X-ray diffraction analysis of the effects of myosin regulatory light chain phosphorylation and butanedione monoxime on skinned skeletal muscle fibers.

The phosphorylation of the myosin regulatory light chain (RLC) is an important modulator of skeletal muscle performance and plays a key role in posttetanic potentiation and staircase potentiation of twitch contractions. The structural basis for these phenomena within the filament lattice has not been thoroughly investigated. Using a synchrotron radiation source at SPring8, we obtained X-ray diffraction patterns from skinned rabbit psoas muscle fibers before and after phosphorylation of myosin RLC in the presence of myosin light chain kinase, calmodulin, and calcium at a concentration below the threshold for tension development ([Ca(2+)] = 10(-6.8)M). After phosphorylation, the first myosin layer line slightly decreased in intensity at ∼0.05 nm(-1)along the equatorial axis, indicating a partial loss of the helical order of myosin heads along the thick filament. Concomitantly, the (1,1/1,0) intensity ratio of the equatorial reflections increased. These results provide a firm structural basis for the hypothesis that phosphorylation of myosin RLC caused the myosin heads to move away from the thick filaments towards the thin filaments, thereby enhancing the probability of interaction with actin. In contrast, 2,3-butanedione monoxime (BDM), known to inhibit contraction by impeding phosphate release from myosin, had exactly the opposite effects on meridional and equatorial reflections to those of phosphorylation. We hypothesize that these antagonistic effects are due to the acceleration of phosphate release from myosin by phosphorylation and its inhibition by BDM, the consequent shifts in crossbridge equilibria leading to opposite changes in abundance of the myosin-ADP-inorganic phosphate complex state associated with helical order of thick filaments.

Jørgensen-Hayashi catalysts supported on poly(ethylene glycol)s as highly efficient and reusable organocatalysts for the enamine-catalyzed asymmetric Michael reaction.

A new kind of recyclable and reusable PEG-supported Jørgensen-Hayashi catalyst is synthesized for the first time and proven to be efficient for the enamine-catalyzed asymmetric Michael reaction with generally moderate to good diastereoselectivity and high to excellent enantioselectivity (up to 6 : 1 dr, 99% ee). The prepared PEG-supported catalyst can be recovered eight times and was found to provide similar diastereoselectivity and enantioselectivity to unsupported functional catalysts.

Molecular cloning and functional characterization of two divergent 4-coumarate†:†coenzyme A ligases from Kudzu (Pueraria lobata).

As part of the efforts to understand isoflavonoid metabolism in Pueraria lobata at the molecular level, the cDNAs encoding two divergent 4-coumarate : coenzyme A ligases (4CLs, designated Pl4CL1 and Pl4CL2, respectively) were isolated from P. lobata roots. Sequence analysis revealed that Pl4CL1 had an N-terminal extension of twenty-one amino acid residues compared to Pl4CL2. Phylogenetic analysis showed that Pl4CL1 and Pl4CL2 fell into angiosperm Class II and Class I, respectively. Through in vitro biochemical assays, both Pl4CLs were found to have the capacity to utilize 4-coumarate and trans-cinnamate as substrates, while neither of them could convert sinapate. Pl4CL2 had a broader substrate specificity than Pl4CL1. The affinity of Pl4CL1 for 4-coumarate was 2.6-fold higher than that of Pl4CL2 (with the Km values of 3.5 µM and 9.1 µM, respectively). Combining the dataset including gene expression profiles, metabolites measurements, and biochemical properties, our results indicated that Pl4CL1, just as other angiosperm Class II 4CLs, might play a role in isoflavone biosynthesis in P. lobata, while Pl4CL2 belongs to angiosperm Class I, and may function as a housekeeping enzyme concerning lignification.

Prolinethiol ether catalysis in an asymmetric Michael reaction: solvent-free synthesis of functionalized monohaloalkenes.

The organocatalytic Michael reaction of ketones with γ-monohalonitrodienes catalyzed by chiral prolinethiol ether under solvent-free conditions was developed. The described method represents a novel approach for accessing highly functionalized monohaloalkenes with α, ß-stereocenters of up to >99% ee.

(E)-1-Nitro-2-(2-nitro-prop-1-en-yl)benzene.

The title compound, C(9)H(8)N(2)O(4), adopts an E conformation about the C=C bond. The CH(phen-yl)-C(phen-yl)-CH-C(-NO(2)) torsion angle is -57.7 (3)°. The crystal structure features weak inter-molecular C-H⋯O inter-actions.

1-Methyl-4-[(1E)-2-nitro-prop-1-en-1-yl]benzene.

The title compound, C(10)H(11)NO(2), adopts an E conformation about the C=C bond. The C=C-C=C torsion angle is 32.5 (3)°. The crystal structure features weak inter-molecular C-H⋯O inter-actions.

(E)-2-(2-Nitro-prop-1-en-yl)thio-phene.

The title compound, C(7)H(7)NO(2)S, adopts an E conformation about the C=C bond. The torsion angle C=C-C-C is -177.7 (3)°. The crystal structure features weak inter-molecular by C-H⋯O inter-actions.

The highly enantioselective Michael addition of ketones to nitrodienes catalyzed by the efficient organocatalyst system of pyrrolidinyl-thioimidazole and chiral thioureido acid.

The highly enantioselective Michael addition reaction of ketones to nitrodienes was promoted efficiently by the accessible and fine-tunable organocatalytic system of pyrrolidinyl-thioimidazole and chiral thioureido acid. The corresponding adducts were afforded in good yields with high diastereoselectivities (up to 99 : 1) and excellent enantioselectivities (up to 99% ee).

1-Nitro-4-(2-nitro-prop-1-en-yl)benzene.

The asymmetric unit of the title compound, C(9)H(8)N(2)O(4), contains two crystallographically independent mol-ecules, both of which adopt an E configuration about the C=C bond. In the crystal, the mol-ecules stack into columns along the c axis through π-π inter-actions, with centroid-centroid distances of 3.695 (3) and 3.804 (3) Å. The columns are further connected into a three-dimensional network by C-H⋯O hydrogen bonds.

Chiral amine/chiral acid as an excellent organocatalytic system for the enantioselective tandem oxa-Michael-aldol reaction.

The asymmetric tandem oxa-Michael-aldol reaction of salicylic aldehyde derivatives with alpha,beta-unsaturated aldehydes catalyzed by a chiral amine/chiral acid organocatalytic system was investigated. The organocatalytic system of (S)-diphenylpyrrolinol trimethylsilyl ether with chiral shift reagent (S)-Mosher acid presented a synergistic effect in the improvement of reaction performance and offered an efficient steric effect in the transformation. The tandem oxa-Michael-aldol reaction proceeded with high yields (up to 90%) and with excellent ee values (up to 99%) to give the corresponding chromene derivatives. The structure of the chiral ammonium salt formed in situ and the corresponding mechanism were also studied by (1)H NMR.

[Effect of Down-Regulatory Nucleostemin Expression on Autophagy Activities in p53 Null HL-60 Leukemia Cells].

OBJECTIVE: Based on previous microarry and bioinformatic analysis results, to investigate the effect of nucleostemin(NS) expression down-regulation on autophagy activity in p53 null HL-60 leukemia cells, so as to provide evidence for studying mechanisms of p53-independent signal pathway of NS in details. METHODS: The autophagy activity of HL-60 cells after down-regulation of NS expression was detected with acidine orange staining, Western blot and transmission electron mcrioscope technique. RESULTS: The expression level of NS in test groups was lower than that in blank control and negative control groups after HL-60 cells were readily transinfected by lentivirus. The result of acidine orange staining showed that the number of acid vesicular organelle in test groups(22.4±0.76)% was higher than that in blank control groups(3.1±0.28)% and negative control groups(6.2±0.64)% (P<0.05). Western blot showed that the ratio of LC3II/LC3I in test groups(1.537±0.072) was higher than that in blank control and negative control groups (1.010±0.039) and (0.608±0.008). The result of transmission electron mcrioscopy also showed that the number of autophagosomes in test group(8.7±3.1) was higher than that in the blank control and negative control groups(4.2±1.2) and (2.3±0.5). CONCLUSION: Autophagy activty can be enhanced after the level of NS was down regulated. The change indicates the signaling transductions screened by bioinformatic analysis may be one of p53-independent pathway of NS, which lays a foundation for contineously studying key points of p53-independent signal pathway of NS.

[Influence of Nucleostemin Expression Down-Regulation on PI3K/AKT/mTOR Signal Pathway in HL-60 Cells].

OBJECTIVE: To explore the effect of nucleostemin(NS) RNAi on the expression of signal molecules in PI3K/AKT/mTOR pathway, a candidate of p53-independent signal pathway in the leukemia HL-60 cells. METHODS: The expression of NS was interfered by transfection of P53-deficient HL-60 cells with the recombinant lentivirus expression vector NS-RNAi-GV248. The exression of NS and signal molecules of PI3K/AKT/mTOR pathway were detected by Western blot. RESULTS: The fluorescence microscopy showed that the recombinant lentivirus vector NS-RNAi-GV248 transfected HL-60 cells successfully with a 80% transfection rate. Western blot showed that the expression of NS protein was inhibited obviously in HL-60 cells, and the expression levels of AKT, p-AKT, p70s6k and p-p70s6k were not statistically different(t1=2.31,P>0.05;t2=3.62,P>0.05;t3=1.60,P>0.05;t4=2.72,P>0.05) in comparison with control; the expression of GßL protein was statistically down-regnlated (t=15.01,P=0.002). CONCLUSION: The changes of GßL protein correlats with NS knockdown. The PI3K/AKT/mTOR pathway may be one of nucleostemin p53-independent signal pathways.

High efficiency gene delivery into laryngeal muscle with bidirectional electroporation.

OBJECTIVE: The impact of polarity change on the efficiency of in vivo electroporative (EP) gene transfection was assessed in rat laryngeal muscle. STUDY DESIGN AND SETTING: High (HV) and low field voltage (LV) were combined with polarity changes to determine transfection in 5 different conditions: 1) without EP (EP[-]), 2) HV+LV (HL), 3) HV+LV followed by HV+LV with no change in polarity (HLHL unidirectional), 4) HV+LV followed by HV+LV with opposite polarity (HLHL bidirectional), 5) HV+LV followed by LV with opposite polarity (HLL bidirectional). RESULTS: HLL bidirectional sequence showed the best result with less interindividual variability and extended expression period. With the exception of repeated high voltage sequences, EP parameters were not likely to induce cell injury or inflammation. CONCLUSION: HLL bidirectional electroporative gene delivery produces high transfection rates with limited tissue trauma. SIGNIFICANCE: Bidirectional EP provides a safe and highly efficient method for therapeutic gene delivery into skeletal muscle.

[Effect of Dexamethasone on Blast Composition in Patients with Myelodysplastic Syndrome and Its Diagnostic Significance].

OBJECTIVE: To analyze the effect of dexamethason (Dex) on blast composition in patients with myelodysplastic syndrome (MDS) and investigate its significance in diagnosis of MDS. METHODS: The flow cytometry (FCM) was used to detect the blast rate and the expression of its antigens in 30 cases of MDS (10 cases were treated with Dex as DX group and 20 cases were treated without Dex as control group). RESULTS: The difference of the CD34(+) cell number detected by FCM was not statistically significant between DX group and control group (P > 0.05); The rate of BM B cell precursors (BCP CD34(+)/CD19(+)/CD10(+) cells) increased in DX group significantly, and BM CD117(+) cells in CD34(+) cells was decreased significantly as compared with control group (P < 0.001). The expression of antigens between granulocyte and monocyte was not significantly different (P > 0.05). CONCLUSION: The dexamethasone can increase the rate of BCP significantly and decreased the rate of BM CD117(+) cells in CD34(+) cells significantly. There is significant influence on the blast composition in MDS patients after dexamethasone treatment and without significant influence on the other phenotypcs.

Tissue performance of bladder following stretched electrospun silk fibroin matrix and bladder acellular matrix implantation in a rabbit model.

The goal of this study was to investigate the tissue performance of bladder following stretched electrospun silk fibroin matrix (SESFM) implantation compared with bladder acellular matrix (BAM). We compared SESFM with BAM based on porosity and pore size. Scaffolds were separately transplanted into opposite walls of the bladder of 30 rabbits after stripping the bladder mucosa and smooth muscle (1.5 × 2.0 cm(2)). Gross anatomical observation, histological analysis and muscle contractility studies were performed at 2, 4, and 8 weeks post-op. SESFM has higher porosity and larger pore size compared with BAM (p < 0.05). At 2 weeks, the presence of vesical calculus was evident in 7/10 rabbits. Histological analysis showed that SESFM and BAM promoted similar degree of urothelium regeneration (p > 0.05). However, SESFM promoted a higher degree of smooth muscle and vessel regeneration compared to BAM (p < 0.05). In addition, muscle strips supported by SESFM displayed higher contractile responses to carbachol, KCl, and phenylephrine compared with BAM. At 8 weeks, both matrices elicited similar mild acute and chronic inflammatory reactions. Our results demonstrated that SESFM has greater ability to promote bladder tissue regeneration with structural and functional properties compared to BAM, and with similar biocompatibility.

Laryngeal muscle surface receptors identified using random phage library.

OBJECTIVES: The ultimate goal of this study is to improve the efficiency of gene transfer in mammalian muscle by developing targeted adenoviral vectors. Altering the tropism of viral vectors to recognize tissue specific antigens is one method to achieve this goal. This approach requires identification of cell-surface receptors and the insertion of target peptide sequences into the adenoviral fiber protein. In this study, phage biopanning was performed on cultured rat skeletal and laryngeal muscle to identify cell-surface receptors. STUDY DESIGN: In vitro cell culture and in vivo animal model. METHODS: M-13 Phage biopanning was used for muscle cell-surface receptor analysis on cultured rat skeletal and laryngeal muscle. Nonbinding and binding phage to cultured skeletal and laryngeal muscle were screened for muscle specific surface peptides. In vivo studies were then performed using muscle specific phage. RESULTS: Skeletal muscle specific binding by the YASTNPM phage was observed by in vivo immunostaining. Phage titering demonstrated a 10(9)-fold increase in skeletal muscle binding compared with nontarget tissue. A peptide sequence (NPSQVKH) specific for laryngeal muscle yielded a 10(7)-fold increase in laryngeal muscle phage titer compared with nontarget tissue. CONCLUSIONS: These results identify muscle cell-surface receptors that may be used as potential targets for genetic modification of adenovirus tropism. Moreover, phage specificity for skeletal and laryngeal muscle indicates specific muscle groups may be targeted.

[Expression of CD71 on cell proliferation in hematologic malignancy and its correlation with Ki-67].

OBJECTIVE: This study was to explore the expression of CD71, as a proliferation indicator, on cell proliferaration in hematologic malignancy and its correlation with Ki-67, so as to assess the feasibility of CD71 instead of Ki-67 for assaying cell proliferation by flow cytometry (FCM). METHODS: (1) Compared with mature B lymphoctyes during stationary phase in peripheral blood from healthy people, the cell cycle and the expression of CD71 and Ki-67 of cell lines from patients with leukemia and lymphoma were examined, the correlation among CD71, S-phase cell fraction (SPF) and Ki-67 were analyzed; (2) Compared with mature B lymphoctyes in bone marrow from non-hematologic disease patients, the expression and correlation of CD71 and Ki-67 of all kinds of leukemic cells and myeloma cells from bone marrow were analyzed by using Ki-67/CD71/CD45/CD123, Ki-67/CD71/CD45/CD20 or Ki-67/CD71/CD45/CD138. RESULTS: (1) in respect to the expression rate of CD71 on tumor cell lines, the expression rate of CD71 on HL-60 cells was (99.77 ± 0.064)%, the expression rate of CD71 on NB4 cells was (99.23 ± 0.12)%, the expression rate on THP-1 cells was (98.90 ± 0.30)% and the expression rate on K562 cells was (97.03 ± 0.15)% in myelogenous leukemia cell lines, the expression rate of CD71 on Raji cells was (99.35 ± 0.21)% and the expression rate on Mino cell was (96.95 ± 0.42)% in lymphoma cell lines, which were also obviously higher than that on cells of the control group (P < 0.05); (2) in respect to the expression rate of CD71 on tumor cells in bone marrow, the expression rate of CD71 on poorly differentiated AML(M1 and M2) cells was (51.50 ± 19.31)%, the expression rate of CD71 on acute promyelocytic leukemia (AML-M3) cells was (35.71 ± 14.02) %, the expression rate of CD71 on acute monocytic leukemia (AML-M5) cells was (30.54 ± 14.38)%, the expression rate of CD71 on acute T lymphoblastic leukemia cells was (68.40 ± 20.83)%, the expression rate of CD71 on acute B lymphoblastic leukemia was (39.67 ± 18.27)%, the expression rate of CD71 on multiple myeloma (MM) cells was (55.49 ± 18.15%), the expression rate of CD71 on chronic lymphocytic leukemia(CLL) was (1.32 ± 0.33%), which were also higher than that on cells in the control group(P < 0.05) except for CLL cells (P > 0.05); (3) CD71 had a positive linear corrlation with SPF in cell lines (r = 0.914, P < 0.05), and also had a positive linear corrlation with Ki-67 in cell lines and carcinoma cells from bone marrow (r = 0.894,r = 0.904, P < 0.05). CONCLUSION: The CD71 can take the place of Ki-67 as an indicator of cell proliferation activity of hematologic malignancies and the determination CD71 by FCM is simpler and better than that of Ki-67 in respest of methodology.

Differential expression of myosin heavy chain isoforms between abductor and adductor muscles in the human larynx.

OBJECTIVE: This study examines the differential expression of myosin heavy chain (MyHC) components in human laryngeal muscle groups. STUDY DESIGN: A battery of monospecific monoclonal antibodies in Western blots was used to determine expression of IIX, extraocular-specific (EOM), and IIB MyHCs for the thyroarytenoid (TA), vocalis (VOC), lateral cricoarytenoid (LCA), cricothyroid (CT), and posterior cricoarytenoid (PCA) muscles obtained from fresh cadaver specimens. RESULTS: Fast IIX MyHC was only expressed in the TA, VOC, and LCA muscles. Fast IIA and slow MyHCs were expressed in all laryngeal muscles including the CT and PCA. The CT with mixed phonatory and respiratory function and the PCA with respiratory function did not express IIX MyHC. The 2 MyHC isoforms associated with the highest speeds of contraction in rat laryngeal muscle, namely, the EOM MyHC and IIB MyHC, were not detected in human laryngeal muscles. Novel MyHC bands were not detected in SDS-PAGE gels or Western blots using a broad specificity MyHC antibody. CONCLUSION: The profile of MyHC expression in human laryngeal muscles differs from that observed in human extraocular and masticator muscles, and other mammalian species. Our data demonstrate that IIX MyHC expression is associated primarily with muscles affecting glottic closure and is absent in CT and PCA. SIGNIFICANCE: A higher percentage of IIX MyHC is expected to impart a high speed of shortening to the TA and LCA muscles. The absence of IIX MyHC in muscles with respiratory (PCA) and mixed respiratory/phonatory function (CT) further supports the inference that the physiologic difference between laryngeal muscles is reflected in the molecular composition of contractile protein.

[Down-regulatory effect of Nucleostemin expression on signal molecule of PI3K/AKT/mTOR pathway in HL-60 cells].

This study was purpose to explore the down-regulatory effect of nucleostemin (NS) expression on signal molecules of PI3K/AKT/mTOR pathway belonged to candidate ways of p53-independent signal pathway in the leukemia cells. The expression of NS was interfered by using recombinant lentivirus expression vector NS-RNAi-GV248 to transfect HL-60 cells of p53 deficiency. The expression of NS and signal molecules of PI3K/AKT/mTOR pathway were detected by using Real-time PCR. The results of showed that the HL-60 cells were transfected by recombinant lentivirus vector NS-RNAi-GV248 successfully and with transfection rate up to 80%. According to results of Real-time PCR detection, the inhibition rate of NS gene was 56.5% in HL-60 cells. And the expression levels of PI3K,AKT and GßL mRNA (0.491 ± 0.084,0.398 ± 0.164, 0.472 ± 0.097 respectively) were obviously down-regulated by silencing NS, and showed statistical difference (P < 0.05) in comparison with control (1.002 ± 0.171, 1.000 ± 0.411, 1.001 ± 0.206 respectively) . It is concluded that the changes of signal molecules of PI3K/AKT/mTOR pathway positively correlate with NS down-regulation, which provides evidence for confirming PI3K/AKT/mTOR signal pathway possible as a type of NS p53-independent pathway.

Myostatin directly regulates skeletal muscle fibrosis.

Skeletal muscle fibrosis is a major pathological hallmark of chronic myopathies in which myofibers are replaced by progressive deposition of collagen and other extracellular matrix proteins produced by muscle fibroblasts. Recent studies have shown that in the absence of the endogenous muscle growth regulator myostatin, regeneration of muscle is enhanced, and muscle fibrosis is correspondingly reduced. We now demonstrate that myostatin not only regulates the growth of myocytes but also directly regulates muscle fibroblasts. Our results show that myostatin stimulates the proliferation of muscle fibroblasts and the production of extracellular matrix proteins both in vitro and in vivo. Further, muscle fibroblasts express myostatin and its putative receptor activin receptor IIB. Proliferation of muscle fibroblasts, induced by myostatin, involves the activation of Smad, p38 MAPK and Akt pathways. These results expand our understanding of the function of myostatin in muscle tissue and provide a potential target for anti-fibrotic therapies.