Structure of the L1 protuberance in the ribosome

The L1 protuberance of the 50S ribosomal subunit is implicated in the release/disposal of deacylated tRNA from the E site. The apparent mobility of this ribosomal region has thus far prevented an accurate determination of its three-dimensional structure within either the 50S subunit or the 70S ribosome. Here we report the crystal structure at 2.65 A resolution of ribosomal protein L1 from Sulfolobus acidocaldarius in complex with a specific 55-nucleotide fragment of 23S rRNA from Thermus thermophilus. This structure fills a major gap in current models of the 50S ribosomal subunit. The conformations of L1 and of the rRNA fragment differ dramatically from those within the crystallographic model of the T. thermophilus 70S ribosome. Incorporation of the L1-rRNA complex into the structural models of the T. thermophilus 70S ribosome and the Deinococcus radiodurans 50S subunit gives a reliable representation of most of the L1 protuberance within the ribosome.     

Nitric oxide plays a central role in determining lateral root development in tomato

Nitric oxide (NO) is a bioactive molecule that functions in numerous physiological processes in plants, most of them involving cross-talk with traditional phytohormones. Auxin is the main hormone that regulates root system architecture. In this communication we report that NO promotes lateral root (LR) development, an auxin-dependent process. Application of the NO donor sodium nitroprusside (SNP) to tomato (Lycopersicon esculentum Mill.) seedlings induced LR emergence and elongation in a dose-dependent manner, while primary root (PR) growth was diminished. The effect is specific for NO since the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO) blocked the action of SNP. Depletion of endogenous NO with CPTIO resulted in the complete abolition of LR emergence and a 40% increase in PR length, confirming a physiological role for NO in the regulation of root system growth and development. Detection of endogenous NO by the specific probe 4,5-diaminofluorescein diacetate (DAF-2 DA) revealed that the NO signal was specifically located in LR primordia during all stages of their development. In another set of experiments, SNP was able to promote LR development in auxin-depleted seedlings treated with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). Moreover, it was found that LR formation induced by the synthetic auxin 1-naphthylacetic acid (NAA) was prevented by CPTIO in a dose-dependent manner. All together, these results suggest a novel role for NO in the regulation of LR development, probably operating in the auxin signaling transduction pathway.Abbreviations CPTIO 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide - DAF-2 DA 4,5-Diaminofluorescein diacetate - LR Lateral root - NAA 1-Naphthylacetic acid - NO Nitric oxide - NPA N-1-Naphthylphthalamic acid - PR Primary root - SNP Sodium nitroprusside     

Ultrasensitive nucleic acid biosensors for early cancer diagnostics

We have developed ultrasensitive nucleic acid detection systems involving an amplification step where the analytical signal correlates directly to the amount of nucleic acid in the solution So far, we have performed nucleic acid quantification on several breast cancer susceptibility genes and were able to detect nucleic acid amounts that ranged from 0.1–1.0 fg of nucleic acid, which is at least 1000 times more sensitive than conventional fluorescent detection methods. The biosensors are so sensitive that they can be used for direct detection of breast cancer susceptibility genes in mRNA without involving a PCR step.     

Changes in the ratio of free NEDD8 to ubiquitin triggers NEDDylation by ubiquitin enzymes

Ubiquitin and UBL (ubiquitin-like) modifiers are small proteins that covalently modify other proteins to alter their properties or behaviours. Ubiquitin modification (ubiquitylation) targets many substrates, often leading to their proteasomal degradation. NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is the UBL most closely related to ubiquitin, and its best-studied role is the activation of CRLs (cullin-RING ubiquitin ligases) by its conjugation to a conserved C-terminal lysine residue on cullin proteins. The attachment of UBLs requires three UBL-specific enzymes, termed E1, E2 and E3, which are usually well insulated from parallel UBL pathways. In the present study, we report a new mode of NEDD8 conjugation (NEDDylation) whereby the UBL NEDD8 is linked to proteins by ubiquitin enzymes in vivo. We found that this atypical NEDDylation is independent of classical NEDD8 enzymes, conserved from yeast to mammals, and triggered by an increase in the NEDD8 to ubiquitin ratio. In cells, NEDD8 overexpression leads to this type of NEDDylation by increasing the concentration of NEDD8, whereas proteasome inhibition has the same effect by depleting free ubiquitin. We show that bortezomib, a proteasome inhibitor used in cancer therapy, triggers atypical NEDDylation in tissue culture, which suggests that a similar process may occur in patients receiving this treatment.     

Continual assembly of desmosomes within stable intercellular contacts of epithelial A-431 cells

Subclones of human carcinoma-derived A-431 cell line stably producing fusion proteins consisting of the enhanced green fluorescent protein and either human desmoglein 2 (Dsg-GFP) or human plakoglobin (GFP-Pg) were used to examine the behavior of desmosomes in living cells. Immunofluorescence microscopy of the fixed cells showed that both fusion proteins, which were expressed in significantly lower levels relative to their endogenous counterparts, were efficiently recruited into desmosomes. Time-lapse confocal imaging of these cells reveals that such GFP-labeled desmosomes (GFP desmosomes) are stable structures which exhibit various dynamic and motile activities. The most notable are independent lateral mobility and fusion. Furthermore, the continual assembly of new nascent desmosomes is observed within stable contacts located at the middle of the epithelial sheet. A new GFP desmosome appears as a closely apposed group of fine patches which after a few minutes aggregate into a single structure. These three dynamic processes resulted in constant changes of desmosome distribution, numbers, and sizes. In addition, fluorescence recovery after photobleaching experiments showed that fine patches of desmosomal proteins may participate in desmosome maintenance. Such a diverse range of dynamic activities of desmosomes apparently produces flexible but tight cell-cell adhesion required for different morphogenetic events in epithelial structures.This work was supported by grant AR44016-04 from the National Institutes of Health     

Clinanthus microstephium,an Amaryllidaceae Species with Cholinesterase Inhibitor Alkaloids: Structure−Activity Analysis of Haemanthamine Skeleton Derivatives

Plants of the Amaryllidaceae family are well‐known (not only) for their ornamental value but also for the alkaloids that they produce. In this report, the first phytochemical study of Clinanthus genus was carried out. The chemical composition of alkaloid fractions from Clinanthus microstephium was analyzed by GC/MS and NMR. Seven known compounds belonging to three structural types of Amaryllidaceae alkaloids were identified. An epimeric mixture of a haemanthamine‐type compound (6‐hydroxymaritidine) was tested as an inhibitor against acetyl‐ and butyrylcholinesterase enzymes (AChE and BChE, respectively), two enzymes relevant in the treatment of Alzheimer's disease, with good results. Structure–activity relationships through molecular docking studies with this alkaloid and other structurally related compounds were discussed.     

A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and the associated proteins (Cas) comprise a system of adaptive immunity against viruses and plasmids in prokaryotes. Cas1 is a CRISPR-associated protein that is common to all CRISPR-containing prokaryotes but its function remains obscure. Here we show that the purified Cas1 protein of Escherichia coli (YgbT) exhibits nuclease activity against single-stranded and branched DNAs including Holliday junctions, replication forks and 5'-flaps. The crystal structure of YgbT and site-directed mutagenesis have revealed the potential active site. Genome-wide screens show that YgbT physically and genetically interacts with key components of DNA repair systems, including recB, recC and ruvB. Consistent with these findings, the ygbT deletion strain showed increased sensitivity to DNA damage and impaired chromosomal segregation. Similar phenotypes were observed in strains with deletion of CRISPR clusters, suggesting that the function of YgbT in repair involves interaction with the CRISPRs. These results show that YgbT belongs to a novel, structurally distinct family of nucleases acting on branched DNAs and suggest that, in addition to antiviral immunity, at least some components of the CRISPR-Cas system have a function in DNA repair.     

Neuroimmunomodulative properties of dipeptidyl peptidase IV/CD26 in a TNBS-induced model of colitis in mice

Causal connections between dipeptidyl peptidase IV, also known as CD26 molecule (DPP IV/CD26) and inflammatory bowel disease (IBD) have been shown, but mechanisms of these interactions are unclear. Our hypothesis was that DPP IV/CD26 could affect the neuroimmune response during inflammatory events. Therefore, we aimed to evaluate its possible role and the relevance of the gut-brain axis in a model of IBD in mice. Trinitrobenzenesulfonic acid-induced (TNBS) colitis was induced in CD26-deficient (CD26(-/-) ) and wild-type (C57BL/6) mice. Pathohistological and histomorphometrical measurements were done. Concentrations and protein expressions of DPP IV/CD26 substrates neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) were determined. Concentrations of IL-6 and IL-10 were evaluated. Investigations were conducted at systemic and local levels. Acute inflammation induced increased serum NPY concentrations in both mice strains, more enhanced in CD26(-/-) mice. Increased NPY concentrations were found in colon and brain of C57BL/6 mice, while in CD26(-/-) animals only in colon. VIP and IL-6 serum and tissue concentrations were increased in both mice strains in acute inflammation, more pronouncedly in CD26(-/-) mice. IL-10 concentrations, after a decrease in serum of both mice strains, increased promptly in CD26(-/-) mice. Decreased IL-10 concentration was found in brain of C57BL/6 mice, while it was increased in colon of CD26(-/-) mice in acute inflammation. DPP IV/CD26 deficiency affects the neuroimmune response at systemic and local levels during colitis development and resolution in mice. Inflammatory changes in the colon reflected on investigated parameters in the brain, suggesting an important role of the gut-brain axis in IBD pathogenesis.