Localization, purification, and characterization of the rabbit sarcoplasmic reticulum associated calmodulin-dependent protein kinase

The Ca2+/calmodulin dependent protein kinase associated with the sarcoplasmic reticulum membranes (SR CaM kinase) plays a specific and important role in the modulation of both Ca2+ uptake and release functions of the sarcoplasmic reticulum itself. In this work we have localized a 60 kD SR CaM kinase in slow and fast twitch rabbit skeletal muscle fractions; the kinase was present in both the longitudinal and the junctional sarcoplasmic reticulum. We then developed a procedure for the purification of the active kinase from the longitudinal sarcoplasmic reticulum and performed biochemical and functional characterization of the enzyme. Differently from what was previously suggested, our analysis shows that the biochemical properties of the purified SR CaM kinase (Ca2+ sensitivity, K0.5 for calmodulin, Km for ATP, IC50 for the specific inhibitory peptide (290-309), autophosphorylation properties) are not significantly different from those of the soluble multifunctional CaM kinase II. Moreover, we show that the purified SR CaM kinase retains the ability to autophosphorylate in a Ca2+/calmodulin-dependent manner, becoming a Ca2+-independent enzyme. In the light of the knowledge of the rabbit SR CaM kinase biochemical properties, we propose and discuss the possibility that, under physiological conditions, the activity of the autophosphorylated kinase persists when the Ca2+ transient is over.     

Toxicological analysis of the marine cyanobacterium Nodularia harveyana

Nodularia harveyana, a dinitrogen-fixing cyanobacterial isolate from the Mediteranean Sea, grown in an outdoor photobioreactor, was assayed for its bioactive compounds. The active substance(s) were lypophilic and showed strong allelopathic actvity against other axenic cyanobacteria, antibiotic activity against Gram-positive pathogenic bacteria and antifungal activity against two plant pathogens. The extracts were toxic (LC₅₀ at 30 μL) for grazers such as a rotifer (Brachionus calyciflorus) and a crustacean (Thamnocephalus platyurus). This revised version was published online in August 2006 with corrections to the Cover Date.     

啤酒酵母菌的细胞融合

啤酒多倍体酵母菌原生质体已成功地与单倍体原生质体进行融合。经细胞壁再生后,稳定的融合重组体被分离出来。这些融合体的基因分析表明,融合体中含有双亲的基因型。孢子形成良好,且每个子囊中含有四个孢子,每个孢子确实是二倍体。这样原生质体融合就提供了一个对啤酒酿造酵母进行遗传分析的方法。但是如果没有一个方便的杂交技术,这个方法将是很困难的。     

Syntheses and initial evaluation of a series of indolo-fused heterocyclic inhibitors of the polymerase enzyme (NS5B) of the hepatitis C virus

Herein, we present initial SAR studies on a series of bridged 2-arylindole-based NS5B inhibitors. The introduction of bridging elements between the indole N1 and the ortho-position of the 2-aryl moiety resulted in conformationally constrained heterocycles that possess multiple additional vectors for further exploration. The binding mode and pharmacokinetic (PK) properties of select examples, including: 13-cyclohexyl-6-oxo-6,7-dihydro-5H-indolo[2,1-d][1,4]benzodiazepine-10-carboxylic acid (7) (IC₅₀ = 0.07 μM, %F = 18), are reported.     

Complexes of 2-thiophenecarbonyl and isonicotinoyl hydrazones of 3-(N-methyl)isatin. A study of their antimicrobial activity

Cobalt(II), nickel(II), copper(II) and zinc(II) complexes of 2-thiophenecarbonyl and isonicotinoyl hydrazones of 3-(N-methyl)isatin (HL(1) and HL(2), respectively) were synthesized and characterized, being the crystal structures of HL(1), HL(2) and [Ni(L(1))(2)].2CHCl(3) elucidated by X-ray diffraction techniques. The in vitro antimicrobial activity of all these compounds was tested against several bacteria and fungi. HL(1)and its complexes exhibited a strong inhibition of the growth of Haemophilus influenzae (MIC 0.15-1.50microg/mL) and good antibacterial properties towards Bacillus subtilis (MIC 3-25microg/mL). The minimal inhibitory concentration (MIC) was defined as the lowest concentration of compound inhibiting the growth of each strain. The antibacterial effectiveness was confirmed against a number of Gram positive bacteria, including methicillin-resistant Staphylococcus aureus. Yeasts and moulds showed a low susceptibility, except the dermatophyte mould Epidermophyton floccosum that is inhibited at concentrations ranging from 6 to 50microg/mL. In general, the antimicrobial activity of the thiophene derivatives was greater than that of the isonicotinic analogues.     

SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of motor neurons in patients with null mutations in the SMN1 gene. The almost identical SMN2 gene is unable to compensate for this deficiency because of the skipping of exon 7 during pre–messenger RNA (mRNA) processing. Although several splicing factors can modulate SMN2 splicing in vitro, the physiological regulators of this disease-causing event are unknown. We found that knockout of the splicing factor SAM68 partially rescued body weight and viability of SMAΔ7 mice. Ablation of SAM68 function promoted SMN2 splicing and expression in SMAΔ7 mice, correlating with amelioration of SMA-related defects in motor neurons and skeletal muscles. Mechanistically, SAM68 binds to SMN2 pre-mRNA, favoring recruitment of the splicing repressor hnRNP A1 and interfering with that of U2AF65 at the 3′ splice site of exon 7. These findings identify SAM68 as the first physiological regulator of SMN2 splicing in an SMA mouse model.     

Supramaximal exercise mobilizes hematopoietic progenitors and reticulocytes in athletes

Marathon runners show increased circulating CD34+ cell counts and postexercise release of interleukin-6 (IL-6), granulocyte-colony stimulating factor (G-CSF) and flt3-ligand (Bonsignore MR, Morici G, Santoro A, Pegano M, Cascio L, Bonnano A, Abate P, Mirabella F, Profita M, Insalaco G, Gioia M, Vignola AM, Majolino I, Testa U, and Hogg JC. J Appl Physiol 93: 1691-1697, 2002). In the present study we hypothesized that supramaximal ("all-out") exercise may acutely affect circulating progenitors and reticulocytes and investigated possible mechanisms involved. Progenitor release was measured by flow cytometry (n = 20) and clonogenic assays (n = 6) in 20 young competitive rowers (13 M, 7 F, age +/- SD: 17.1 +/- 2.1 yr, peak O2 consumption: 56.5 +/- 11.4 ml.min(-1).kg(-1)) at rest and shortly after 1,000 m "all-out." Release of reticulocytes, cortisol, muscle enzymes, neutrophil elastase, and several cytokines/growth factors was measured. Supramaximal exercise doubled circulating CD34+ cells (rest: 7.6 +/- 3.0, all-out: 16.3 +/- 9.1 cells/mul, P < 0.001), and increased immature reticulocyte fractions; AC133+ cells doubled, suggesting release of angiogenetic precursors. Erythrocyte burst forming units and colony forming units for granulocytes-monocytes and all blood series increased postexercise by 3.4-, 5.5-, and 4.8-fold, respectively (P < 0.01 for all). All-out rowing acutely increased plasma cortisol, neutrophil elastase, flt3-ligand, hepatocyte growth factor, VEGF, and transforming growth factor-beta1, and decreased erythropoietin; K-ligand, stromal-derived factor-1, IL-6, and G-CSF were unchanged. Therefore, all-out exercise is a physiological stimulus for progenitor release in athletes. Release of reticulocytes and proangiogenetic cells and mediators suggests tissue hypoxia as possibly involved in progenitor mobilization.     

Decreased gene expression from T7 promoters may be due to impaired production of active T7 RNA polymerase

Background  

Protein expression vectors that utilize the bacteriophage T7 polymerase/promoter system are capable of very high levels of protein production. Frequently, however, expression from these vectors does not reliably achieve optimal levels of protein production. Strategies have been proposed previously that successfully maintain high expression levels, however we sought to determine the cause of induction failure.     

Histidine decarboxylase, DOPA decarboxylase, and vesicular monoamine transporter 2 expression in neuroendocrine tumors: immunohistochemical study and gene expression analysis.

Histidine decarboxylase (HDC) and vesicular monoamine transporter 2 (v-MAT2) are involved in the biosynthesis and storage of histamine. DOPA decarboxylase (DDC) is involved in the biosynthesis of a variety of amines and shares a high degree of homology with HDC. HDC and v-MAT2 immunoreactivities (IR) have recently been detected in well-differentiated neuroendocrine tumors (WDNETs) and poorly differentiated neuroendocrine carcinomas (PDNECs) of various sites and have been proposed as general endocrine markers. We evaluated HDC and v-MAT2 IR in a series of 117 WDNETs and PDNECs from different sites. Western blotting analysis was performed to verify the specificity of anti-DDC and anti-HDC antibodies. Real-time RT-PCR was performed using specific probes for HDC and DDC on 42 cases, examined also for DDC IR. HDC and v-MAT2 IR were observed in the majority of WDNETs and PDNECs of all sites and HDC-IR cases were always also DDC-IR. In contrast, high levels of HDC mRNA were detected only in the gastroenteropancreatic WDNETs, which did not show increased DDC mRNA levels. On the other hand, bronchial carcinoids and lung PDNECs showed high DDC mRNA levels, but nearly undetectable HDC mRNA levels. Western blotting analysis showed a cross-reaction between anti-HDC and anti-DDC antibodies. HDC should not be considered as a general endocrine marker and HDC IR in bronchial carcinoids and PDNECs of the lung can probably be attributed to a cross-reaction with DDC.