Crystal structures of unligated and CN-ligated Glycera dibranchiata monomer ferric hemoglobin components III and IV

Erythrocytes of the marine annelid, Glycera dibranchiata, contain a mixture of monomeric and polymeric hemoglobins. There are three major monomer hemoglobin components, II, III, IV (also called GMH2, 3, and 4), that have been highly purified and well characterized. We have now crystallized GMH3 and GMH4 and determined their structures to 1.4-1.8 A resolution. The structures were determined for these two monomer hemoglobins in the oxidized (Fe3+, ferric, or met-) forms in both the unligated and cyanide-ligated states. This work differs from two published, refined structures of a Glycera dibranchiata monomer hemoglobin, which has a sequence that is substantially different from any bona fide major monomer hemoglobins (GMH2, 3, or 4). The high-resolution crystal structures (presented here) and the previous NMR structure of CO-ligated GMH4, provide a basis for interpreting structure/function details of the monomer hemoglobins. These details include: (1) the strong correlation between temperature factor and NMR dynamics for respective protein forms; (2) the unique nature of the HisE7Leu primary sequence substitutions in GMH3 and GMH4 and their impact on cyanide ion binding kinetics; (3) the LeuB10Phe difference between GMH3 and GMH4 and its impact on ligand binding; and (4) elucidation of changes in the structural details of the distal and proximal heme pockets upon cyanide binding.     

Rate of formation of AGEs during ascorbate glycation and during aging in human lens tissue

The similarity of the yellow chromophores isolated from human cataracts with those from ascorbic acid modified calf lens proteins was recently published [Biochim. Biophys. Acta 1537 (2001) 14]. The data presented here additionally quantify age-dependent increases in individual yellow chromophores and fluorophores in the water-insoluble fraction of normal human lens. The water-insoluble fraction of individual normal human lens was isolated, solubilized by sonication and digested with a battery of proteolytic enzymes under argon to prevent oxidation. The level of A(330)-absorbing yellow chromophores, 350/450 nm fluorophores and total water-insoluble (WI) protein were quantified in each lens. The total yellow chromophores and fluorophores accumulated in parallel with the increase in the water-insoluble protein fraction during aging. The digest from each single human lens was then subjected to Bio-Gel P-2 size-exclusion chromatography. The fractions obtained were further separated by a semi-preparative prodigy C-18 high-performance liquid chromatography (RP-HPLC). Bio-Gel P-2 chromatography showed four major fractions, each of which increased with age. RP-HPLC of the amino acid peak resolved five major A(330)-absorbing peaks and eight fluorescent peaks, and each peak increased coordinately with age. A late-eluting peak, which contained hydrophobic amino acids increased significantly after age 60.Aliquots from an in vitro glycation of calf lens proteins by ascorbic acid were removed and subjected to the same enzymatic digestion. Ascorbic acid-modified calf lens protein digests showed an almost identical profile of chromophores, which also increased in a time-dependent manner. The late-eluting peak, however, did not increase with the time of glycation and may not be an advanced glycation endproduct (AGE) product. The data indicate that the total water-insoluble proteins, individual yellow chromophores and fluorophores increased equally both with aging in normal human lens and during ascorbate glycation in vitro. The major protein modifications, which accumulate during aging, therefore, appear to be AGEs. Whereas the late-eluting peak, which showed poor correlation to ascorbylation, may represent UV filter compounds bound to lens proteins.     

Regulation by thyroid hormone of the synthesis of a cytosolic thyroid hormone binding protein during liver regeneration.

To understand the regulation by thyroid hormone, 3,3',5-triiodo-L-thyronine (T3), of the synthesis of a cytosolic thyroid hormone binding protein (p58-M2) during liver regeneration, the synthesis of p58-M2 was evaluated. The synthesis of p58-M2 was measured by metabolic labeling of primary cultures derived from the regenerating liver of euthyroid, hypo- or hyperthyroid rats. During regeneration, the increase in the liver/body weight ratio is approximately 25% higher in hyper- than in hypothyroid rats. However, T3 has no effect on the rate of overall liver regeneration observed in four days. In mature liver, T3 increased the synthesis of p58-M2 by approximately 2.5-fold. During regeneration, however, the change in the synthesis of p58-M2 varied with the thyroid status. In euthyroid rats, the synthesis of p58-M2 continued to increase up to 2-fold during liver regeneration. In hyperthyroid rats, after an initial increase by 1.5-fold on day 1, the synthesis of p58-M2 subsequently declined during regeneration. In hypothyroid rats, the synthesis of p58-M2 remained virtually unchanged during regeneration. These results indicate that T3 regulates the synthesis of p58-M2 in mature and regenerating liver.     

心磷脂-细胞色素C体系CD谱的研究

 线粒体内膜中含有特异的心磷脂是细胞色素C氧化酶活性的必需脂。本工作测定了心磷脂脂质体对细胞色素C溶液园二色(CD)谱的影响,发现心磷脂可引起血色素铁的氧化,并使其轴向配位场强的对称性下降。提示心磷脂可能参与酶和底物之间的电子转移过程。     

Hypothermic Machine Perfusion Reduces Delayed Graft Function and Improves One-Year Graft Survival of Kidneys from Expanded Criteria Donors: A Meta-Analysis

Background

Expanded criteria donors (ECDs) are currently accepted as potential sources to increase the donor pool and to provide more chances of kidney transplantation for elderly recipients who would not survive long waiting periods. Hypothermic machine perfusion (HMP) is designed to mitigate the deleterious effects of simple cold storage (CS) on the quality of preserved organs, particularly when the donor is in a marginal status.

Methods

We compared the transplant outcomes in patients receiving ECD kidneys with either HMP or CS graft preservation. Articles from the MEDLINE, EMBASE and Cochrane Library databases were searched and all studies reporting outcomes from HMP versus CS methods of kidney preservation were included in this meta-analysis. The parameters analyzed included the incidence of delayed graft function (DGF), primary non-function (PNF) and one-year graft and patient survival.

Results

A total of seven studies qualified for the review, involving 2374 and 8716 kidney grafts with HMP or CS preservation respectively, all from ECD donors. The incidence of delayed graft function (DGF) was significantly reduced with an odd ratio(OR) of 0.59 (95% CI 0.54–0.66, P<0.001) and one-year graft survival was significantly improved with an OR of 1.12 (95% CI 1.03–1.21, P = 0.005) in HMP preservation compared to CS. However, there was no difference in the incidence of PNF (OR 0.54, 95% CI 0.21–1.40, P = 0.20), and one-year patient survival (OR 0.98, 95% CI 0.94–1.02, P = 0.36) between HMP and CS preservation.

Conclusions

HMP was associated with a reduced incidence of DGF and an with increased one-year graft survival, but it was not associated with the incidence of PNF and one-year patient survival.     

Adipose Vascular Endothelial Growth Factor Regulates Metabolic Homeostasis through Angiogenesis

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Highlights? Two-way modulations of adipose VEGF were generated with aP2-Cre transgene ? Adipose VEGF KO reduces vasculature, increases hypoxia and inflammation in fat ? Adipose VEGF KO accelerates the development of metabolic disease in high-fat diet ? Induced adipose VEGF has opposite effect on fat and restores metabolic homeostasis     

Transgenic Overexpression of Ephrin B1 in Bone Cells Promotes Bone Formation and an Anabolic Response to Mechanical Loading in Mice

To test if ephrin B1 overexpression enhances bone mass, we generated transgenic mice overexpressing ephrin B1 under the control of a 3.6 kb rat collagen 1A1 promoter (Col3.6-Tg^efnb1). Col3.6-Tg^efnb1 mice express 6-, 12- and 14-fold greater levels of full-length ephrin B1 protein in bone marrow stromal cells, calvarial osteoblasts, and osteoclasts, respectively. The long bones of both genders of Col3.6-Tg^efnb1 mice have increased trabecular bone volume, trabecular number, and trabecular thickness and decreased trabecular separation. Enhanced bone formation and decreased bone resorption contributed to this increase in trabecular bone mass in Col3.6-Tg^efnb1 mice. Consistent with these findings, our in vitro studies showed that overexpression of ephrin B1 increased osteoblast differentiation and mineralization, osterix and collagen 1A1 expression in bone marrow stromal cells. Interaction of ephrin B1 with soluble clustered EphB2-Fc decreased osteoclast precursor differentiation into multinucleated cells. Furthermore, we demonstrated that the mechanical loading-induced increase in EphB2 expression and newly formed bone were significantly greater in the Col3.6-Tg^efnb1 mice than in WT littermate controls. Our findings that overexpression of ephrin B1 in bone cells enhances bone mass and promotes a skeletal anabolic response to mechanical loading suggest that manipulation of ephrin B1 actions in bone may provide a means to sensitize the skeleton to mechanical strain to stimulate new bone formation.     

Role of Musclin in the Pathogenesis of Hypertension in Rat

Musclin is a novel skeletal muscle-derived secretory factor found in the signal sequence trap of mouse skeletal muscle cDNAs. Musclin possesses a region homologous to the natriuretic peptide family. Thus, musclin is found to bind with the natriuretic peptide clearance receptors. However, the role of musclin in vascular regulation remains unclear. In this study, we aim to investigate the direct effect of musclin on vascular tone and to analyze its role in hypertension using the spontaneously hypertensive rats (SHR). In aortic strips isolated from SHR, musclin induced contractions in a dose-dependent manner. We found that the musclin-induced vasoconstriction was more marked in SHR than in normal rats (WKY). Moreover, this contraction was reduced by blockade of natriuretic peptide receptor C using the ab14355 antibody. Therefore, mediation of the natriuretic peptide receptor in musclin-induced vasoconstriction can be considered. In addition, similar to the natriuretic peptide receptor, expression of the musclin gene in blood vessels was higher in SHR than in WKY. Injection of musclin markedly increased the blood pressure in rats that can be inhibited by anti-musclin antibodies. Musclin-induced vasoconstriction was more pronounced in SHR than in WKY as in its expression. Taken together, these results suggest that musclin is involved in blood pressure regulation. The higher expression of musclin in hypertension indicates that musclin could be used as a new target for the treatment of hypertension in the future.     

Molecular Characterization and Functions of Fatty Acid and Retinoid Binding Protein Gene (Ab-far-1) in Aphelenchoides besseyi

Rice white tip nematode, Aphelenchoides besseyi, is a kind of plant parasitic nematodes that cause serious losses in rice and many other crops. Fatty acid and retinoid binding protein (FAR) is a specific protein in nematodes and is related to development, reproduction, infection to the host, and disruption of plant defense reactions, so the inhibition of FAR function is the potential approach to control A. besseyi. The full-length of Ab-far-1 cDNA is 805 bp, including 546 bp of ORF that encodes 181 amino acids. Software analysis revealed that the Ab-FAR-1 was rich in α-helix structure, contained a predicted consensus casein kinase II phosphorylation site and a hydrophobic secretory signal peptide, but did not have glycosylation sites. The Ab-FAR-1 had 52% homology to Gp-FAR-1 protein. The Ab-FAR-1 and Gp-FAR-1 were grouped in the same branch according to the phylogenetic tree. Fluorescence-based ligand binding analysis confirmed that the recombinant Ab-FAR-1 (rAb-FAR-1) bound with the fluorescent analogues 11-((5-dimethylaminonaphthalene-1-sulfonyl) amino) undecannoic acid (DAUDA), cis-parinaric acid and retinol, but the oleic acid would compete with the binding site. Quantitative PCR (qPCR) was used to assess the expression level of Ab-far-1 at different development stages of A. besseyi, the highest expression was found in the females, followed by eggs, juveniles and males. Using in situ hybridization technique, Ab-far-1 mRNA was present in the hypodermis of juveniles and adults, the ovaries of females and the testes of males. When A. besseyi was treated with Ab-far-1 dsRNA for 48 h, the silencing efficiency of Ab-far-1 was the best and the number of nematodes on the carrot was the least. Thus FAR plays important roles in the development and reproduction of nematodes. This study is useful and helpful to figure out a new way to control the plant parasitic nematodes.     

MiR-30-Regulated Autophagy Mediates Angiotensin II-Induced Myocardial Hypertrophy

Dysregulated autophagy may lead to the development of disease. Role of autophagy and the diagnostic potential of microRNAs that regulate the autophagy in cardiac hypertrophy have not been evaluated. A rat model of cardiac hypertrophy was established using transverse abdominal aortic constriction (operation group). Cardiomyocyte autophagy was enhanced in rats from the operation group, compared with those in the sham operation group. Moreover, the operation group showed up-regulation of beclin-1 (an autophagy-related gene), and down-regulation of miR-30 in cardiac tissue. The effects of inhibition and over-expression of the beclin-1 gene on the expression of hypertrophy-related genes and on autophagy were assessed. Angiotensin II-induced myocardial hypertrophy was found to be mediated by over-expression of the beclin-1 gene. A dual luciferase reporter assay confirmed that beclin-1 was a target gene of miR-30a. miR-30a induced alterations in beclin-1 gene expression and autophagy in cardiomyocytes. Treatment of cardiomyocytes with miR-30a mimic attenuated the Angiotensin II-induced up-regulation of hypertrophy-related genes and decreased in the cardiomyocyte surface area. Conversely, treatment with miR-30a inhibitor enhanced the up-regulation of hypertrophy-related genes and increased the surface area of cardiomyocytes induced by Angiotensin II. In addition, circulating miR-30 was elevated in patients with left ventricular hypertrophy, and circulating miR-30 was positively associated with left ventricular wall thickness. Collectively, these above-mentioned results suggest that Angiotensin II induces down-regulation of miR-30 in cardiomyocytes, which in turn promotes myocardial hypertrophy through excessive autophagy. Circulating miR-30 may be an important marker for the diagnosis of left ventricular hypertrophy.