and enzymatic characterization of the isoamylase1 homo-oligomer and the isoamylase1–isoamylase2 hetero-oligomer from rice endosperm

The present study established that there are two distinct polymeric forms of isoamylase1 (ISA1) in rice endosperm: presumably a homo-pentamer of ISA1 and a hetero-hexamer composed of five ISA1 and one ISA2. The molecular sizes of the homo- and hetero-oligomers, which could be fractionated by hydrophobic chromatography, were approximately 420–480 and 510–550 kDa, respectively. The hetero-oligomer exhibited higher affinities for various branched polyglucans, especially for phytoglycogen, which had a K _m value that was approximately 12 times lower relative to that with the homo-oligomer, although no marked differences were found in chain preferences for debranching of amylopectin and phytoglycogen between these forms. The hetero-oligomer was active even when incubated at 50°C for 10 min, while the homo-multimer was completely inactivated at 40°C in 10 min. When the ISA1 homo-oligomer was incubated with the ISA2 protein expressed in Escherichia coli and applied onto a nondenature polyacrylamide gel, additional debranching activity bands which were specific for the purified ISA1–ISA2 preparation were also detected, indicating that ISA1 and ISA2 combine to form a hetero-oligomer. These results suggest that the hetero-oligomer plays a predominant role in the amylopectin biosynthesis in rice endosperm although the homo-oligomer can complement the function of the hetero-oligomer at least to some extent.Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Molecular Cloning of the Dog β1 and β2 Adrenergic Receptors

Abstract

We report the isolation of the genes encoding the β1 and β2 adrenergic receptors from dog genomic DNA. Sequence analysis of both genes revealed intronless open reading frames of 473 and 415 amino acid residues, receptively. Heterologous expression of both receptors in CHO cells indicated that both receptors are functionally similar to the human homologs. Comparing the dog β1 and β2 adrenergic receptors, the β1 receptor appears to bind to G proteins more tightly than the β2 receptor. Heterologously expressed receptors provide a convenient system for evaluating novel receptor agonists and antagonists.     

PCBP2 enhances the antiviral activity of IFN-α against HCV by stabilizing the mRNA of STAT1 and STAT2

Interferon-α (IFN-α) is a natural choice for the treatment of hepatitis C, but half of the chronically infected individuals do not achieve sustained clearance of hepatitis C virus (HCV) during treatment with IFN-α alone. The virus can impair IFN-α signaling and cellular factors that have an effect on the viral life cycles. We found that the protein PCBP2 is down-regulated in HCV-replicon containing cells (R1b). However, the effects and mechanisms of PCBP2 on HCV are unclear. To determine the effect of PCBP2 on HCV, overexpression and knockdown of PCBP2 were performed in R1b cells. Interestingly, we found that PCBP2 can facilitate the antiviral activity of IFN-α against HCV, although the RNA level of HCV was unaffected by either the overexpression or absence of PCBP2 in R1b cells. RIP-qRT-PCR and RNA half-life further revealed that PCBP2 stabilizes the mRNA of STAT1 and STAT2 through binding the 3'Untranslated Region (UTR) of these two molecules, which are pivotal for the IFN-α anti-HCV effect. RNA pull-down assay confirmed that there were binding sites located in the C-rich tracts in the 3'UTR of their mRNAs. Stabilization of mRNA by PCBP2 leads to the increased protein expression of STAT1 and STAT2 and a consistent increase of phosphorylated STAT1 and STAT2. These effects, in turn, enhance the antiviral effect of IFN-α. These findings indicate that PCBP2 may play an important role in the IFN-α response against HCV and may benefit the HCV clinical therapy.     

Interleukin-1α treatment of meniscal explants stimulates the production and release of aggrecanase-generated, GAG-substituted aggrecan products and also the release of pre-formed, aggrecanase-generated G1 and m-calpain-generated G1-G2

Pro-inflammatory cytokines induce meniscal matrix degradation and inhibition of endogenous repair mechanisms, but the pathogenic mechanisms behind this are mostly unknown. Therefore, we investigated details of interleukin-1 (IL-1α)-induced aggrecan turnover in mature meniscal tissue explants. Fibro-cartilagenous disks (3 mm diameter × 1 mm thickness) were isolated from the central, weight-bearing region of menisci from 2-year-old cattle. After 3 or 6 days of IL-1α-treatment, GAG loss (DMMB assay), biosynthetic activity ([³⁵SO₄]-sulfate and [³H]-proline incorporation), gene expression (quantitative RT-PCR) and the abundance (zymography, Western blot) of matrix-degrading enzymes and specific aggrecan products were determined. Meniscal fibrocartilage had a 4-fold lower GAG content (per wet weight) than adjacent articular cartilage, and expressed MMPs-1, -2, -3 and ADAMTS4 constitutively, whereas ADAMTS5 m-RNA was essentially undetectable. Significant IL-1 effects were a decrease in biosynthetic activity, an increase in GAG release and in the expression/abundance of MMP-2, MMP-3 and ADAMTS4. Fresh tissue contained aggrecan core protein products similar to those previously described for bovine articular cartilage of this age. IL-1 induced the release of aggrecanase-generated CS-substituted products including both high (>250 kDa) and low molecular weight (about 75 kDa) species. TIMP-3 (but not TIMP-1 and -2 or a broad spectrum MMP inhibitor) inhibited IL-1-dependent GAG loss. In addition, IL-1 induced the release of preformed pools of three known G1-bearing products. We conclude that aggrecanases are responsible for IL-1-stimulated GAG release from meniscal explants, and that IL-1 also stimulates release of G1-bearing products, by a process possibly involving hyaluronan fragmentation.     

Roles of Adrenergic α1 and Dopamine D1 and D2 Receptors in the Mediation of the Desynchronization Effects of Modafinil in a Mouse EEG Synchronization Model

Background

Synchronized electroencephalogram (EEG) activity is observed in pathological stages of cognitive impairment and epilepsy. Modafinil, known to increase the release of catecholamines, is a potent wake-promoting agent, and has shown some abilities to desynchronize EEG,but its receptor mechanisms by which modafinil induces desynchoronization remain to be elucidated. Here we used a pharmacological EEG synchronization model to investigate the involvement of adrenergic α₁ receptors (R, α₁R) and dopamine (DA) D₁ and D₂ receptors (D₁Rs and D₂Rs) on modafinil-induced desynchronization in mice.

Methodology/Principal Findings

Mice were treated with cholinergic receptor antagonist scopolamine and monoamine depletor reserpine to produce experimental EEG synchronization characterized by continuous large-amplitude synchronized activity, with prominent increased delta and decreased theta, alpha, and beta power density. The results showed that modafinil produced an EEG desynchronization in the model. This was characterized by a general decrease in amplitude of all the frequency bands between 0 and 20 Hz, a prominent reduction in delta power density, and an increase in theta power density. Adrenergic α₁R antagonist terazosin (1 mg/kg, i.p.) completely antagonized the EEG desynchronization effects of modafinil at 90 mg/kg. However, DA D₁R and D₂R blockers partially attenuated the effects of modafinil. The modafinil-induced decrease in the amplitudes of the delta, theta, alpha, and beta waves and in delta power density were completely abolished by pretreatment with a combination of the D₁R antagonist SCH 23390 (30 µg/kg) and the D₂R antagonist raclopride (2 mg/kg, i.p.).

Conclusions/Significance

These results suggest that modafinil-mediated desynchronization may be attributed to the activation of adrenergic α₁R, and dopaminergic D₁R and D₂R in a model of EEG synchronization.     

Crystal Structures of the Network-Forming Short-Arm Tips of the Laminin β1 and γ1 Chains

The heterotrimeric laminins are a defining component of basement membranes and essential for tissue formation and function in all animals. The three short arms of the cross-shaped laminin molecule are composed of one chain each and their tips mediate the formation of a polymeric network. The structural basis for laminin polymerisation is unknown. We have determined crystal structures of the short-arm tips of the mouse laminin β1 and γ1 chains, which are grossly similar to the previously determined structure of the corresponding α5 chain region. The short-arm tips consist of a laminin N-terminal (LN) domain that is attached like the head of a flower to a rod-like stem formed by tandem laminin-type epidermal growth factor-like (LE) domains. The LN domain is a β-sandwich with elaborate loop regions that differ between chains. The γ1 LN domain uniquely contains a calcium binding site. The LE domains have little regular structure and are stabilised by cysteines that are disulphide-linked 1-3, 2-4, 5-6 and 7-8 in all chains. The LN surface is not conserved across the α, β and γ chains, but within each chain subfamily there is a striking concentration of conserved residues on one face of the β-sandwich, while the opposite face invariably is shielded by glycans. We propose that the extensive conserved patches on the β and γ LN domains mediate the binding of these two chains to each other, and that the α chain LN domain subsequently binds to the composite β-γ surface. Mutations in the laminin β2 LN domain causing Pierson syndrome are likely to impair the folding of the β2 chain or its ability to form network interactions.     

The N-terminal globular domain of the laminin α1 chain binds to α1β1 and α2β1 integrins and to the heparan sulfate-containing domains of perlecan

The N-terminal domains VI plus V (62 kDa) and V alone (43 kDa) of the laminin α1 chain were obtained as recombinant products and shown to be folded into a native form by electron microscopy and immunological assays. Domain VI alone, which corresponds to an LN module, did not represent an autonomously folding unit in mammalian cells, however. Fragment α1VI/V, but not fragment α1V, bound to purified α1β1 and α2β1 integrins, to heparin, and to heparan sulfate-substituted domains I and V of perlecan. This localized the binding activities to the LN module, which contains two basic sequences suitable for heparin interactions.     

Substrate profiling and aldehyde dismutase activity of the Kvβ2 subunit of the mammalian Kv1 potassium channel

Voltage-dependent potassium channels (Kv) are involved in various cellular signalling processes by governing the membrane potential of excitable cells. The cytosolic face of these α subunit-containing channels is associated with β subunits that can modulate channel responses. Surprisingly, the β subunit of the mammalian Kv1 channels, Kvβ2, has a high level of sequence homology with the aldo–keto reductase (AKR) superfamily of proteins. Recent studies have shown that Kvβ2 can catalyze the reduction of aldehydes and, most significantly, that channel function is modulated when Kvβ2-bound NADPH is concomitantly oxidized. As a result, the redox chemistry of this subunit is crucial to understanding its role in K⁺ channel modulation. The present study has extended knowledge of the substrate profile of this subunit using a single turnover fluorimetric assay. Kvβ2 was found to catalyse the reduction of aromatic aldehyde substrates such as 2, 3 and 4-nitrobenzaldehydes, 4-hydroxybenzaldehyde, pyridine 2-aldehyde and benzaldehyde. The presence of an electron withdrawing group at the position para to the aldehyde in aromatic compounds facilitated reduction. Aliphatic aldehydes proved to be poor substrates. We devised a simple HPLC-based assay to identify Kvβ2 reaction products. Using this assay we showed, for the first time, that Kvβ2 can catalyze a slow aldehyde dismutation reaction using 4-nitrobenzaldehyde as substrate and have identified the products of this reaction. The ability of Kvβ2 to carry out both an aldehyde reduction and a dismutation reaction is discussed in the light of current thinking on the role of redox chemistry in channel modulation.     

Structure and mechanism of the 2′,3′ phosphatase component of the bacterial Pnkp-Hen1 RNA repair system

Pnkp is the end-healing and end-sealing component of an RNA repair system present in diverse bacteria from many phyla. Pnkp is composed of three catalytic modules: an N-terminal polynucleotide 5′ kinase, a central 2′,3′ phosphatase and a C-terminal ligase. The phosphatase module is a Mn²⁺-dependent phosphodiesterase–monoesterase that dephosphorylates 2′,3′-cyclic phosphate RNA ends. Here we report the crystal structure of the phosphatase domain of Clostridium thermocellum Pnkp with Mn²⁺ and citrate in the active site. The protein consists of a core binuclear metallo-phosphoesterase fold (exemplified by bacteriophage λ phosphatase) embellished by distinctive secondary structure elements. The active site contains a single Mn²⁺ in an octahedral coordination complex with Asp187, His189, Asp233, two citrate oxygens and a water. The citrate fills the binding site for the scissile phosphate, wherein it is coordinated by Arg237, Asn263 and His264. The citrate invades the site normally occupied by a second metal (engaged by Asp233, Asn263, His323 and His376), and thereby dislocates His376. A continuous tract of positive surface potential flanking the active site suggests an RNA binding site. The structure illuminates a large body of mutational data regarding the metal and substrate specificity of Clostridium thermocellum Pnkp phosphatase.     

Sequence and developmental expression of amphioxus AmphiNk2–1: insights into the evolutionary origin of the vertebrate thyroid gland and forebrain

 We characterized an amphioxus NK-2 homeobox gene (AmphiNk2–1), a homologue of vertebrate Nkx2–1, which is involved in the development of the central nervous system and thyroid gland. At the early neurula stage of amphioxus, AmphiNk2–1 expression is first detected medially in the neural plate. By the mid-neurula stage, expression is localized ventrally in the nerve cord and also begins in the endoderm. During the late neurula stage, the ventral neural expression becomes transiently segmented posteriorly and is then down-regulated except in the cerebral vesicle at the anterior end of the central nervous system. Within the cerebral vesicle AmphiNk2–1 is expressed in a broad ventral domain, probably comprising both the floor plate and basal plate regions; this pattern is comparable to Nkx2–1 expression in the mouse diencephalon. In the anterior part of the gut, expression becomes intense in the endostyle (the right wall of the pharynx), which is the presumed homologue of the vertebrate thyroid gland. More posteriorly, there is transitory expression in the midgut and hindgut. In sum, the present results help to support homologies (1) between the amphioxus endostyle and the vertebrate thyroid gland and (2) between the amphioxus cerebral vesicle and the vertebrate diencephalic forebrain. Received: 4 September 1998 / Accepted: 24 October 1998     

Changes in the Anatomic and Microscopic Structure and the Expression of HIF-1α and VEGF of the Yak Heart with Aging and Hypoxia

The study aimed to identify the changes of anatomic and microscopic structure and the expression and localization of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) in the myocardium and coronary artery of the yak heart adapted to chronic hypoxia with aging. Thirty-two yaks (1 day, 6 months, 1 year, 2 years, and 5 year old) were included, and immunoelectronmicroscopy, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) were used. Right ventricular hypertrophy was not present in yaks with aging. There was no intima thickening phenomenon in the coronary artery. The ultrastructure of myofibrils, mitochondria, and collagen fibers and the diameter and quantity of collagen changed significantly with aging. The enzymatic activity of complexes I, II, and V increased with age. Immunogold labeling showed the localization of HIF-1α protein in the cytoplasm and nuclei of endothelial cells and cytoplasm of cardiac muscle cells, and VEGF protein in the nuclei and perinuclei areas of smooth muscle cells of coronary artery, and in the cytoplasm and nuclei of endothelial cells. ELISA results showed that HIF-1α secretion significantly increased in the myocardium and coronary artery from an age of 1 day to 2 years of yaks and decreased in old yaks. However, VEGF protein always increased with aging. The findings of this study suggest that 6 months is a key age of yak before which there are some adaptive changes to deal with low-oxygen environment, and there is a maturation of the yak heart from the age of 6 months to 2 years.     

Assignment of the 1H, 13C and 15N resonances of the calponin homology-2 domain of α-actinin-4

We report the assignment of the 110 amino acid second calponin homology domain of human α-actinin-4. The two calponin homology domains of α-actinin combine to regulate F-actin binding.     

Structural and functional insights into the heme-binding domain of the human soluble guanylate cyclase α2 subunit and heterodimeric α2β1

Soluble guanylate cyclase (sGC) mediates NO signaling for a wide range of physiological effects in the cardiovascular system and the central nervous system. The α1β1 isoform is ubiquitously distributed in cytosolic fractions of tissues, whereas α2β1 is mainly found in the brain. The major occurrence and the unique characteristic of human sGC α2β1 indicate a special role in the mediation of neuronal communication. We have efficiently purified and characterized the recombinant heme-binding domain of the human sGC α2 subunit (hsGC α2(H)) and heterodimeric α2β1 (hsGC β1(H)-α2(H)) by UV-vis spectroscopy, circular dichrosim spectroscopy, EPR spectroscopy, and homology modeling. The heme dissociation and related NO/CO binding/dissociation of both hsGC α2(H) and hsGC β1(H)-α2(H) were investigated. The two truncated proteins interact with heme noncovalently. The CO binding affinity of hsGC α2(H) is threefold greater than that of human sGC α1(H), whereas the dissociation constant k (1) for dissociation of NO from hsGC α2(H) is sevenfold larger than that for dissociation of NO from hsGC α1(H), although k (2) is almost identical. The results indicate that in comparison with the α1β1 isoform, the brain α2β1 isoform exhibits a distinctly different CO/NO affinity and binding rate in favor of NO signaling, and this is consistent with its physiological role in the activation and desensitization. Molecular modeling and sequence alignments are consistent with the hypothesis that His105 contributes to the different CO/NO binding properties of different isoforms. This valuable information is helpful to understand the molecular mechanism by which human sGC α2β1 mediates NO/CO signaling.     

Effects of Mutating Leucine to Threonine in the M2 Segment of α1 and β1 Subunits of GABAAα1β1 Receptors

The conserved leucine residues at the 9′ positions in the M2 segments of α₁ (L264) and β₁ (L259) subunits of the human GABA_A receptor were replaced with threonine. Normal or mutant α₁ subunits were co-expressed with normal or mutant β₁ subunits in Sf9 cells using the baculovirus/Sf9 expression system. Cells in which one or both subunits were mutated had a higher ``resting' chloride conductance than cells expressing wild-type α₁β₁ receptors. This chloride conductance was blocked by 10 mm penicillin, a recognized blocker of GABA_A channels, but not by bicuculline (100 μm) or picrotoxin (100 μm) which normally inhibit the chloride current activated by GABA: nor was it potentiated by pentobarbitone (100 μm). In cells expressing wild-type β₁ with mutated α₁ subunits, an additional chloride current could be elicited by GABA but the rise time and decay were slower than for wild-type α₁β₁ receptors. In cells expressing mutated β₁ subunits with wild-type or mutated α₁ subunits (αβ(L9′T) and α(L9′T)β(L9′T)), no response to GABA could be elicited: this was not due to an absence of GABA_A receptors in the plasmalemma because the cells bound [³H]-muscimol. It was concluded that in GABA_A channels containing the L9′T mutation in the β₁ subunit, GABA-binding does not cause opening of channels, and that the L9′T mutation in either or both subunits gives an open-channel state of the GABA_A receptor in the absence of ligand. Received: 17 April 1996/Revised: 5 July 1996