Recombinant Collagen Engineered to Bind to Discoidin Domain Receptor Functions as a Receptor Inhibitor

A bacterial collagen-like protein Scl2 has been developed as a recombinant collagen model system to host human collagen ligand-binding sequences, with the goal of generating biomaterials with selective collagen bioactivities. Defined binding sites in human collagen for integrins, fibronectin, heparin, and MMP-1 have been introduced into the triple-helical domain of the bacterial collagen and led to the expected biological activities. The modular insertion of activities is extended here to the discoidin domain receptors (DDRs), which are collagen-activated receptor tyrosine kinases. Insertion of the DDR-binding sequence from human collagen III into bacterial collagen led to specific receptor binding. However, even at the highest testable concentrations, the construct was unable to stimulate DDR autophosphorylation. The recombinant collagen expressed in Escherichia coli does not contain hydroxyproline (Hyp), and complementary synthetic peptide studies showed that replacement of Hyp by Pro at the critical Gly-Val-Met-Gly-Phe-Hyp position decreased the DDR-binding affinity and consequently required a higher concentration for the induction of receptor activation. The ability of the recombinant bacterial collagen to bind the DDRs without inducing kinase activation suggested it could interfere with the interactions between animal collagen and the DDRs, and such an inhibitory role was confirmed in vitro and with a cell migration assay. This study illustrates that recombinant collagen can complement synthetic peptides in investigating structure-activity relationships, and this system has the potential for the introduction or inhibition of specific biological activities.     

Nitric oxide-forming reaction between the iron-N-methyl-D-glucamine dithiocarbamate complex and nitrite

The objective of this study was to elucidate the origin of the nitric oxide-forming reactions from nitrite in the presence of the iron-N-methyl-D-glucamine dithiocarbamate complex ((MGD)(2)Fe(2+)). The (MGD)(2)Fe(2+) complex is commonly used in electron paramagnetic resonance (EPR) spectroscopic detection of NO both in vivo and in vitro. Although it is widely believed that only NO can react with (MGD)(2)Fe(2+) complex to form the (MGD)(2)Fe(2+).NO complex, a recent article reported that the (MGD)(2)Fe(2+) complex can react not only with NO, but also with nitrite to produce the characteristic triplet EPR signal of (MGD)(2)Fe(2+).NO (Hiramoto, K., Tomiyama, S., and Kikugawa, K. (1997) Free Radical Res. 27, 505-509). However, no detailed reaction mechanisms were given. Alternatively, nitrite is considered to be a spontaneous NO donor, especially at acidic pH values (Samouilov, A., Kuppusamy, P., and Zweier, J. L. (1998) Arch Biochem. Biophys. 357, 1-7). However, its production of nitric oxide at physiological pH is unclear. In this report, we demonstrate that the (MGD)(2)Fe(2+) complex and nitrite reacted to form NO as follows: 1) (MGD)(2)Fe(2).NO complex was produced at pH 7.4; 2) concomitantly, the (MGD)(3)Fe(3+) complex, which is the oxidized form of (MGD)(2)Fe(2+), was formed; 3) the rate of formation of the (MGD)(2)Fe(2+).NO complex was a function of the concentration of [Fe(2+)](2), [MGD], [H(+)] and [nitrite].     

Two Family B DNA Polymerases from Aeropyrum pernix, an Aerobic Hyperthermophilic Crenarchaeote

DNA polymerase activities in fractionated cell extract of Aeropyrum pernix, a hyperthermophilic crenarchaeote, were investigated. Aphidicolin-sensitive (fraction I) and aphidicolin-resistant (fraction II) activities were detected. The activity in fraction I was more heat stable than that in fraction II. Two different genes (polA and polB) encoding family B DNA polymerases were cloned from the organism by PCR using degenerated primers based on the two conserved motifs (motif A and B). The deduced amino acid sequences from their entire coding regions contained all of the motifs identified in family B DNA polymerases for 3'-->5' exonuclease and polymerase activities. The product of polA gene (Pol I) was aphidicolin resistant and heat stable up to 80 degrees C. In contrast, the product of polB gene (Pol II) was aphidicolin sensitive and stable at 95 degrees C. These properties of Pol I and Pol II are similar to those of fractions II and I, respectively, and moreover, those of Pol I and Pol II of Pyrodictium occultum. The deduced amino acid sequence of A. pernix Pol I exhibited the highest identities to archaeal family B DNA polymerase homologs found only in the crenarchaeotes (group I), while Pol II exhibited identities to homologs found in both euryarchaeotes and crenarchaeotes (group II). These results provide further evidence that the subdomain Crenarchaeota has two family B DNA polymerases. Furthermore, at least two DNA polymerases work in the crenarchaeal cells, as found in euryarchaeotes, which contain one family B DNA polymerase and one heterodimeric DNA polymerase of a novel family.     

Three Proliferating Cell Nuclear Antigen-Like Proteins Found in the Hyperthermophilic Archaeon Aeropyrum pernix: Interactions with the Two DNA Polymerases

Proliferating cell nuclear antigen (PCNA) is an essential component in the eukaryotic DNA replication machinery, in which it works for tethering DNA polymerases on the DNA template to accomplish processive DNA synthesis. The PCNA also interacts with many other proteins in important cellular processes, including cell cycle control, DNA repair, and an apoptotic pathway in the domain EUCARYA: We identified three genes encoding PCNA-like sequences in the genome of Aeropyrum pernix, a crenarchaeal archaeon. We cloned and expressed these genes in Escherichia coli and analyzed the gene products. All three PCNA homologs stimulated the primer extension activities of the two DNA polymerases, polymerase I (Pol I) and Pol II, identified in A. pernix to various extents, among which A. pernix PCNA 3 (ApePCNA3) provided a most remarkable effect on both Pol I and Pol II. The three proteins were confirmed to exist in the A. pernix cells. These results suggest that the three PCNAs work as the processivity factor of DNA polymerases in A. pernix cells under different conditions. In Eucarya, three checkpoint proteins, Hus1, Rad1, and Rad9, have been proposed to form a PCNA-like ring structure and may work as a sliding clamp for the translesion DNA polymerases. Therefore, it is very interesting that three active PCNAs were found in one archaeal cell. Further analyses are necessary to determine whether each PCNA has specific roles, and moreover, how they reveal different functions in the cells.     

Azelnidipine Inhibits Cultured Rat Aortic Smooth Muscle Cell Death Induced by Cyclic Mechanical Stretch

Acute aortic dissection is the most common life-threatening vascular disease, with sudden onset of severe pain and a high fatality rate. Clarifying the detailed mechanism for aortic dissection is of great significance for establishing effective pharmacotherapy for this high mortality disease. In the present study, we evaluated the influence of biomechanical stretch, which mimics an acute rise in blood pressure using an experimental apparatus of stretching loads in vitro, on rat aortic smooth muscle cell (RASMC) death. Then, we examined the effects of azelnidipine and mitogen-activated protein kinase inhibitors on mechanical stretch-induced RASMC death. The major findings of the present study are as follows: (1) cyclic mechanical stretch on RASMC caused cell death in a time-dependent manner up to 4 h; (2) cyclic mechanical stretch on RASMC induced c-Jun N-terminal kinase (JNK) and p38 activation with peaks at 10 min; (3) azelnidipine inhibited RASMC death in a concentration-dependent manner as well as inhibited JNK and p38 activation by mechanical stretch; and (4) SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor) protected against stretch-induced RASMC death; (5) Antioxidants, diphenylene iodonium and tempol failed to inhibit stretch-induced RASMC death. On the basis of the above findings, we propose a possible mechanism where an acute rise in blood pressure increases biomechanical stress on the arterial walls, which induces RASMC death, and thus, may lead to aortic dissection. Azelnidipine may be used as a pharmacotherapeutic agent for prevention of aortic dissection independent of its blood pressure lowering effect.     

Similarity, in molecular structure and function, between the plant toxin purothionin and the mammalian pore-forming proteins.

Many proteins containing domains of a cysteine-rich repeated motif, such as epidermal growth factor (EGF), have been reported. Here we report strong similarity between the amino acid sequence of a plant toxin--i.e., purothionin and its homologues--and with those of a domain found in mammalian pore-forming cytoplasmic proteins: components of complement and perforin of cytotoxic T-lymphocytes or natural killer-like cytotoxic cells. These similar sequences were found to be identical to the so-called EGF-like cysteine-rich repeated motif itself. Electron-microscopic observations indicated that, like complement and perforin, purothionin forms pores in the cytoplasmic membrane of target cells, resulting in their death within a few hours. On the basis of these sequence comparisons and physiological functions, we propose a scheme for the evolution of proteins containing modules of the cysteine-rich repeat motif.     

Localized melting of duplex DNA by Cdc6/Orc1 at the DNA replication origin in the hyperthermophilic archaeon Pyrococcus furiosus

The initiation step is a key process to regulate the frequency of DNA replication. Although recent studies in Archaea defined the origin of DNA replication (oriC) and the Cdc6/Orc1 homolog as an origin recognition protein, the location and mechanism of duplex opening have remained unclear. We have found that Cdc6/Orc1 binds to oriC and unwinds duplex DNA in the hyperthermophilic archaeon Pyrococcus furiosus, by means of a P1 endonuclease assay. A primer extension analysis further revealed that this localized unwinding occurs in the oriC region at a specific site, which is 12-bp long and rich in adenine and thymine. This site is different from the predicted duplex unwinding element (DUE) that we reported previously. We also discovered that Cdc6/Orc1 induces topological changes in supercoiled oriC DNA, and that this process is dependent on the AAA+ domain. These results indicate that topological alterations of oriC DNA by Cdc6/Orc1 introduce a single-stranded region at the 12-mer site, that could possibly serve as an entry point for Mcm helicase.     

Comprehensive Search for DNA Polymerase in the Hyperthermophilic Archaeon,Pyrococcus furiosus

DNA polymerase activities were scanned in a Pyrococcus furiosus cell extract to identify all of the DNA polymerases in this organism. Three main fractions containing DNA polymerizing activity were subjected to Western blot analyses, which revealed that the main activities in each fraction were derived from three previously identified DNA polymerases. PCNA (proliferating cell nuclear antigen), the sliding clamp of DNA polymerases, did not bind tightly to any of the three DNA polymerases. A primer usage preference was also shown for each purified DNA polymerase. Considering their biochemical properties, the roles of the three DNA polymerases during DNA replication in the cells are discussed.     

Inhibitory effects of whisky polyphenols on melanogenesis in mouse B16 melanoma cells

Whisky exerts an inhibitory effect on melanogenesis in B16 cells, the anti-melanogenic activity being positively correlated with the aging period and anti-oxidative activity of whisky. We examined the correlation between the inhibition of melanogenesis and the concentration of each compound in various whiskies to evaluate the importance of 11 different whisky polyphenols, including ellagic acid, gallic acid and lyoniresinol, in the anti-melanogenic activity of whisky. The concentration of all the compounds was positively correlated with the anti-melanogenic activity of whisky. Ellagic acid, gallic acid and lyoniresinol were the predominant polyphenols in the whiskies measured by HPLC. These three compounds also significantly inhibited the melanogenesis and tyrosinase activity in B16 cells. Ellagic acid, gallic acid and lyoniresinol were confirmed as the major participants in the anti-melanogenic activity of whisky.