Kinesin heads fused to hinge-free myosin tails drive efficient motility

The rat kinesin motor domain was fused at residues 433, 411, 376 or 367, respectively, to the C-terminal 1185, 1187, 1197 or 1185 residues of the brush border myosin tail. In motility assays, K433myt and K411myt, which preserve the head-proximal kinesin hinge, and K367myt, which deletes it, drove rapid microtubule sliding ( approximately 0.6 microms(-1)) that was optimal when the head-pairs were spaced apart by adding 1:1 headless myosin tails. K376myt, which partially deletes the head-proximal hinge, showed poor motility in sliding assays but wild type processivity, velocity and stall force in single molecule optical trapping. Accordingly, the head-proximal kinesin hinge is functionally dispensable.     

The P450 monooxygenase BcABA1 is essential for abscisic acid biosynthesis in Botrytis cinerea

The phytopathogenic ascomycete Botrytis cinerea is known to produce abscisic acid (ABA), which is thought to be involved in host-pathogen interaction. Biochemical analyses had previously shown that, in contrast to higher plants, the fungal ABA biosynthesis probably does not proceed via carotenoids but involves direct cyclization of farnesyl diphosphate and subsequent oxidation steps. We present here evidence that this "direct" pathway is indeed the only one used by an ABA-overproducing strain of B. cinerea. Targeted inactivation of the gene bccpr1 encoding a cytochrome P450 oxidoreductase reduced the ABA production significantly, proving the involvement of P450 monooxygenases in the pathway. Expression analysis of 28 different putative P450 monooxygenase genes revealed two that were induced under ABA biosynthesis conditions. Targeted inactivation showed that one of these, bcaba1, is essential for ABA biosynthesis: DeltaBcaba1 mutants contained no residual ABA. Thus, bcaba1 represents the first identified fungal ABA biosynthetic gene.     

Plasmodium induces swelling-activated ClC-2 anion channels in the host erythrocyte

Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum depends on delivery of nutrients. Moreover, infection challenges cell volume constancy of the host erythrocyte requiring enhanced activity of cell volume regulatory mechanisms. Patch clamp recording demonstrated inwardly and outwardly rectifying anion channels in infected but not in control erythrocytes. The molecular identity of those channels remained elusive. We show here for one channel type that voltage dependence, cell volume sensitivity, and activation by oxidation are identical to ClC-2. Moreover, Western blots and FACS analysis showed protein and functional ClC-2 expression in human erythrocytes and erythrocytes from wild type (Clcn2(+/+)) but not from Clcn2(-/-) mice. Finally, patch clamp recording revealed activation of volume-sensitive inwardly rectifying channels in Plasmodium berghei-infected Clcn2(+/+) but not Clcn2(-/-) erythrocytes. Erythrocytes from infected mice of both genotypes differed in cell volume and inhibition of ClC-2 by ZnCl(2) (1 mm) induced an increase of cell volume only in parasitized Clcn2(+/+) erythrocytes. Lack of ClC-2 did not inhibit P. berghei development in vivo nor substantially affect the mortality of infected mice. In conclusion, activation of host ClC-2 channels participates in the altered permeability of Plasmodium-infected erythrocytes but is not required for intraerythrocytic parasite survival.     

Interference of hepatitis A virus replication by small interfering RNAs

The rate of acute liver failure due to hepatitis A virus (HAV) has not decreased, and therapy of severe infections is still of major interest. Using a DNA-based HAV replicon cell culture system, we demonstrate that small interfering RNAs (siRNAs) targeted against viral sequences or a reporter gene contained in the viral genome specifically inhibit HAV RNA replication in HuhT7 cells. Combinations of siRNAs were more effective suppressors of HAV RNA replication. Also, siRNAs targeted against HAV 2C and 3D inhibited the expression of the respective protein. Expressions of endogenous beta-actin and double-stranded-specific RNA-activated serin/threonine kinase (PKR) were unaltered, demonstrating that the siRNA inhibitory effect was not connected to interferon inhibition, but rather was specifically targeted against HAV RNA. These results suggest that RNA interference might ultimately be useful in treatment of severe HAV infection with or without chronic liver diseases.     

Yersinia enterocolitica type III secretion chaperone SycH. Recombinant expression, purification, characterisation, and crystallisation

All pathogenic Yersinia species (Y. enterocolitica, Y. pestis, and Y. pseudotuberculosis) share a type three secretion system (TTSS) that allows translocation of effector proteins into host cells. Yersinia enterocolitica SycH is a chaperone assisting the transport of the effector YopH and two regulatory components of the TTSS, YscM1 and YscM2. We have recombinantly expressed SycH in Escherichia coli. Purification of tag-free SycH to near homogeneity was achieved by combining ammonium sulfate precipitation, anion exchange chromatography, and gel filtration. Functionality of purified SycH was proven by demonstrating binding to YopH. SycH crystals were grown that diffracted to 2.94A resolution. Preliminary crystallographic data and biochemical findings suggest that SycH forms homotetramers. SycH may therefore represent a novel class of TTSS chaperones. In addition, we found that YopH was enzymatically active in the presence of SycH. This implies that the function of the secretion chaperone SycH is not to keep YopH in a globally unfolded state prior to secretion.     

The Caenorhabditis elegans SCC-3 homologue is required for meiotic synapsis and for proper chromosome disjunction in mitosis and meiosis

The product of the Caenorhabditis elegans ORF F18E2.3 is homologous to the cohesin component Scc3p. By antibody staining the product of F18E2.3 is found in interphase and early meiotic nuclei. At pachytene it localizes to the axes of meiotic chromosomes but is no longer detectable on chromatin later in meiosis or in mitoses. Depletion of the gene product by RNAi results in aberrant mitoses and meioses. In meiosis, homologous pairing is defective during early meiotic prophase and at diakinesis there occur univalents consisting of loosely connected sister chromatids or completely separated sisters. The recombination protein RAD-51 accumulates in nuclear foci at higher numbers during meiotic prophase and disappears later than in wild-type worms, suggesting a defect in the repair of meiotic double-stranded DNA breaks. Embryos showing nuclei of variable size and anaphase bridges, indicative of mitotic segregation defects, are frequently observed. In the most severely affected gonads, nuclear morphology cannot be related to any specific stage. The cytological localization and the consequences of the lack of the protein indicate that C. elegans SCC-3 is essential for sister chromatid cohesion both in mitosis and in meiosis.     

The Nitrilase ZmNIT2 converts indole-3-acetonitrile to indole-3-acetic acid

We isolated two nitrilase genes, ZmNIT1 and ZmNIT2, from maize (Zea mays) that share 75% sequence identity on the amino acid level. Despite the relatively high homology to Arabidopsis NIT4, ZmNIT2 shows no activity toward beta-cyano-alanine, the substrate of Arabidopsis NIT4, but instead hydrolyzes indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA). ZmNIT2 converts IAN to IAA at least seven to 20 times more efficiently than AtNIT1/2/3. Quantitative real-time polymerase chain reaction revealed the gene expression of both nitrilases in maize kernels where high concentrations of IAA are synthesized tryptophan dependently. Nitrilase protein and endogenous nitrilase activity are present in maize kernels together with the substrate IAN. These results suggest a role for ZmNIT2 in auxin biosynthesis.     

Use of complex DNA and antibody microarrays as tools in functional analyses

While the deciphering of basic sequence information on a genomic scale is yielding complete genomic sequences in ever-shorter intervals, experimental procedures for elucidating the cellular effects and consequences of the DNA-encoded information become critical for further analyses. In recent years, DNA microarray technology has emerged as a prime candidate for the performance of many such functional assays. Technically, array technology has come a long way since its conception some 15 years ago, initially designed as a means for large-scale mapping and sequencing.The basic arrangement, however, could be adapted readily to serve eventually as an analytical tool in a large variety of applications. On their own or in combination with other methods, microarrays open up many new avenues of functional analysis.