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.     

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.     

Complementation and characterization of the nested Rz and Rz1 reading frames in the genome of bacteriophage λ

Transposon insertions in the Rz gene of bacteriophage λ block lysis if the medium contains divalent cations at concentrations greater than 5?mM, but otherwise cause no change in phenotype. The Rz protein is thought to have an endopeptidase activity, previously reported in λ lysates, which might be involved in cleavage of oligopeptide crosslinks between glycosidic strands in the peptidoglycan and the Lpp lipoproteins of the outer bacterial membrane. Recently, a small lipoprotein has been reported as the product of a short reading frame, designated Rz1, in the +1 register within Rz. This protein has been detected in membranes of induced λ lysogens. To determine whether Rz1 has a function in the λ vegetative cycle, amber nonsense alleles of Rz and Rz1 have been constructed by site-directed mutagenesis and used for complementation and suppression analysis. Both Rzam and Rz1am alleles have phenotypes identical to those of the original Rz insertion alleles, and complement and are fully suppressed in a supE host, indicating that the two genes are independent, trans-acting genes encoding proteins required for lysis in the presence of cations. Moreover, supF suppresses Rzam but not the Rz1am mutation, and the defective Rz1am product in the supF host shows a partially dominant character and significantly retards lysis even in the absence of additional cations in the medium. Rz and Rz1 represent a unique example of two genes located in different reading frames in the same nucleotide sequence, which encode different proteins that are both required in the same physiological pathway.     

Cross-species investigation of the functions of the Rhodobacter PufX polypeptide and the composition of the RC–LH1 core complex

In well-characterised species of the Rhodobacter (Rba.) genus of purple photosynthetic bacteria it is known that the photochemical reaction centre (RC) is intimately-associated with an encircling LH1 antenna pigment protein, and this LH1 antenna is prevented from completely surrounding the RC by a single copy of the PufX protein. In Rba. veldkampii only monomeric RC–LH1 complexes are assembled in the photosynthetic membrane, whereas in Rba. sphaeroides and Rba. blasticus a dimeric form is also assembled in which two RCs are surrounded by an S-shaped LH1 antenna. The present work established that dimeric RC–LH1 complexes can also be isolated from Rba. azotoformans and Rba. changlensis, but not from Rba. capsulatus or Rba. vinaykumarii. The compositions of the monomers and dimers isolated from these four species of Rhodobacter were similar to those of the well-characterised RC–LH1 complexes present in Rba. sphaeroides. Pigment proteins were also isolated from strains of Rba. sphaeroides expressing chimeric RC–LH1 complexes. Replacement of either the Rba. sphaeroides LH1 antenna or PufX with its counterpart from Rba. capsulatus led to a loss of the dimeric form of the RC–LH1 complex, but the monomeric form had a largely unaltered composition, even in strains in which the expression level of LH1 relative to the RC was reduced. The chimeric RC–LH1 complexes were also functional, supporting bacterial growth under photosynthetic conditions. The findings help to tease apart the different functions of PufX in different species of Rhodobacter, and a specific protein structural arrangement that allows PufX to fulfil these three functions is proposed.     

Analysis of the Relationship Between Ultrasound of Breast Cancer DOT-SDI and the Expression of MVD,VEGF and HIF-1α

Ultrasonic light scattering tomography system is a new imaging technique for breast function, which associates with diffused optical tomography (DOT) with ultrasonic examination. A recent study found microvessel density (MVD), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1 alpha (HIF-1α) play a major role in angiogenesis of breast cancer. This study analyses the relationship between breast cancer ultrasound synthesis diagnostic index (SDI) and the expression of MVD, VEGF and HIF-1α by testing the expression level of the breast cancer gene MVD, VEGF and HIF-1α. In this study, we disclose the relationship between ultrasound of breast cancer DOT-SDI and the expression of MVD, VEGF and HIF-1α.     

Mapping of the regulatory type Iα and catalytic β subunits of cAMP-dependent protein kinase and interleukin 1α and 1β in the pig

The genes coding for the regulatory type I subunit (PRKAR1A) and the catalytic subunit (PRKACB) of cAMP-dependent protein kinase and the genes for interleukin 1 (IL1A) and interleukin 1 (IL1B) were localized in the pig by means of radioactive in situ hybridization. PRKAR1A was mapped to 12p1.4 and PRKARB to 6q3.1 q3.3. The genes for IL1A and IL1B were both assigned to Chromosome (Chr) 3, in the region q1.2 q1.3 and q1.1 q1.4, respectively. The cDNA nucleotide sequences of these porcine genes were compared with those of human, mouse, and cattle. The location of the genes was discussed in relation to the position of their homologous loci in these mammalian species.     

Interleukin-1β and receptor antagonist (IL-1Ra) gene polymorphisms and the prediction of the risk of end-stage renal disease

Abstract

Cytokines play an important role in the pathogenesis of kidney disease and its progression to end-stage renal disease (ESRD). Inflammation is regulated by the genes of the interleukin 1 (IL-1) gene cluster. Therefore, it was hypothesized that a polymorphism in this gene cluster may be associated with the risk of ESRD. Polymorphisms in the IL-1 gene cluster were examined in a cohort of 222 ESRD patients and 206 controls of similar ethnicity. These individuals were genotyped for IL-1 β (promoter –511 and exon-5 +3953) genes and a variable number of tandem repeats (VNTR) in the IL-1 receptor antagonist gene (IL-1Ra). There was significant difference in genotype frequencies between ESRD patients and control group for IL-1β (promoter region and exon-5) and IL-1Ra gene polymorphism (p<0.001, 0.006 and?<?0.001, respectively). A significant difference was observed in IL-1Ra for 1/1 (410/410) and 1/2 (410/240) genotypes, and the risk for ESRD was higher in those carrying the 1/1 genotype (p=0.014, OR?=?1.692, and p<0.001, OR?=?0.163). Also identified was a novel, rare allele of a single copy of 86 bp in ESRD patients as compared with the controls. The haplotype ‘T-E2-1’ frequency distribution between patients and controls revealed greater than threefold risk (p=0.001, OR?=?3.572, 95% CI?=?1.589–8.032). Genetic linkage between the IL-1β promoter region and exon-5 and between the IL-1β promoter and IL-1Ra of IL-1 gene demonstrated a strong association among the variants in controls (D′?=?0.42, p<0.001, and D′?=?0.39, p=0.001). Thus, the three polymorphisms within the IL-1 cluster are associated with ESRD. This finding is perhaps one of the strongest associations between genotype and ESRD reported, and it suggests that the IL-1 gene cluster affects the risk of development of ESRD.     

Nuclear import of exogenous FGF1 requires the ER-protein LRRC59 and the importins Kpnα1 and Kpnβ1

Fibroblast growth factor 1 (FGF1) taken up by cells into endocytic vesicles can be translocated across vesicular membranes into the cytosol and the nucleus where it has a growth regulatory activity. Previously, leucine-rich repeat containing 59 (LRRC59) was identified as an intracellular binding partner of FGF1, but its biological role remained unknown. Here, we show that LRRC59 is strictly required for nuclear import of exogenous FGF1. siRNA-mediated depletion of LRRC59 did not inhibit the translocation of FGF1 into cytosol, but blocked the nuclear import of FGF1. We also found that an nuclear localization sequence (NLS) in FGF1, Ran GTPase, karyopherin-α1 (Kpnα1), and Kpnβ1 were required for nuclear import of FGF1. Nuclear import of exogenous FGF2, which depends on CEP57/Translokin, was independent of LRRC59, but was dependent on Kpnα1 and Kpnβ1, while the nuclear import of FGF1 was independent of CEP57. LRRC59 is a membrane-anchored protein that localizes to the endoplasmic reticulum (ER) and the nuclear envelope (NE). We found that LRRC59 possesses NLS-like sequences in its cytosolic part that can mediate nuclear import of soluble LRRC59 variants, and that the localization of LRRC59 to the NE depends on Kpnβ1. We propose that LRRC59 facilitates transport of cytosolic FGF1 through nuclear pores by interaction with Kpns and movement of LRRC59 along the ER and NE membranes.     

Activation of decapping involves binding of the mRNA and facilitation of the post-binding steps by the Lsm1-7–Pat1 complex

Decapping is a critical step in the conserved 5′-to-3′ mRNA decay pathway of eukaryotes. The hetero-octameric Lsm1-7–Pat1 complex is required for normal rates of decapping in this pathway. This complex also protects the mRNA 3′-ends from trimming in vivo. To elucidate the mechanism of decapping, we analyzed multiple lsm1 mutants, lsm1-6, lsm1-8, lsm1-9, and lsm1-14, all of which are defective in decapping and 3′-end protection but unaffected in Lsm1-7–Pat1 complex integrity. The RNA binding ability of the mutant complex was found to be almost completely lost in the lsm1-8 mutant but only partially impaired in the other mutants. Importantly, overproduction of the Lsm1-9p- or Lsm1-14p-containing (but not Lsm1-8p-containing) mutant complexes in wild-type cells led to a dominant inhibition of mRNA decay. Further, the mRNA 3′-end protection defect of lsm1-9 and lsm1-14 cells, but not the lsm1-8 cells, could be partly suppressed by overproduction of the corresponding mutant complexes in those cells. These results suggest the following: (1) Decapping requires both binding of the Lsm1-7–Pat1 complex to the mRNA and facilitation of the post-binding events, while binding per se is sufficient for 3′-end protection. (2) A major block exists at the post-binding steps in the lsm1-9 and lsm1-14 mutants and at the binding step in the lsm1-8 mutant. Consistent with these ideas, the lsm1-9, 14 allele generated by combining the mutations of lsm1-9 and lsm1-14 alleles had almost fully lost the RNA binding activity of the complex and behaved like the lsm1-8 mutant.     

ATP and MO25α Regulate the Conformational State of the STRADα Pseudokinase and Activation of the LKB1 Tumour Suppressor

Pseudokinases lack essential residues for kinase activity, yet are emerging as important regulators of signal transduction networks. The pseudokinase STRAD activates the LKB1 tumour suppressor by forming a heterotrimeric complex with LKB1 and the scaffolding protein MO25. Here, we describe the structure of STRADα in complex with MO25α. The structure reveals an intricate web of interactions between STRADα and MO25α involving the αC-helix of STRADα, reminiscent of the mechanism by which CDK2 interacts with cyclin A. Surprisingly, STRADα binds ATP and displays a closed conformation and an ordered activation loop, typical of active protein kinases. Inactivity is accounted for by nonconservative substitution of almost all essential catalytic residues. We demonstrate that binding of ATP enhances the affinity of STRADα for MO25α, and conversely, binding of MO25α promotes interaction of STRADα with ATP. Mutagenesis studies reveal that association of STRADα with either ATP or MO25α is essential for LKB1 activation. We conclude that ATP and MO25α cooperate to maintain STRADα in an “active” closed conformation required for LKB1 activation. It has recently been demonstrated that a mutation in human STRADα that truncates a C-terminal region of the pseudokinase domain leads to the polyhydramnios, megalencephaly, symptomatic epilepsy (PMSE) syndrome. We demonstrate this mutation destabilizes STRADα and prevents association with LKB1. In summary, our findings describe one of the first structures of a genuinely inactive pseudokinase. The ability of STRADα to activate LKB1 is dependent on a closed “active” conformation, aided by ATP and MO25α binding. Thus, the function of STRADα is mediated through an active kinase conformation rather than kinase activity. It is possible that other pseudokinases exert their function through nucleotide binding and active conformations.     

Substitutions of the Conserved Thr42 Increased the Roles of the ε-Subunit of Maize CF1 as CF1 Inhibitor and Proton Gate

The conserved residue Thr42 of -subunit of the chloroplast ATP synthase of maize (Zea mays L.) was substituted with Cys, Arg, and Ile, respectively, through site-directed mutagenesis. The over-expressed and refolded -proteins were purified by chromatography on DEAE-cellulose and FPLC on mono-Q column, which were as biologically active (inhibiting Ca²⁺-ATPase activity and blocking proton gate) as the native subunit isolated from chloroplasts. The T42C and T42R showed higher inhibitory activities on the soluble CF₁(–) Ca²⁺-ATPase than the WT. The T42I inhibited the Ca²⁺-ATPase activity of soluble CF₁ and restored photophosphorylation activity of membrane-bound CF₁ deficient in the most efficiently. Far-ultraviolet CD spectra showed that the portions of -helix and -sheet structures of the three mutants were somewhat different from WT. Thus the conserved residue Thr42 may be important for maintaining the structure and function of the -subunit and the basic functions of the -subunit as far as an inhibitor of Ca²⁺-ATPase and the proton gate are related.     

The development of the antigenic structures of human α1-antichymotrypsin and C 1 q

Summary The cross-reactions of human ₁-antichymotrypsin and C 1 q with their homologues in the plasmas of the chimpanzee, several Old World monkeys and nine non-primate eutheria were investigated by standard procedures. The results show that cross-reactions are limited to the first species mentioned. Comparative Ouchterlony tests and absorption controls revealed the presence of two (human) determinants on both human and chimpanzee molecules, while the cercopithecoids analyzed carried only one of them on their homologue. The results are discussed briefly with reference to earlier findings from this laboratory.

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Zusammenfassung Die Kreuzreaktionen des menschlichen ₁-Antichymotrypsin und des C 1 q mit seinen Homologen im Plasma des Schimpansen, einiger Altweltaffen und demjenigen von 9 Nichtprimaten (Eutheria) wurden mit Standardmethoden untersucht. Die Ergebnisse zeigen, daß Kreuzreaktionen auf die zuerst genannten Species beschränkt sind. Vergleichende Ouchterlony-Tests und Absorptionskontrollen ließen die Anwesenheit zweier (menschlicher) Determinaten auf den Molekülen des Menschen und des Schimpansen erkennbar werden, während die untersuchten Cercopithecoidea nur eine dieser Determinanten besitzen. Die Ergebnisse werden kurz im Zusammenhang mit früheren Befunden aus unserem Laboratorium diskutiert.

     

Affinity of different α1-agonists and antagonists for the α1-adrenoceptors of rabbit and rat liver membranes

In membranes prepared from rabbit liver, competition with [³H] prazosin by different α₁-agonists and antagonists revealed different affinities in comparison to the results obtained on rat liver membranes, and showed a good correlation with the affinity of the same compounds for the cloned α_1c-adrenoceptor subtype. The potencies observed on rat liver membranes were well correlated with the affinity observed for the cloned α_1b-adrenoceptors. These results confirm that rabbit and rat liver membranes preparations can be utilized to evaluate the affinity of compounds for these α₁-adrenergic subtypes.