N terminus of calpain 1 is a mitochondrial targeting sequence

The ubiquitous m- and mu-calpains are thought to be localized in the cytosolic compartment, as is their endogenous inhibitor calpastatin. Previously, mu-calpain was found to be enriched in mitochondrial fractions isolated from rat cerebral cortex and SH-SY5Y neuroblastoma cells, but the submitochondrial localization of mu-calpain was not determined. In the present study, submitochondrial fractionation and digitonin permeabilization studies indicated that both calpain 1 and calpain small subunit 1, which together form mu-calpain, are present in the mitochondrial intermembrane space. The N terminus of calpain 1 contains an amphipathic alpha-helical domain, and is distinct from the N terminus of calpain 2. Calpain 1, but not calpain 2, was imported into mitochondria. Removal of the N-terminal 22 amino acids of calpain 1 blocked the mitochondrial calpain import, while addition of this N-terminal region to calpain 2 or green fluorescent protein enabled mitochondrial import. The N terminus of calpain 1 was not processed following mitochondrial import, but was removed by autolysis following calpain activation. Calpain small subunit 1 was not directly imported into mitochondria, but was imported in the presence of calpain 1. The presence of a mitochondrial targeting sequence in the N-terminal region of calpain 1 is consistent with the localization of mu-calpain to the mitochondrial intermembrane space and provides new insight into the possible functions of this cysteine protease.     

Novel chimeras of botulinum neurotoxins A and E unveil contributions from the binding, translocation, and protease domains to their functional characteristics

Hyperexcitability disorders of cholinergically innervated muscles are treatable with botulinum neurotoxin (BoNT) A. The seven serotypes (A-G) potently block neurotransmission by binding to presynaptic receptors, undergoing endocytosis, transferring to the cytosol, and inactivating proteins essential for vesicle fusion. Although BoNT/A and BoNT/E cleave SNAP-25, albeit at distinct sites, BoNT/E blocks neurotransmission faster and more potently. To identify the domains responsible for these characteristics, the C-terminal heavy chain portions of BoNT/A and BoNT/E were exchanged to create chimeras AE and EA. After high yield expression in Escherichia coli, these single chain chimeras were purified by two-step chromatography and activated by conversion to disulfide-linked dichains. In vitro, each entered neurons, cleaved SNAP-25, and blocked neuromuscular transmission while causing flaccid paralysis in vivo. Acidification-dependent translocation of the light chain to the cytosol occurred more rapidly for BoNT/E and EA than for BoNT/A and AE because the latter pair remained susceptible for longer to inhibitors of the vesicular proton pump, and BoNT/A proved less sensitive. The receptor-binding and protease domains do not seem to be responsible for the speeds of intoxication; rather the N-terminal halves of their heavy chains are implicated, with dissimilar rates of cytosolic transfer of the light chains being due to differences in pH sensitivity. AE produced the most persistent muscle weakening and therefore has therapeutic potential. Thus, proof of principle is provided for tailoring the pharmacological properties of these toxins by protein engineering.     

Enhanced fitness due to higher fecundity,increased defence against a specialist and tolerance towards a generalist herbivore in an invasive annual plant

Aims The superior performance of many non-indigenous species in a new range can be attributed to different factors such as pre-adaptation to environmental conditions in new areas or to factors inherent to displacement mechanisms such as loss of co-evolved pathogens and herbivores that increase the speed of evolutionary change towards a shift in allocation from defence to growth and reproduction. To assess the importance of the different mechanisms governing the success of Conyza canadensis, a globally successful invader, we simultaneously tested several recent hypotheses potentially explaining the factors leading to biological invasion.Methods We tested (i) whether plants from the non-native range showed a higher fitness than plants from the native North American range, (ii) whether they differed in resistance against an invasive generalist herbivore, the slug Arion lusitanicus and against a recently established specialist aphid herbivore, Uroleucon erigeronense and (iii) experimentally assessed whether C. canadensis releases allelopathic chemicals that have harmful effects on competing species in the non-native range. We compared populations along a similar latitudinal gradient both in the native North American and invasive European range and analysed patterns of adaptive clinal variation in biomass production.Important findings The invasion success of C. canadensis in Europe cannot be attributed to a single trait, but to a combination of factors. Invasive plants benefited from increased growth and above all, increased reproduction (a key trait in an annual plant) and were less attacked by a co-migrated specialist enemy. The observed loss of defence against generalist slugs did not translate into a decreased fitness as invasive C. canadensis plants showed a high re-growth potential. In contrast to earlier in vitro studies, we detected no allelopathic effects on the competing flora in the non-native range. The latitudinal cline in vegetative biomass production in the non-native range observed in our common garden study indicates a high adaptive potential. However, only further genetic studies will provide conclusive evidence whether the differentiation in the non-native range is caused by pre-adaptation and sorting-out processes of putatively repeatedly introduced populations of this composite, long-distance disperser with highly volatile seeds or evolved de novo as a rapid response to new selection pressures in the non-native range.     

Synthesis, characterization, X-ray structure and DNA photocleavage by cis-dichloro bis(diimine) Co(III) complexes

Complexes of the type [Co(LL)2Cl2]Cl, where LL = N,N'-ethylenediamine (en), 2,2'-bipyridine (bpy), 1,10-phenanthroline (phen), 1,10-phenanthroline-5,6-dione (phendione) and dipyrido[3,2-a:2',3'-c]phenazine (dppz) have been synthesized and characterized by elemental analyses, IR, UV-visible and NMR spectroscopy. Crystal structure of [Co(phendione)2Cl2]Cl x 0.5 HCl x 3.5 H2O has been solved and refined to R = 0.0552. The crystal is monoclinic with space group C2/c; a = 25.730(2) A, b = 12.375(1) A, c = 18.979(2) A, beta = 119.925(1) degrees and Z = 8. The DNA binding characteristics of the complexes, investigated by covalent binding assay, viscosity measurements and competitive binding fluorescence measurements show that the complexes interact with DNA covalently except the complex containing the planar dppz ligand which intercalates within the base pairs of DNA. The complexes containing en, phen and phendione cleave plasmid pBR 322 DNA upon irradiation under aerobic conditions while the complex containing the dppz ligand cleaves DNA upon irradiation under inert atmosphere. Molecular modeling studies show that the minimized structure of [Co(phendione)2Cl2]+, maintained the octahedral structure while binding to the N7 of guanines and the ligand fits into the major groove without disrupting the helical structure of the B-DNA.     

Design and synthesis of novel furoquinoline based inhibitors of multiple targets in the PI3K/Akt-mTOR pathway

We herein report the design and synthesis of furoquinoline based novel molecules (16-36) and their in vitro multiple targeted inhibitory potency against PI3K/Akt phosphorylation and mTOR using cell based and cell-free kinase assay. In particular, compound 23 in addition to PI3K-mTOR inhibitory potency, it has shown potent inhibition of hypoxia-induced accumulation of HIF-1alpha protein in U251-HRE cell line. The inhibitory activities of compound 23 were confirmed by Western blot analysis, using human non-small cell lung carcinoma H-460 cell line and glioblastoma U251 cell lines.     

ced-4 and proto-oncogene tfg-1 antagonistically regulate cell size and apoptosis in C. elegans

BACKGROUND: Cell-size-control systems, coupled with apoptotic- and cell-proliferation-regulatory mechanisms, determine the overall dimensions of organs and organisms, and their dysregulation can lead to tumor formation. The interrelationship between cell-growth-regulatory mechanisms and apoptosis during normal development and cancer is not understood. The TRK-fused gene (TFG) promotes tumorigenesis when present in chromosomal rearrangements from various human-cancer types by unknown mechanisms. Apaf1/CED-4 is essential for apoptosis but has not been shown to function in cell-growth control. RESULTS: We found that loss of TFG-1, the TFG ortholog in Caenorhabditis elegans, results in supernumerary apoptotic corpses, whereas its overexpression is sufficient to inhibit developmentally programmed cell death. TFG-1 is also required for cells and nuclei to grow to normal size. Furthermore, we found that CED-4 is required for cell-growth inhibition in animals lacking TFG-1. However, caspases, the downstream effectors of CED-4-mediated apoptosis, are not required in TFG-1- or CED-4-regulated cell-size control. CED-4 acts to inhibit cell growth by antagonizing the effects of other conserved cell-size-regulating proteins, including cAMP response element binding (CREB) protein, translation-initiation factor eIF2B, and the nucleolar p53-interacting protein nucleostemin. CONCLUSIONS: These findings show that TFG-1 suppresses apoptosis and is essential for normal cell-size control, suggesting that abnormalities in the cell-growth-promoting and apoptosis-inhibiting functions of TFG might be responsible for its action in tumorigenesis. Also, they reveal that CED-4 plays a pivotal role in activating apoptosis and restricting cell and nuclear size, thereby determining the appropriate overall size of an animal. Thus, these findings reveal links between the control mechanisms for apoptosis and cell growth.     

Synthesis of highly selective fluorescent peptide probes for metal ions: tuning selective metal monitoring with secondary structure

Metal selective fluorescent peptide probes (dansyl-Cys-X-Gly-His-X-Gly-Glu-NH2, X = Pro or Gly) were developed by synthesizing peptides containing His, Cys, and Glu residues with Pro-Gly sequence to stabilize a turn structure and Gly-Gly sequence to adopt a random coil. The probe containing two Gly-Gly sequences exhibited marked selectivity only for Cu2+ over 13 metal ions including competitive transition and Group I and II metal ions under physiological buffer condition. In contrast, the probe containing double Pro-Gly sequences showed high selectivity for Zn2+. The peptide probe containing one Pro-Gly sequence exhibited selectivity for Zn2+ and Cu2+. CD spectra indicated that the secondary structure of the probes played an important role in the selective metal monitoring and a pre-organized secondary structure is not required for the selective detection of Cu2+ ion, but is required for the detection of Zn2+. We investigated and characterized the binding affinity, binding stoichiometry, reversibility, and pH sensitivity of the peptide probes.     

Discovery of potent furan piperazine sodium channel blockers for treatment of neuropathic pain

The synthesis and pharmacological characterization of a novel furan-based class of voltage-gated sodium channel blockers is reported. Compounds were evaluated for their ability to block the tetrodotoxin-resistant sodium channel Na(v)1.8 (PN3) as well as the Na(v)1.2 and Na(v)1.5 subtypes. Benchmark compounds from this series possessed enhanced potency, oral bioavailability, and robust efficacy in a rodent model of neuropathic pain, together with improved CNS and cardiovascular safety profiles compared to the clinically used sodium channel blockers mexiletine and lamotrigine.     

Effects of plumieride, an iridoid on spermatogenesis in male albino rats.

Oral feeding of male rats with plumieride (15 mg/rat/day) for the period of 60 days did not cause any significant change in the body weight of treated rats. However, the weights of testes, epididymides, seminal vesicle and ventral prostate were significantly reduced when compared to control values. The production of step-19 spermatids was reduced by 87.26% in plumieride treated rats. The population of preleptotene and pachytene spermatocytes were decreased by 64.26% and 55.13% respectively. Spermatogonia and sertoli cell population was also affected. Plumieride treatment resulted in an arrest of spermatogenesis without any systemic side effect. Sperm motility as well as sperm density was reduced significantly. The number of mature Leydig cells was decreased and complete suppression of fertility was observed. A significant fall in the protein and sialic acid contents of the testes, epididymides, seminal vesicle and ventral prostate as well as glycogen content of testes was also noticed. Fructose in seminal vesicle was lowered whereas testicular cholesterol was elevated. There was no significant change in RBC and WBC count, haemoglobin, haematocrit and sugar in the whole blood and total protein, cholesterol, phospholipid and triglycerides in the serum. Conclusion: Plumieride administration arrests spermatogenesis in male rats without noticeable side effects. For the clinical use more experiments should be carried out in a phased programme.     

In vivo immunogold labeling confirms large-scale chromatin folding motifs

The difficulty in localizing specific cellular proteins by immuno-electron microscopy techniques limits applications of electron microscopy to cell biology. We found that in vivo immunogold labeling improves epitope accessibility, ultrastructural preservation and three-dimensional visualization, and allows correlated light and electron microscopy. We detected large-scale chromatin folding motifs within intact interphase nuclei of CHO cells and visualized the ultrastructure of DNA replication 'factories' labeled with GFP-proliferating cell nuclear antigen (PCNA).