Functional changes in the structure of the SRP GTPase on binding GDP and Mg2+GDP.

Ffh is a component of a bacterial ribonucleoprotein complex homologous to the signal recognition particle (SRP) of eukaryotes. It comprises three domains that mediate both binding to the hydrophobic signal sequence of the nascent polypeptide and the GTP-dependent interaction of Ffh with a structurally homologous GTPase of the SRP receptor. The X-ray structures of the two-domain 'NG' GTPase of Ffh in complex with Mg2+GDP and GDP have been determined at 2.0 A resolution. The structures explain the low nucleotide affinity of Ffh and locate two regions of structural mobility at opposite sides of the nucleotide-binding site. One of these regions includes highly conserved sequence motifs that presumably contribute to the structural trigger signaling the GTP-bound state. The other includes the highly conserved interface between the N and G domains, and supports the hypothesis that the N domain regulates or signals the nucleotide occupancy of the G domain.     

Inactivation of the streptokinase gene prevents Streptococcus equisimilis H46A from acquiring cell-associated plasmin activity in the presence of plasminogen

Abstract The effects of some physico-chemical parameters on production of extracellular α-L-arabinofuranosidase by Aspergillus nidulans were examined. Highest levels of α-L-arabinofuranosidase were generated with cultures grown on 1% (w/v) purified beet pulp arabinan at 30°C and at an initial pH of 7.0. The enzyme was shown to be very sensitive to the action of proteases. Zymogram overlay of a protein profile obtained by SDS-PAGE revealed the occurrence of a band ( M _r 36 000) exhibiting α-L-arabinofuranosidase activity. The isoelectric pH of the enzyme lay near 4.3. Temperature and pH optima for the activity of crude α-L-arabinofuranosidase preparations were 55°C and 5.5, respectively. Enzyme activity was greatly reduced by thiol reagents such as Hg²⁺ and p -hydroxymercuribenzoate and showed a K _m value of 2.7 mM on p -nitrophenyl α-L-arabinofuranoside as substrate.     

Structural coupling of the EF hand and C‐terminal GTPase domains in the mitochondrial protein Miro

Miro is a highly conserved calcium‐binding GTPase at the regulatory nexus of mitochondrial transport and autophagy. Here we present crystal structures comprising the tandem EF hand and carboxy terminal GTPase (cGTPase) domains of Drosophila Miro. The structures reveal two previously unidentified ‘hidden’ EF hands, each paired with a canonical EF hand. Each EF hand pair is bound to a helix that structurally mimics an EF hand ligand. A key nucleotide‐sensing element and a Pink1 phosphorylation site both lie within an extensive EF hand–cGTPase interface. Our results indicate structural mechanisms for calcium, nucleotide and phosphorylation‐dependent regulation of mitochondrial function by Miro.     

Endobrevin/VAMP8 mediates exocytotic release of hexosaminidase from rat basophilic leukaemia cells

Mast cells are important players in innate immunity and mediate allergic responses. Upon stimulation, they release biologically active mediators including histamine, cytokines and lysosomal hydrolases. We used permeabilized rat basophilic leukaemia cells as model to identify R-SNAREs (soluble NSF (N-ethylmaleimide-sensitive fusion protein)) mediating exocytosis of hexosaminidase from mast cells. Of a complete set of recombinant mammalian R-SNAREs, only vesicle associated membrane protein (VAMP8)/endobrevin consistently blocked hexosaminidase release, which was also insensitive to treatment with clostridial neurotoxins. Thus, VAMP8, which also mediates fusion of late endosomes and lysosomes, plays a major role in hexosaminidase release, strengthening the view that mast cell granules share properties of both secretory granules and lysosomes.     

Tolerance of the resting cysts of Colpoda inflata (Ciliophora,Colpodea) and Meseres corlissi (Ciliophora,Spirotrichea) to desiccation and freezing

The survival of ciliate resting cysts, in the presence and absence of soil, was studied under two environmental stresses: desiccation and freezing. Laboratory strains of the common species Colpoda inflata and the rare species Meseres corlissi were used in these experiments, which yielded the following results: 1) Freezing of cysts in soil with a residual moisture level exceeding ～30% was destructive for both species. 2) Survival of Meseres corlissi cysts depended largely on the presence of soil. 3) In the absence of soil, Colpoda inflata cysts had greater tolerance to desiccation and freezing than Meseres corlissi cysts. Possible consequences for the distribution of natural populations are discussed.     

Interaction between transmembrane TNF and TNFR1/2 mediates the activation of monocytes by contact with T cells

Monocytes and monocytic cells produce proinflammatory cytokines upon direct cell contact with activated T cells. In the autoimmune disease rheumatoid arthritis, the pivotal role of TNF-alpha implies that the interaction between transmembrane TNF-alpha (mTNF) and the TNF receptors (TNFR1 and TNFR2) might participate in the T cell contact-dependent activation of monocytes. Accordingly, treatment of rheumatoid arthritis by administration of a TNF-alpha-blocking Ab was found to significantly decrease TNF-alpha production by monocytes. Several lines of evidence indicated that signaling through TNFR1/2 and through mTNF (reverse signaling) is involved in TNF-alpha production by monocytes after T cell contact: 1) blocking mTNF on activated T cells leads to a significant reduction in TNF-alpha production; 2) down-regulation of TNFR1/2 on monocytes by transfection with small interfering RNA results in diminished TNF-alpha production; 3) blocking or down-regulating TNFR2 on activated T cells inhibits TNF-alpha production, indicating that mTNF on the monocyte surface mediates signaling; 4) ligation of mTNF on monocytes by surface TNFR2 transfected into resting T cells induces TNF-alpha production due to reverse signaling by mTNF; and 5) ligation of mTNF on monocytes by a soluble TNFR2:Ig receptor construct induces TNF-alpha production due to reverse signaling. In conclusion, we identified mTNF and TNFR1/2 as interaction partners contributing to TNF-alpha production in monocytes. Both pathways initiated by mTNF-TNFR interaction are likely to be inhibited by treatment with anti-TNF-alpha Abs.     

20-Aminosteroids as a novel class of selective and complete androgen receptor antagonists and inhibitors of prostate cancer cell growth

Here, the synthesis and the evaluation of novel 20-aminosteroids on androgen receptor (AR) activity is reported. Compounds 11 and 18 of the series inhibit both the wild type and the T877A mutant AR-mediated transactivation indicating AR antagonistic function. Interestingly, minor structural changes such as stereoisomers of the amino lactame moiety exhibit preferences for antagonism among wild type and mutant AR. Other tested nuclear receptors are only weakly or not affected. In line with this, the prostate cancer cell growth of androgen-dependent but not of cancer cells lacking expression of the AR is inhibited. Further, the expression of the prostate specific antigen used as a diagnostic marker is also repressed. Finally steroid 18 enhances cellular senescence that might explain in part the growth inhibition mediated by this derivative. Steroids 11 and 18 are the first steroids that act as complete AR antagonists and exhibit AR specificity.     

Oligoribonuclease is a common downstream target of lithium-induced pAp accumulation in Escherichia coli and human cells

We identified Oligoribonuclease (Orn), an essential Escherichia coli protein and the only exonuclease degrading small ribonucleotides (5mer to 2mer) and its human homologue, small fragment nuclease (Sfn), in a screen for proteins that are potentially regulated by 3′-phosphoadenosine 5′-phosphate (pAp). We show that both enzymes are sensitive to micromolar amounts of pAp in vitro. We also demonstrate that Orn can degrade short DNA oligos in addition to its activity on RNA oligos, similar to what was documented for Sfn. pAp was shown to accumulate as a result of inhibition of the pAp-degrading enzyme by lithium, widely used to treat bipolar disorder, thus its regulatory targets are of significant medical interest. CysQ, the E.coli pAp-phosphatase is strongly inhibited by lithium and calcium in vitro and is a main target of lithium toxicity in vivo. Our findings point to remarkable conservation of the connection between sulfur- and RNA metabolism between E.coli and humans.