Stress-induced cardiomyopathy (SIC), also known as takotsubo cardiomyopathy (TTC), is a relatively newly-described condition, which has been increasingly reported in the literature. It is characterized by acute onset of symptoms and electrocardiogram changes mimicking myocardial infarction, with transient but completely reversible left ventricular (LV) dysfunction. SIC commonly occurs following physical or emotional stress. The present study discusses the case of a 68-year-old female patient who had suffered from infection of the upper respiratory tract for 10 days before admission to the hospital with symptoms of chest stuffiness and dyspnea that persisted for 2 days. Coronary angiography showed normal coronary artery function, while LV angiography demonstrated systolic apical ballooning. Based on these observed characteristics, the patient was diagnosed with SIC and was successfully treated.
stress-induced cardiomyopathy; infection; takotsubo cardiomyopathy
Cardiac tissue engineering (CTE) has developed rapidly, but a great challenge remains in finding practical scaffold materials for the construction of engineered cardiac tissues. Carbon nanohorns (CNHs) may be a potential candidate due to their special structure and properties. The purpose of this study was to assess the effect of CNHs on the biological behavior of neonatal rat ventricular myocytes (NRVMs) for CTE applications. CNHs were incorporated into collagen to form growth substrates for NRVMs. Transmission electron microscopy (TEM) observations demonstrated that CNHs exhibited a good affinity to collagen. Moreover, it was found that CNH-embedded substrates enhanced adhesion and proliferation of NRVMs. Immunohistochemical staining, western blot analysis, and intracellular calcium transient measurements indicated that the addition of CNHs significantly increased the expression and maturation of electrical and mechanical proteins (connexin-43 and N-cadherin). Bromodeoxyuridine staining and a Cell Counting Kit-8 assay showed that CNHs have the ability to inhibit the proliferation of cardiac fibroblasts. These findings suggest that CNHs can have a valuable effect on the construction of engineered cardiac tissues and may be a promising scaffold for CTE.
Electronic supplementary material
The online version of this article (doi:10.1186/s11671-016-1464-z) contains supplementary material, which is available to authorized users.
Carbon nanohorns; Cardiac tissue engineering; Neonatal rat ventricular myocytes; Cardiac fibroblasts; Maturation
Biofilms play important roles in microbial communities and are related to infectious diseases. Here, we report direct evidence that a bacterial nox gene encoding NADH oxidase is involved in biofilm formation. A dramatic reduction in biofilm formation was observed in a Streptococcus sanguinis nox mutant under anaerobic conditions without any decrease in growth. The membrane fluidity of the mutant bacterial cells was found to be decreased and the fatty acid composition altered, with increased palmitic acid and decreased stearic acid and vaccenic acid. Extracellular DNA of the mutant was reduced in abundance and bacterial competence was suppressed. Gene expression analysis in the mutant identified two genes with altered expression, gtfP and Idh, which were found to be related to biofilm formation through examination of their deletion mutants. NADH oxidase-related metabolic pathways were analyzed, further clarifying the function of this enzyme in biofilm formation.
Thrombosis is a leading cause of death worldwide . Recombinant tissue-type plasminogen activator (tPA) is the FDA-approved thrombolytic drug for ischemic strokes, myocardial infarction and pulmonary embolism. tPA is a multi-domain serine protease of the trypsin-family  and catalyses the critical step in fibrinolysis , converting the zymogen plasminogen to the active serine protease plasmin, which degrades the fibrin network of thrombi and blood clots. tPA is rapidly inactivated by endogenous plasminogen activators inhibitor-1 (PAI-1)  (Fig. 1). Engineering on tPA to reduce its inhibition by PAI-1 without compromising its thrombolytic effect is a continuous effort . Tenecteplase (TNK-tPA) is a newer generation of tPA variant showing slower inhibition by PAI-1 . Extensive studies to understand the molecular interactions between tPA and PAI-1 have been carried out , , , , , , , , , , , , however, the precise details at atomic resolution remain unknown. We report the crystal structure of tPA·PAI-1 complex here. The methods required to achieve these data include: (1) recombinant expression and purification of a PAI-1 variant (14-1B) containing four mutations (N150H, K154T, Q319L, and M354I), and a tPA serine protease domain (tPA-SPD) variant with three mutations (C122A, N173Q, and S195A, in the chymotrypsin numbering) ; (2) formation of a tPA-SPD·PAI-1 Michaëlis complex in vitro ; and (3) solving the three-dimensional structure for this complex by X-ray crystallography [deposited in the PDB database as 5BRR]. The data explain the specificity of PAI-1 for tPA and uPA , , and provide structural basis to design newer generation of PAI-1-resistant tPA variants as thrombolytic agents .
Tpa; Serine protease; PAI-1; Serpin; Michaëlis complex; Crystal structure; Fibrinolysis; Thrombolytic agents; Structural biology
Nuclear receptor subfamily 2 group E member 1 (Nr2e1) has been regarded as an essential regulator of the growth of neural stem cells. However, its function elsewhere is unknown. In the present study, we generated Nr2e1 knockdown MIN6 cells and studied whether Nr2e1 knockdown affected basal beta cell functions such as proliferation, cell death, and insulin secretion. We showed that knockdown of Nr2e1 in MIN6 cells resulted in increased sensitivity to lipotoxicity, decreased proliferation, a partial G0/G1 cell-cycle arrest, and higher rates of apoptosis. Moreover, Nr2e1 deficiency exaggerates palmitate-induced impairment in insulin secretion. At the molecular level, Nr2e1 deficiency augments palmitate-induced oxidative stress. Nr2e1 deficiency also resulted in decreases in antioxidant enzymes and expression level of Nrf2. Together, this study indicated a potential protective effect of Nr2e1 on beta cells, which may serve as a target for the development of novel therapies for diabetes.
Porcine diarrhea outbreaks caused by porcine epidemic diarrhea virus (PEDV) has occurred in China with significant losses of piglets since 2010. In this study, the complete S and ORF3 genes of 15 field PEDV isolates in mid-eastern China from 2011 to 2013 were detected and compared with other reference strains. Based on S gene, all of the PEDV strains could be assigned to 3 genogroups. Only 1 isolate, JS120103, belonged to genogroup 1 and showed a close relationship with previous Chinese strains DX and JS-2004-2, European strain CV777, and Korean strain DR13. The other 14 isolates belonged to genogroup 3 and showed a close relationship with other Chinese strains isolated after 2010. The S genes of those isolates were 9 nucleotides longer in length than JS120103 and the other reference strains in genogroup 1, with 15 bp insertion and 6 bp deletion. Homology analyses revealed that all of the Chinese field isolates, except JS120103, are 97.6% to 100% (95.8% to 100%) identical in nucleotide (deduced amino acid) sequence to each other. Meanwhile, based on the ORF3 gene, all of the PEDV isolates could be separated into 3 genogroups. Eleven of the 15 field isolates in this study belonged to genogroup 3 and were 95.8% to 100% identical in nucleotide sequence or 95.6% to 100% in deduced amino acid sequence to each other. Our results indicate that the variant PEDV strain spread wildly in mid-eastern China. This will be useful to take into consideration in the control and prevention of this disease.
Bergenia purpurascens has tonic, haemostatic and anti-tussive actions. Anti-inflammatory and anti-bacterial activities of Bergenia purpurascens have not been reported so far. The objective of this paper is to provide experimental basis for the clinical application of Bergenia purpurascens through the pharmacodynamic study on its anti-inflammatory and anti-bacterial effects.
Experimental models of xylene-induced ear edema in mice, cotton pellet granuloma in rats, and acetic acid-induced peritoneal capillary permeability in mice were used to investigate the anti-inflammatory effect of Bergenia purpurascens; bacteriostatic and bactericidal effects of Bergenia purpurascens extract on Staphylococcus aureus (SA), methicillin-resistant Staphylococcus aureus (MRSA), and β-lactamase positive Staphylococcus aureus (ESBLs-SA), were observed in vitro.
The results show that Bergenia purpurascens extract could markedly inhibit xylene-induced mouse ear edema, cotton pellet granulation tissue hyperplasia, and increased capillary permeability. Bergenia purpurascens extract has an inhibitory effect on SA, MRSA and ESBLs-SA.
We conclude that Bergenia purpurascens extract has certain anti-inflammatory and anti-bacterial effects.
Bergenia purpurascens extract; anti inflammation; SA; MRSA; ESBLs-SA; antibacterial
The main functions of Radix Glycyrrhizae include regulating middle warmer, moistening lung, relieving toxicity, harmonizing property of drugs which is a traditional Chinese medicine widely used in clinical settings. The objective of the paper is to isolate and identify the constituents in ethanol extract of Radix Glycyrrhizae, and to study their anticancer activity.
Materials and Methods
Column chromatography, ODS column chromatography, preparative thin layer chromatography and NMR spectroscopy techniques were used to isolate compounds from ethanol extract of Radix Glycyrrhizae; optical microscopy and flow cytometry were used to determine the anticancer effect of Radix Glycyrrhizae extract.
Four compounds were isolated from the ethanol extract of Radix Glycyrrhizae, namely oleanolic acid, isoliquiritin, glycyrrhetinic acid and licochalcone A. Optical microscopic observation showed that the growth of gastric cancer cell line SGC-7901 was inhibited in the experimental groups, and apoptotic morphological changes were observed in adherent cells; flow cytometry with PI staining showed that Radix Glycyrrhizae extract could induce SGC-7901 cell apoptosis within a concentration range of 0.5–1.5 mg/mL, compared with the control group, the apoptosis was positively correlated with the drug concentration, which exhibited an apparent dose-dependence.
We conclude Ethanol extract of Radix Glycyrrhizae has an anti-proliferative activity on SGC-7901 cells.
Radix Glycyrrhizae; oleanolic acid; isoliquiritin; glycyrrhetinic acid; licochalcone A; SGC-7901 cell
Although PEGylation plays an important role in drug delivery, knowledge about the distribution behavior of PEGylated drugs in ischemic myocardia is rather limited compared to nanoparticles. This work therefore aims to characterize the targeting behavior of the anti-myocardial ischemic mono-PEGylated conjugates of Radix Ophiopogonis polysaccharide (ROP) in two clinically relevant animal models, ie, the myocardial infarction (MI) model and the ischemia/reperfusion (IR) model. To determine the effect of the molecular size of conjugates, two representative conjugates (20- and 40-kDa polyethylene glycol mono-modified ROPs), with hydrodynamic size being approximately and somewhat beyond 10 nm, respectively, were studied in parallel at three time points postdose after a method for determining them quantitatively in biosamples was established. The results showed that the cardiac distribution of the two conjugates was significantly enhanced in both MI and IR rats due to the enhanced permeability and retention effect induced by ischemia. In general, the cardiac targeting efficacy of the conjugates in MI and IR rats was approximately 2; however, different changing in targeting efficacy with time was observed between MI and IR rats and also between the conjugates. Although the enhanced permeability and retention effect-based targeting efficacy for mono-PEGylated ROPs was not high, they, as dissolved macromolecules, are prone to diffusion in the cardiac interstitium space, and thus, facilitate the drug to reach perfusion-deficient and nonperfused areas. These findings are helpful in choosing the cardiac targeting strategy.
heart distribution; myocardial ischemia; polyethylene glycol; Radix Ophiopogonis polysaccharide
Lake Chaohu, which is a large, shallow, hypertrophic freshwater lake in southeastern China, has been experiencing lake-wide toxic Microcystis blooms in recent decades. To illuminate the relationships between microcystin (MC) production, the genotypic composition of the Microcystis community and environmental factors, water samples and associated environmental data were collected from June to October 2012 within Lake Chaohu. The Microcystis genotypes and MC concentrations were quantified using quantitative real-time PCR (qPCR) and HPLC, respectively. The results showed that the abundances of Microcystis genotypes and MC concentrations varied on spatial and temporal scales. Microcystis exists as a mixed population of toxic and non-toxic genotypes, and the proportion of toxic Microcystis genotypes ranged from 9.43% to 87.98%. Both Pearson correlation and stepwise multiple regressions demonstrated that throughout the entire lake, the abundances of total and toxic Microcystis and MC concentrations showed significant positive correlation with the total phosphorus and water temperature, suggesting that increases in temperature together with the phosphorus concentrations may promote more frequent toxic Microcystis blooms and higher concentrations of MC. Whereas, dissolved inorganic carbon (DIC) was negatively correlated with the abundances of total and toxic Microcystis and MC concentrations, indicating that rising DIC concentrations may suppress toxic Microcystis abundance and reduce the MC concentrations in the future. Therefore, our results highlight the fact that future eutrophication and global climate change can affect the dynamics of toxic Microcystis blooms and hence change the MC levels in freshwater.
Microcystis; microcystin; 16S rDNA; mcyD; qPCR; environmental factors; Lake Chaohu
Objective: To study chromosome 1p/19q loss of heterozygosity (LOH) and Sox17 protein expression in oligodendrogliomas and correlate this loss with clinicopathological features. Methods: This study included 100 cases of oligodendrogliomas at the First Affiliated Hospital of Xinjiang Medical University from 2003 to 2014. The cases included paraffin-embedded tissues from 50 low-grade oligodendrogliomas and 50 anaplastic oligodendrogliomas. Chromosome 1p/19q LOH was detected by fluorescence in situ hybridization (FISH) and Sox17 protein expression was analyzed by immunohistochemistry. Clinicopathological characteristics of the oligodendrogliomas were compared and prognosis analyzed using Cox regression and Kaplan-Meier analyses. Results: The LOH positivity rate of 1p/19q was 52% in 50 cases of low-grade oligodendrogliomas and 68% in 50 cases of anaplastic oligodendrogliomas (P = 0.102). The rates of Sox17 expression were significantly different in oligodendrogliomas (82%) and anaplastic oligodendrogliomas (62%, P = 0.026). Single factor analysis determined that 1p/19q LOH (P = 0.000), Sox17 protein expression (P = 0.000), location (P = 0.001), chemotherapy (P = 0.000), and radiation therapy (P = 0.001) were associated with oligodendroglioma patient prognosis. Cox multiple factors regression analysis determined that 1p/19q LOH and Sox17 expression were independent prognostic factors of oligodendrogliomas. Conclusion: In this study, oligodendroglioma patients with 1p/19q LOH and Sox17 protein expression had a better prognosis. Thus, analysis of 1p/19q LOH and Sox17 protein expression could significantly enhance diagnostic accuracy, guide treatment, and improve the prognosis.
Oligodendroglioma; 1p/19q LOH; Sox17; prognosis; clinical features
Radix Ophiopogonis polysaccharide (ROP), a highly hydrophilic macromolecule, has a unique anti-ischemic action in the myocardium. One of the main problems with its use is its relatively short half-life in vivo. To solve this problem, injectable long-acting drug delivery systems, which combine mono-PEGylation (PEG, polyethylene glycol) with the in situ formation of poly(d,l-lactide-co-glycolide) copolymer (PLGA) depots, were tested in this study.
Through a moderate coupling reaction between 20 kDa amino-terminated methoxy-PEG and excessive ROP with activated hydroxyls, a long-circulating and bioactive mono-PEGylated ROP was prepared and characterized. A reasonable and applicable range of PLGA formulations loaded with the mono-PEGylated ROP were prepared, characterized, and evaluated in vivo.
Relative to ROP, the half-life of which was only 0.5 hours, the conjugate alone, following subcutaneous administration, showed markedly prolonged retention in the systemic circulation, with a mean residence time in vivo of approximately 2.76 days. In combination with in situ-forming PLGA depots, the residence time of the conjugate in vivo was prolonged further. In particular, a long-lasting and steady plasma exposure for nearly a month was achieved by the formulation comprising 40% 30 kDa PLGA in N-methyl-2-pyrrolidone.
Long-lasting and steady drug exposure could be achieved using mono-PEGylation in combination with in situ formation of PLGA depots. Such a combination with ROP would be promising for long-term prophylaxis and/or treatment of myocardial ischemia. For high-dose and highly hydrophilic macromolecular drugs like ROP, more than one preparation technology might be needed to achieve week-long or month-long delivery per dosing.
Radix Ophiopogonis polysaccharide; polyethylene glycol; poly(d,l-lactide-co-glycolide) copolymer; conjugation; in situ-forming system
NPM, TFF3 and TACC1 are molecular markers that play important roles in cell differentiation. Herein, we investigated their prognostic impact in patients with primary gastric cancer (GC) and determined whether they could be used as markers of more aggressive gastric carcinomas by detecting the extent of expression in human gastric carcinoma samples.
Tumor tissue specimens from 142 GC patients were retrospectively retrieved and immunohistochemically evaluated. Correlations between NPM, TFF3 and TACC1 over-expression and clincopathologic parameters, and their prognostic values were investigated with χ2, Kaplan-Meier method, and Cox uni- and multivariate survival models. NPM, TFF3 and TACC1 expression was significantly higher in GC patients with poorly differentiated histologic type than that in patients with well differentiated histologic type. NPM expression was significantly higher in patients with hepatic metastasis or recurrence than that in patients without metastasis. TFF3 expression was significantly higher in patients with positive lymph node metastasis than that in patients with negative lymph node metastasis. Age, lymph node metastasis, and TFF3 and TACC1 over-expression were significantly correlated with low survival (P<0.05, P<0.05, P = 0.005 and P = 0.009, respectively). Multivariate analysis showed that lymph node metastasis and TFF3 and TACC1 over-expression were independent prognostic factors.
TFF3 and TACC1 over-expression in epithelial cells of surgically resected GC tissues was an independent predictor of short survival in GC patients. The prognosis was poorer in patients with positive expression of both TFF3 and TACC1 than that in patients with positive expression of TFF3 or TACC1 alone, or with negative expression of TFF3 and TACC1.
The functions of Src family kinases are tightly regulated through Src Homology (SH) domain-mediated protein-protein interactions. We previously reported the biophysical characteristics of the Apoptosis-Linked Gene 2-Interacting protein X (Alix) in complex with the Haemopoietic cell kinase (Hck) SH3 domain. In the current study, we have combined ITC, NMR, SAXS and molecular modeling to determine a 3D model of the complex. We demonstrate that Hck SH3 recognizes an extended linear prolin-rich region of Alix. This particular binding mode enables Hck SH3 to sense a specific non-canonical residue situated in the SH3 RT-loop of the kinase. The resulting model helps clarify the mechanistic insights of Alix-Hck interaction.
apoptosis-linked gene 2-interacting protein X (Alix); protein-protein interaction; Src homology 3 domain (SH3 domain); Src family kinase (SFK); NMR; SAXS
To determine whether HPV DNA can be detected in the plasma of patients with HPV(+) oropharyngeal carcinoma (OP) and to monitor its temporal change during radiotherapy (RT).
Methods and Materials
We used PCR to detect HPV DNA in the culture media of HPV(+) SCC90, VU147T and the plasma of SCC90 and HeLa tumor bearing mice, non-tumor controls and those bearing HPV(-) tumors. We used real time quantitative PCR (qPCR) to quantify plasma HPV DNA in 40 HPV(+) OP, 24 HPV(-) head and neck cancer (HNC) patients and 10 non-cancer volunteers. Tumor HPV status was confirmed by p16INK4a staining and HPV16/18 PCR or HPV ISH. 14 patients had serial plasma samples for HPV DNA quantification during RT.
HPV DNA was detectable in the plasma samples of SCC90- and HeLa-bearing mice but not in controls. It was detected in 65% of pretreatment plasma samples from HPV(+) OP patients using E6/7 qPCR. None of the HPV(-) HNC or non-cancer controls had detectable HPV DNA. Pretreatment plasma HPV DNA copy number correlated significantly with nodal metabolic tumor volume (assessed on FDG-PET). Serial measurements in 14 patients showed rapid decline in HPV DNA that became undetectable at RT completion. In 3 patients, HPV DNA rose to discernable level at the time of metastasis.
Xenograft studies indicated that plasma HPV DNA is released from HPV(+) tumors. Circulating HPV DNA is detectable in most HPV(+) OP patients. Plasma HPV DNA may be a valuable tool for identifying relapse.
Human papillomavirus; oropharyngeal carcinoma; radiotherapy; plasma; circulating DNA
Each cotton fiber is a single-celled seed trichome or hair, and over 20,000 fibers may develop semi-synchronously on each seed. The molecular basis for seed hair development is unknown but is likely to share many similarities with leaf trichome development in Arabidopsis. Leaf trichome initiation in Arabidopsis thaliana is activated by GLABROUS1 (GL1) that is negatively regulated by TRIPTYCHON (TRY). Using laser capture microdissection and microarray analysis, we found that many putative MYB transcription factor and structural protein genes were differentially expressed in fiber and non-fiber tissues. Gossypium hirsutum MYB2 (GhMYB2), a putative GL1 homolog, and its downstream gene, GhRDL1, were highly expressed during fiber cell initiation. GhRDL1, a fiber-related gene with unknown function, was predominately localized around cell walls in stems, sepals, seed coats, and pollen grains. GFP:GhRDL1 and GhMYB2:YFP were co-localized in the nuclei of ectopic trichomes in siliques. Overexpressing GhRDL1 or GhMYB2 in A. thaliana Columbia-0 (Col-0) activated fiber-like hair production in 4–6% of seeds and had on obvious effects on trichome development in leaves or siliques. Co-overexpressing GhRDL1 and GhMYB2 in A. thaliana Col-0 plants increased hair formation in ∼8% of seeds. Overexpressing both GhRDL1 and GhMYB2 in A. thaliana Col-0 try mutant plants produced seed hair in ∼10% of seeds as well as dense trichomes inside and outside siliques, suggesting synergistic effects of GhRDL1 and GhMYB2 with try on development of trichomes inside and outside of siliques and seed hair in A. thaliana. These data suggest that a different combination of factors is required for the full development of trichomes (hairs) in leaves, siliques, and seeds. A. thaliana can be developed as a model a system for discovering additional genes that control seed hair development in general and cotton fiber in particular.
Prescriber preference has been used as an instrumental variable (IV) in a prior study of nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) versus selective cyclooxygenase-2 (COX-2) inhibitors, with preference expressed as the drug constituting the immediately preceding prescription by the same prescriber (instantaneous preference). We sought to compare the correlations between different IV measures with exposure.
Study Design & Setting
In an ambulatory electronic medical record database of university-based physicians, we compared correlations with exposure among three measures of prescriber preference: instantaneous preference, and the proportion of that prescriber’s prescriptions in the past three and six months that were for a NSAID.
We identified 37,934 initial NSAID/COX-2 prescriptions. The correlation with exposure was 0.283 (95% confidence interval 0.274 – 0.292) for instantaneous preference, 0.197 (0.187 – 0.206) for three-month preference, and 0.170 (0.160 – 0.180) for six-month preference.
Instantaneous NSAID/COX-2 prescribing preference was most strongly correlated, and therefore the strongest IV. Future research should focus on the robustness of IV methods to violations of underlying assumptions, extension of IV methods to more than two groups, ratio measures of association, second and subsequent prescriptions per person, and timevarying exposures.
confounding factors (epidemiology); bias (epidemiology); models, statistical; pharmacoepidemiology
Current studies on the Saccharomyces cerevisiae protein Dap1p have demonstrated a heme related function within the ergosterol biosynthetic pathway. Here we present data to further the understanding of the role of heme in the proper biological functioning of Dap1p in cellular processes. Firstly, we examined the role of Dap1p in stabilizing the P450 enzyme, Erg11p, a key protein involved with facilitating ergosterol biosynthesis. Our data indicates that the absence of Dap1p does not affect Erg11p mRNA or protein expression levels, nor the protein degradation rates. Secondly, in order to probe the role of heme in the biological functioning of Dap1p, we measured ferric and ferrous heme binding affinities for Dap1p and the mutant Dap1pY138F, as well as equilibrium midpoint reduction potentials of the Fe(III)/Fe(II) couples. Our results show that both wild-type and mutant proteins bind heme in a 1:1 fashion, possessing tight ferric heme affinities, KD values of 400 pM and 200 nM, respectively, but exhibiting weak ferrous affinities, 2 µM and 10 µM respectively. Additionally, the measured reduction potential of Dap1p, which was found to be 307 mV, is similar to that of other mono-tyrosinate hemoproteins. Although the weaker affinity of Dap1pY138F for ferric heme lowers the production of ergosterol with respect to wild-type Dap1p, it is still sufficient to rescue the growth sensitivity of dap1Δ to fluconazole and MMS, suggesting that the activity of Dap1p is directly related to its ability to bind heme.
Yeast damage associated response protein (Dap1p) and mouse progesterone receptor membrane component-1 protein (mPGRMC1p) belong to a highly conserved class of putative membrane-associated progesterone binding proteins (MAPR), with Dap1p and inner zone antigen (IZA), the rat homologue of mPGRMC1p, recently being reported to bind heme. While primary structure analysis reveals similarities to the cytochrome b5 motif, neither of the two axial histidines responsible for ligation to the heme are present in any of the MAPR proteins. In the current paper, EPR, MCD, CD, UV-vis and general biochemical methods have been used to characterize the nature of heme binding in both Dap1p and a His-tagged, membrane anchor-truncated mPGRMC1p. As isolated, Dap1p is a tetramer which can be converted to a dimer upon addition of 150 mM salt. The heme is non-covalently attached, with a maximal, in vitro, heme loading of approximately 30%, for both proteins. CD and fluorescence spectroscopies indicate a well ordered structure, suggesting the low heme loading is probably not due to improperly folded protein. EPR confirmed a five coordinate, high-spin, ferric resting state for both proteins, indicating one axial amino acid ligand, in contrast to the six coordinate, low-spin, ferric state of cytochrome b5. The MCD spectrum confirmed this conclusion for Dap1p and indicated the axial ligand is most likely a tyrosine and not a histidine, nor a cysteine, however an aspartic acid residue could not be conclusively ruled out. Potential axial ligands, which are conserved in all MAPR’s, were mutated (Y78F, D118A and Y138F) and purified to homogeneity. The mutants Y78F and D118A were found to bind heme, however, Y138F did not. This result is consistent with the MCD data and indicates that Tyr138 is most likely the axial ligand to the heme in Dap1p.
The identification of chromosomal homology will shed light on such mysteries of genome evolution as DNA duplication, rearrangement and loss. Several approaches have been developed to detect chromosomal homology based on gene synteny or colinearity. However, the previously reported implementations lack statistical inferences which are essential to reveal actual homologies.
In this study, we present a statistical approach to detect homologous chromosomal segments based on gene colinearity. We implement this approach in a software package ColinearScan to detect putative colinear regions using a dynamic programming algorithm. Statistical models are proposed to estimate proper parameter values and evaluate the significance of putative homologous regions. Statistical inference, high computational efficiency and flexibility of input data type are three key features of our approach.
We apply ColinearScan to the Arabidopsis and rice genomes to detect duplicated regions within each species and homologous fragments between these two species. We find many more homologous chromosomal segments in the rice genome than previously reported. We also find many small colinear segments between rice and Arabidopsis genomes.
We report improved whole-genome shotgun sequences for the genomes of indica and japonica rice, both with multimegabase contiguity, or almost 1,000-fold improvement over the drafts of 2002. Tested against a nonredundant collection of 19,079 full-length cDNAs, 97.7% of the genes are aligned, without fragmentation, to the mapped super-scaffolds of one or the other genome. We introduce a gene identification procedure for plants that does not rely on similarity to known genes to remove erroneous predictions resulting from transposable elements. Using the available EST data to adjust for residual errors in the predictions, the estimated gene count is at least 38,000–40,000. Only 2%–3% of the genes are unique to any one subspecies, comparable to the amount of sequence that might still be missing. Despite this lack of variation in gene content, there is enormous variation in the intergenic regions. At least a quarter of the two sequences could not be aligned, and where they could be aligned, single nucleotide polymorphism (SNP) rates varied from as little as 3.0 SNP/kb in the coding regions to 27.6 SNP/kb in the transposable elements. A more inclusive new approach for analyzing duplication history is introduced here. It reveals an ancient whole-genome duplication, a recent segmental duplication on Chromosomes 11 and 12, and massive ongoing individual gene duplications. We find 18 distinct pairs of duplicated segments that cover 65.7% of the genome; 17 of these pairs date back to a common time before the divergence of the grasses. More important, ongoing individual gene duplications provide a never-ending source of raw material for gene genesis and are major contributors to the differences between members of the grass family.
Comparative genome sequencing of indica and japonica rice reveals that duplication of genes and genomic regions has played a major part in the evolution of grass genomes