Inhibition of plasmin attenuates murine acute graft-versus-host disease mortality by suppressing the matrix metalloproteinase-9-dependent inflammatory cytokine storm and effector cell trafficking.

The systemic inflammatory response observed during acute graft-versus-host disease (aGVHD) is driven by proinflammatory cytokines, a 'cytokine storm'. The function of plasmin in regulating the inflammatory response is not fully understood, and its role in the development of aGVHD remains unresolved. Here we show that plasmin is activated during the early phase of aGVHD in mice, and its activation correlated with aGVHD severity in humans. Pharmacological plasmin inhibition protected against aGVHD-associated lethality in mice. Mechanistically, plasmin inhibition impaired the infiltration of inflammatory cells, the release of membrane-associated proinflammatory cytokines including tumor necrosis factor-α (TNF-α) and Fas-ligand directly, or indirectly via matrix metalloproteinases (MMPs) and alters monocyte chemoattractant protein-1 (MCP-1) signaling. We propose that plasmin and potentially MMP-9 inhibition offers a novel therapeutic strategy to control the deadly cytokine storm in patients with aGVHD, thereby preventing tissue destruction.

Dynamic chromosome reorganization in the osprey ( Pandion haliaetus , Pandionidae, Falconiformes): relationship between chromosome size and the chromosomal distribution of centromeric repetitive DNA sequences.

The osprey (Pandion haliaetus) has a diploid number of 74 chromosomes, consisting of a large number of medium-sized macrochromosomes and relatively few microchromosomes; this differs greatly from the typical avian karyotype. Chromosome painting with chicken DNA probes revealed that the karyotype of P. haliaetus differs from the chicken karyotype by at least 14 fission events involving macrochromosomes (chicken chromosomes 1-9 and Z) and at most 15 fusions of microchromosomes, suggesting that considerable karyotype reorganization occurred in P. haliaetus in a similar manner previously reported for Accipitridae. A distinct difference was observed, however, between Accipitridae and Pandionidae with respect to the pattern of chromosome rearrangements that occurred after fissions of macrochromosomes. Metacentric or submetacentric chromosomes 1-5 in P. haliaetus appear to have been formed by centric fusion of chromosome segments derived from macrochromosomal fissions. By contrast, many pairs of bi-armed chromosomes in Accipitridae species seem to result from pericentric inversions that occurred in the fission-derived chromosomes. Two families of repetitive sequences were isolated; the 173-bp PHA-HaeIII sequence occurred on all chromosomes, whereas intense signals from the 742-bp PHA-NsiI sequence were localized to all acrocentric chromosomes, with weak signals on most of the bi-armed chromosomes. Two repetitive sequences cohybridized in the centromeric heterochromatin; however, the sequences differed in unit size, nucleotide sequence and GC content. The results suggest that the 2 sequence families originated from different ancestral sequences and were homogenized independently in centromeres, and that a chromosome size-dependent compartmentalization may have been lost in P. haliaetus.

Divergence of repetitive DNA sequences in the heterochromatin of medaka fishes: Molecular cytogenetic characterization of constitutive heterochromatin in two medaka species: Oryzias hubbsi and O. celebensis (Adrianichthyidae, Beloniformes).

The large biarmed chromosomes of Oryzias celebensis [2n = 36, fundamental arm number (FN) = 48] are considered to have resulted from fusions of acrocentric chromosomes in the ancestral karyotype of Oryzias, which is retained in O. hubbsi (2n = 48, FN = 48). To understand the molecular evolution of heterochromatin associated with karyotype reorganization in medaka fishes, we cloned 3 and 6 novel families of heterochromatin-related repetitive DNA sequences from O. hubbsi and O. celebensis, respectively, and characterized them using molecular cytogenetics. Two AT-rich repetitive sequences isolated from the genomic DNA of O. hubbsi, a 164-bp satellite DNA (OHU-RsaI-Scen) and a 177-bp telomere-specific repeat (OHU-RsaI-Stelo), were shown to be major components of the constitutive heterochromatin of centromeres and telomeres, respectively. A GC-rich 326-bp sequence, named OHU-AluI-M1, was colocalized with the 18S-28S ribosomal RNA gene cluster to a single autosomal pair of chromosomes and the W chromosome. In O. celebensis, 2 major satellite DNA sequences (the AT-rich 157-bp OCE-AluI-Scen sequence and the 186-bp OCE-HinfI-Scen sequence) were identified in the centromeric regions of almost all chromosomes. The 197-bp OCE-HinfI-S6 sequence was located in the centromeric and distal and/or interstitial heterochromatin of almost all chromosomes, and the 191-bp OCE-HinfI-S8 sequence was located in 6 pairs of chromosomes. Constitutive heterochromatin on the short arm of large submetacentric chromosome 5 was composed of at least 3 different repetitive sequences: the 171-bp OCE-AluI-S18 sequence, the 197-bp OCE-HinfI-S6 sequence and the 172-bp OCE-HinfI-S11 sequence. All families of repeated sequences showed no nucleotide sequence similarity with each other and high species-specificity among 7 different species. These results suggest that the heterochromatin of O. hubbsi and O. celebensis consists of various types of repetitive sequence and that the sequences evolved independently and were then amplified site-specifically in each lineage after karyotype reorganization occurred in the ancestral karyotype.

Karyotype reorganization with conserved genomic compartmentalization in dot-shaped microchromosomes in the Japanese mountain hawk-eagle (Nisaetus nipalensis orientalis, Accipitridae).

The karyotype of the Japanese mountain hawk-eagle (Nisaetus nipalensis orientalis) (2n = 66) consists of a large number of medium-sized and small chromosomes but only 4 pairs of dot-shaped microchromosomes, in contrast to the typical avian karyotype with a small number of macrochromosomes and many indistinguishable microchromosomes. To investigate the drastic karyotype reorganization in this species, we performed a molecular cytogenetic characterization employing chromosome in situ hybridization and molecular cloning of centromeric heterochromatin. Cross-species chromosome painting with chicken chromosome-specific probes 1-9 and Z and a paint pool of 20 microchromosome pairs revealed that the N. n. orientalis karyotype differs from chicken by at least 13 fissions of macrochromosomes and 15 fusions between microchromosomes and between micro- and macrochromosomes. A novel family of satellite DNA sequences (NNO-ApaI) was isolated, consisting of a GC-rich 173-bp repeated sequence element. The NNO-ApaI sequence was localized to the C-positive centromeric heterochromatin of 4 pairs of microchromosomes, which evolved concertedly by homogenization between the microchromosomes. These results suggest that the 4 pairs of dot-shaped microchromosomes have retained their genomic compartmentalization from other middle-sized and small chromosomes.

Homoeologous chromosomes of Xenopus laevis are highly conserved after whole-genome duplication.

It has been suggested that whole-genome duplication (WGD) occurred twice during the evolutionary process of vertebrates around 450 and 500 million years ago, which contributed to an increase in the genomic and phenotypic complexities of vertebrates. However, little is still known about the evolutionary process of homoeologous chromosomes after WGD because many duplicate genes have been lost. Therefore, Xenopus laevis (2n=36) and Xenopus (Silurana) tropicalis (2n=20) are good animal models for studying the process of genomic and chromosomal reorganization after WGD because X. laevis is an allotetraploid species that resulted from WGD after the interspecific hybridization of diploid species closely related to X. tropicalis. We constructed a comparative cytogenetic map of X. laevis using 60 complimentary DNA clones that covered the entire chromosomal regions of 10 pairs of X. tropicalis chromosomes. We consequently identified all nine homoeologous chromosome groups of X. laevis. Hybridization signals on two pairs of X. laevis homoeologous chromosomes were detected for 50 of 60 (83%) genes, and the genetic linkage is highly conserved between X. tropicalis and X. laevis chromosomes except for one fusion and one inversion and also between X. laevis homoeologous chromosomes except for two inversions. These results indicate that the loss of duplicated genes and inter- and/or intrachromosomal rearrangements occurred much less frequently in this lineage, suggesting that these events were not essential for diploidization of the allotetraploid genome in X. laevis after WGD.

Plasmin inhibitor reduces T-cell lymphoid tumor growth by suppressing matrix metalloproteinase-9-dependent CD11b(+)/F4/80(+) myeloid cell recruitment.

Activation of the fibrinolytic system during lymphoma progression is a well-documented clinical phenomenon. But the mechanism by which the fibrinolytic system can modulate lymphoma progression has been elusive. The main fibrinolytic enzyme, plasminogen (Plg)/plasmin (Plm), can activate matrix metalloproteinases (MMPs), such as MMP-9, which has been linked to various malignancies. Here we provide the evidence that blockade of Plg reduces T-cell lymphoma growth by inhibiting MMP-9-dependent recruitment of CD11b(+)F4/80(+) myeloid cells locally within the lymphoma tissue. Genetic Plg deficiency and drug-mediated Plm blockade delayed T-cell lymphoma growth and diminished MMP-9-dependent CD11b(+)F4/80(+) myeloid cell infiltration into lymphoma tissues. A neutralizing antibody against CD11b inhibited T-cell lymphoma growth in vivo, which indicates that CD11b(+) myeloid cells have a role in T-cell lymphoma growth. Plg deficiency in T-cell lymphoma-bearing mice resulted in reduced plasma levels of the growth factors vascular endothelial growth-A and Kit ligand, both of which are known to enhance myeloid cell proliferation. Collectively, the data presented in this study demonstrate a previously undescribed role of Plm in lymphoproliferative disorders and provide strong evidence that specific blockade of Plg represents a promising approach for the regulation of T-cell lymphoma growth.

Body fat distribution and noncommunicable diseases in populations: overview of the 2008 WHO Expert Consultation on Waist Circumference and Waist-Hip Ratio.

A World Health Organization (WHO) Expert Consultation on Waist Circumference (WC) and Waist-Hip Ratio (WHR) was convened in Geneva from 8 to 11 December 2008 to consider approaches to developing international guidelines for indices and action levels in order to characterize health risks associated with these measures of body fat distribution-alternative or complementary to the existing WHO guidelines for assessments of generalized obesity on the basis of body mass index. Six background papers prepared for the Consultation are compiled in this issue. These six papers examine a range of health outcomes and issues, including whether there is a basis for choosing WC over WHR and whether different action levels by gender, age, ethnicity, country or region are warranted. Although guidelines involving WC and WHR are potentially useful and clearly required, the challenges in identifying cutoffs for international guidelines should not be underestimated or oversimplified. The final report and outcomes of the Expert Consultation will be published by WHO.

A multiplex PCR assay for molecular sexing of the naked mole-rat (Heterocephalus glaber).

For molecular sexing of the naked mole-rat (Heterocephalus glaber), we designed a PCR primer set to amplify part of the Y-linked DBY gene. When this primer set was applied to the samples of known sex with the 16S rRNA gene (16S rDNA) primers as control, PCR products were successfully obtained as two DNA bands in males, a male-specific 163 bp DBY band and a 446 bp band of 16S rDNA shared with females, whereas females showed only the common band. This result shows that this multiplex PCR assay is useful for sex identification of H. glaber.

The ZW micro-sex chromosomes of the Chinese soft-shelled turtle (Pelodiscus sinensis, Trionychidae, Testudines) have the same origin as chicken chromosome 15.

The Chinese soft-shelled turtle (Pelodiscus sinensis, Trionychidae, Testudines) has ZZ/ZW-type micro-sex chromosomes where the 18S-28S ribosomal RNA genes (18S-28S rDNA) are located. The W chromosome is morphologically differentiated from the Z chromosome by partial deletion and amplification of 18S-28S rDNA and W-specific repetitive sequences. We recently found a functional gene (TOP3B) mapped on the P. sinensis Z chromosome, which is located on chicken (Gallus gallus, GGA) chromosome 15. Then we cloned turtle homologues of 4 other GGA15-linked genes (GIT2, NF2, SBNO1, SF3A1) and localized them to P. sinensis chromosomes. The 4 genes all mapped on the Z chromosome, and 2 of them (SBNO1, SF3A1) were also localized to the W chromosome. Our mapping data suggest that at least one large inversion occurred between GGA15 and the P. sinensis Z chromosome, and that there are homologous regions in the distal portions of both the short and long arms between the Z and W chromosomes. W chromosomal differentiation in P. sinensis probably proceeded by the deletion of the proximal chromosomal region followed by 18S-28S rDNA amplification, after a paracentric inversion occurred at the breakpoints between the distal region of 18S-28S rDNA and the proximal region of SBNO1 on the Z chromosome.

Karyological characterization of the butterfly lizard (Leiolepis reevesii rubritaeniata, Agamidae, Squamata) by molecular cytogenetic approach.

Karyological characterization of the butterfly lizard (Leiolepis reevesii rubritaeniata) was performed by conventional Giemsa staining, Ag-NOR banding, FISH with the 18S-28S and 5S rRNA genes and telomeric (TTAGGG)n sequences, and CGH. The karyotype was composed of 2 distinct components, macrochromosomes and microchromosomes, and the chromosomal constitution was 2n = 2x = 36 (L(4)(m) + L(2)(sm) + M(2)(m) + S(4)(m) + 24 microchromosomes). NORs and the 18S-28S rRNA genes were located at the secondary constriction of the long arm of chromosome 1, and the 5S rRNA genes were localized to the pericentromeric region of chromosome 6. Hybridization signals of (TTAGGG)n sequences were observed at the telomeric ends of all chromosomes and interstitially at the same position as the 18S-28S rRNA genes, suggesting that in the Leiolepinae tandem fusion probably occurred between chromosome 1 and a microchromosome where the 18S-28S rRNA genes were located. CGH analysis, however, failed to identify sex chromosomes, suggesting that this species may have a TSD system or exhibit GSD with morphologically undetectable cryptic sex chromosomes. Homologues of 6 chicken Z-linked genes (ACO1/IREBP, ATP5A1, CHD1, DMRT1, GHR, RPS6) were all mapped to chromosome 2p in the same order as on the snake chromosome 2p.

Pharmacokinetic analysis of factors determining elimination pathways for sulfate and glucuronide metabolites of xenobiotics. III: mechanisms for sinusoidal efflux of 4-methylumbelliferone sulfate.

1. To elucidate the mechanisms involved in the sinusoidal efflux of sulfate and glucuronide metabolites of 4-methylumbelliferone (4MU), isolated rat liver perfusion studies were performed under several conditions. 2. The effect of sodium azide on the hepatic handling of both conjugates was examined. The net sinusoidal efflux clearance (CL(eff)) based on the unbound concentration in the liver did not change for 4MU glucuronide (4MUG) or significantly increase for 4MU sulfate (4MUS), suggesting that the sinusoidal efflux of both conjugates is not mediated by the transport systems dependent on adenosine triphosphate. 3. Under Cl(-)-depleted conditions, the CL(eff) of 4MUG significantly decreased, but the saturation of its sinusoidal efflux rather than the transport system dependent on Cl(-) might be involved because the hepatic concentration of 4MUG was extensively higher than that of the control study due to the extremely attenuated biliary excretion. The CL(eff) of 4MUS also significantly decreased, but its hepatic concentration was not different from that in the control study, suggesting that the transport system using Cl(-) is involved in the sinusoidal efflux of 4MUS. 4. The effect of glutathione was examined. CL(eff) of 4MUG was not affected by the additional glutathione, but CL(eff) of 4MUS decreased significantly, suggesting that some transport system sensitive to glutathione is involved in the sinusoidal efflux of 4MUS, but not of 4MUG. 5. Transporters such as Oatp1, Oatp2 and/or Npt1 might be involved in the sinusoidal efflux of 4MUS, but 4MUG is secreted from the sinusoidal membrane via the systems that are totally different from those for 4MUS.

A life course approach to diet, nutrition and the prevention of chronic diseases.

OBJECTIVE: To briefly review the current understanding of the aetiology and prevention of chronic diseases using a life course approach, demonstrating the life-long influences on the development of disease. DESIGN: A computer search of the relevant literature was done using Medline-'life cycle' and 'nutrition' and reviewing the articles for relevance in addressing the above objective. Articles from references dated before 1990 were followed up separately. A subsequent search using Clio updated the search and extended it by using 'life cycle', 'nutrition' and 'noncommunicable disease' (NCD), and 'life course'. Several published and unpublished WHO reports were key in developing the background and arguments. SETTING: International and national public health and nutrition policy development in light of the global epidemic in chronic diseases, and the continuing nutrition, demographic and epidemiological transitions happening in an increasingly globalized world. RESULTS OF REVIEW: There is a global epidemic of increasing obesity, diabetes and other chronic NCDs, especially in developing and transitional economies, and in the less affluent within these, and in the developed countries. At the same time, there has been an increase in communities and households that have coincident under- and over-nutrition. CONCLUSIONS: The epidemic will continue to increase and is due to a lifetime of exposures and influences. Genetic predisposition plays an unspecified role, and with programming during fetal life for adult disease contributing to an unknown degree. A global rise in obesity levels is contributing to a particular epidemic of type 2 diabetes as well as other NCDs. Prevention will be the most cost-effective and feasible approach for many countries and should involve three mutually reinforcing strategies throughout life, starting in the antenatal period.

Control of electron transfer in nitric-oxide synthases. Swapping of autoinhibitory elements among nitric-oxide synthase isoforms.

To clarify the role of the autoinhibitory insert in the endothelial (eNOS) and neuronal (nNOS) nitric-oxide synthases, the insert was excised from nNOS and chimeras with its reductase domain; the eNOS and nNOS inserts were swapped and put into the normally insertless inducible (iNOS) isoform and chimeras with the iNOS reductase domain; and an RRKRK sequence in the insert suggested by earlier peptide studies to be important (Salerno, J. C., Harris, D. E., Irizarry, K., Patel, B., Morales, A. J., Smith, S. M., Martasek, P., Roman, L. J., Masters, B. S., Jones, C. L., Weissman, B. A., Lane, P., Liu, Q., and Gross, S. S. (1997) J. Biol. Chem. 272, 29769-29777) was mutated. Insertless nNOS required calmodulin (CaM) for normal NOS activity, but the Ca(2+) requirement for this activity was relaxed. Furthermore, insert deletion enhanced CaM-free electron transfer within nNOS and chimeras with the nNOS reductase, emphasizing the involvement of the insert in modulating electron transfer. Swapping the nNOS and eNOS inserts gave proteins with normal NOS activities, and the nNOS insert acted normally in raising the Ca(2+) dependence when placed in eNOS. Insertion of the eNOS insert into iNOS and chimeras with the iNOS reductase domain significantly lowered NOS activity, consistent with inhibition of electron transfer by the insert. Mutation of the eNOS RRKRK to an AAAAA sequence did not alter the eNOS Ca(2+) dependence but marginally inhibited electron transfer. The salt dependence suggests that the insert modulates electron transfer within the reductase domain prior to the heme/reductase interface. The results clarify the role of the reductase insert in modulating the Ca(2+) requirement, electron transfer rate, and overall activity of nNOS and eNOS.

The metal binding properties of the zinc site of yeast copper-zinc superoxide dismutase: implications for amyotrophic lateral sclerosis.

We have investigated factors that influence the properties of the zinc binding site in yeast copper-zinc superoxide dismutase (CuZnSOD). The properties of yeast CuZnSOD are essentially invariant from pH 5 to pH 9. However, below this pH range there is a change in the nature of the zinc binding site which can be interpreted as either (1) a change in metal binding affinity from strong to weak, (2) the expulsion of the metal bound at this site, or (3) a transition from a normal distorted tetrahedral ligand orientation to a more symmetric arrangement of ligands. This change is strongly reminiscent of a similar pH-induced transition seen for the bovine protein and, based on the data presented herein, is proposed to be a property that is conserved among CuZnSODs. The transition demonstrated for the yeast protein is not only sensitive to the pH of the buffering solution but also to the occupancy and redox status of the adjacent copper binding site. Furthermore, we have investigated the effect of single site mutations on the pH- and redox-sensitivity of Co2+ binding at the zinc site. Each of the mutants H46R, H48Q, H63A, H63E, H80C, G85R, and D83H is capable of binding Co2+ to a zinc site with a distorted tetrahedral geometry similar to that of wild-type. However, they do so only if Cu+ is bound at the copper site or if the pH in raised to near physiological levels, indicating that the change at the zinc binding site seen in the wild-type is conserved in the mutants, albeit with an altered pKa. The mutants H71C and D83A did not bind Co2+ in a wild-type-like fashion under any of the conditions tested. This study reveals that the zinc binding site is exquisitely sensitive to changes in the protein environment. Since three of the mutant yeast proteins investigated here contain mutations analogous to those that cause ALS (amyotrophic lateral sclerosis) in humans, this finding implicates improper metal binding as a mechanism by which CuZnSOD mutants exert their toxic gain of function.

Persistent hypoglossal artery associated with arteriovenous malformation: a case report.

A rare case of persistent hypoglossal artery in conjunction with arteriovenous malformation was presented. MRA could delineate persistent hypoglossal artery and arteriovenous malformation very clearly. The patient suffered from intracranial hemorrhage from in the 37th week of pregnancy. MRI, MRA, angiography, and CT of this rare condition are reported.

Mutation of the five conserved histidines in the endothelial nitric-oxide synthase hemoprotein domain. No evidence for a non-heme metal requirement for catalysis.

Five conserved histidine residues are found in the human endothelial nitric-oxide synthase (NOS) heme domain: His-420, His-421, and His-461 are close to the heme, whereas His-146 and His-214 are some distance away. To investigate whether the histidines form a non-heme iron-binding site, we have expressed the H146A, H214A, H420A, H421A, and H461A mutants. The H420A mutant could not be isolated, and the H146A and H421A mutants were inactive. The H214A mutant resembled the wild-type enzyme in all respects. The H461A mutant had a low-spin heme, but high concentrations of L-Arg and tetrahydrobiopterin led to partial recovery of activity. Laser atomic emission showed that the only significant metal in NOS other than calcium and iron is zinc. The activities of the NOS isoforms were not increased by incubation with Fe(2+), but were inhibited by high Fe(2+) or Zn(2+) concentrations. The histidine mutations altered the ability of the protein to dimerize and to bind heme. However, the protein metal content, the inability of exogenous Fe(2+) to increase catalytic activity, and the absence of evidence that the conserved histidines form a metal site provide no support for a catalytic role for a non-heme redox-active metal.