Characterization of a NifS-like chloroplast protein from Arabidopsis. Implications for its role in sulfur and selenium metabolism

NifS-like proteins catalyze the formation of elemental sulfur (S) and alanine from cysteine (Cys) or of elemental selenium (Se) and alanine from seleno-Cys. Cys desulfurase activity is required to produce the S of iron (Fe)-S clusters, whereas seleno-Cys lyase activity is needed for the incorporation of Se in selenoproteins. In plants, the chloroplast is the location of (seleno) Cys formation and a location of Fe-S cluster formation. The goal of these studies was to identify and characterize chloroplast NifS-like proteins. Using seleno-Cys as a substrate, it was found that 25% to 30% of the NifS activity in green tissue in Arabidopsis is present in chloroplasts. A cDNA encoding a putative chloroplast NifS-like protein, AtCpNifS, was cloned, and its chloroplast localization was confirmed using immunoblot analysis and in vitro import. AtCpNIFS is expressed in all major tissue types. The protein was expressed in Escherichia coli and purified. The enzyme contains a pyridoxal 5' phosphate cofactor and is a dimer. It is a type II NifS-like protein, more similar to bacterial seleno-Cys lyases than to Cys desulfurases. The enzyme is active on both seleno-Cys and Cys but has a much higher activity toward the Se substrate. The possible role of AtCpNifS in plastidic Fe-S cluster formation or in Se metabolism is discussed.     

Amiloride derivatives block ion channel activity and enhancement of virus-like particle budding caused by HIV-1 protein Vpu

The Vpu protein of human immunodeficiency virus type 1 forms cation-selective ion channels and enhances the process of virion budding and release. Mutagenesis studies have shown that the N-terminal transmembrane domain primarily controls both of these activities. Here we report that the Vpu ion channel is inhibited by the amiloride derivatives 5-(N,N-hexamethylene)amiloride and 5-(N,N-dimethyl)amiloride but not by amiloride itself, nor by amantadine. Hexamethyleneamiloride also inhibits budding of virus-like particles from HeLa cells expressing HIV-1 Gag and Vpu proteins. These results confirm the link between Vpu ion channel activity and the budding process and also suggest that amiloride derivatives might have useful anti-HIV-1 properties.     

beta-Tubulin C354 mutations that severely decrease microtubule dynamics do not prevent nuclear migration in yeast

Microtubule dynamics are influenced by interactions of microtubules with cellular factors and by changes in the primary sequence of the tubulin molecule. Mutations of yeast beta-tubulin C354, which is located near the binding site of some antimitotic compounds, reduce microtubule dynamicity greater than 90% in vivo and in vitro. The resulting intrinsically stable microtubules allowed us to determine which, if any, cellular processes are dependent on dynamic microtubules. The average number of cytoplasmic microtubules decreased from 3 in wild-type to 1 in mutant cells. The single microtubule effectively located the bud site before bud emergence. Although spindles were positioned near the bud neck at the onset of anaphase, the mutant cells were deficient in preanaphase spindle alignment along the mother-bud axis. Spindle microtubule dynamics and spindle elongation rates were also severely depressed in the mutants. The pattern and extent of cytoplasmic microtubule dynamics modulation through the cell cycle may reveal the minimum dynamic properties required to support growth. The ability to alter intrinsic microtubule dynamics and determine the in vivo phenotype of cells expressing the mutant tubulin provides a critical advance in assessing the dynamic requirements of an essential gene function.     

Targeted disruption of the Akap4 gene causes defects in sperm flagellum and motility

A-kinase anchoring proteins (AKAPs) tether cyclic AMP-dependent protein kinases and thereby localize phosphorylation of target proteins and initiation of signal-transduction processes triggered by cyclic AMP. AKAPs can also be scaffolds for kinases and phosphatases and form macromolecular complexes with other proteins involved in signal transduction. Akap4 is transcribed only in the postmeiotic phase of spermatogenesis and encodes the most abundant protein in the fibrous sheath, a novel cytoskeletal structure present in the principal piece of the sperm flagellum. Previous studies indicated that cyclic AMP-dependent signaling processes are important in the regulation of sperm motility, and gene targeting was used here to test the hypothesis that AKAP4 is a scaffold for protein complexes involved in regulating flagellar function. Sperm numbers were not reduced in male mice lacking AKAP4, but sperm failed to show progressive motility and male mice were infertile. The fibrous sheath anlagen formed, but the definitive fibrous sheath did not develop, the flagellum was shortened, and proteins usually associated with the fibrous sheath were absent or substantially reduced in amount. However, the other cytoskeletal components of the flagellum were present and appeared fully developed. We conclude that AKAP4 is a scaffold protein required for the organization and integrity of the fibrous sheath and that effective sperm motility is lost in the absence of AKAP4 because signal transduction and glycolytic enzymes fail to become associated with the fibrous sheath.     

Roles of the conserved aspartate and arginine in the catalytic mechanism of an archaeal beta-class carbonic anhydrase

The roles of an aspartate and an arginine, which are completely conserved in the active sites of beta-class carbonic anhydrases, were investigated by steady-state kinetic analyses of replacement variants of the beta-class enzyme (Cab) from the archaeon Methanobacterium thermoautotrophicum. Previous kinetic analyses of wild-type Cab indicated a two-step zinc-hydroxide mechanism of catalysis in which the k(cat)/K(m) value depends only on the rate constants for the CO(2) hydration step, whereas k(cat) also depends on rate constants from the proton transfer step (K. S. Smith, N. J. Cosper, C. Stalhandske, R. A. Scott, and J. G. Ferry, J. Bacteriol. 182:6605-6613, 2000). The recently solved crystal structure of Cab shows the presence of a buffer molecule within hydrogen bonding distance of Asp-34, implying a role for this residue in the proton transport step (P. Strop, K. S. Smith, T. M. Iverson, J. G. Ferry, and D. C. Rees, J. Biol. Chem. 276:10299-10305, 2001). The k(cat)/K(m) values of Asp-34 variants were decreased relative to those of the wild type, although not to an extent which supports an essential role for this residue in the CO(2) hydration step. Parallel decreases in k(cat) and k(cat)/K(m) values for the variants precluded any conclusions regarding a role for Asp-34 in the proton transfer step; however, the k(cat) of the D34A variant was chemically rescued by replacement of 2-(N-morpholino)propanesulfonic acid buffer with imidazole at pH 7.2, supporting a role for the conserved aspartate in the proton transfer step. The crystal structure of Cab also shows Arg-36 with two hydrogen bonds to Asp-34. Arg-36 variants had both k(cat) and k(cat)/K(m) values that were decreased at least 250-fold relative to those of the wild type, establishing an essential function for this residue. Imidazole was unable to rescue the k(cat) of the R36A variant; however, partial rescue of the kinetic parameter was obtained with guanidine-HCl indicating that the guanido group of this residue is important.     

Two-photon fluorescence lifetime imaging of the skin stratum corneum pH gradient

Two-photon fluorescence lifetime imaging is used to identify microdomains (1-25 microm) of two distinct pH values within the uppermost layer of the epidermis (stratum corneum). The fluorophore used is 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), whose lifetime tau (pH 4.5, tau = 2.75 ns; pH 8.5, tau = 3.90 ns) is pH dependent over the pH range of the stratum corneum (pH 4.5 to pH 7.2). Hairless mice (SKH1-hrBR) are used as a model for human skin. Images (< or =50 microm x 50 microm) are acquired every 1.7 microm from the stratum corneum surface to the first viable layer (stratum granulosum). Acidic microdomains (average pH 6.0) of variable size (~1 microm in diameter with variable length) are detected within the extracellular matrix of the stratum corneum, whereas the intracellular space of the corneocytes in mid-stratum corneum (25 microm diameter) approaches neutrality (average pH 7.0). The surface is acidic. The average pH of the stratum corneum increases with depth because of a decrease in the ratio of acidic to neutral regions within the stratum corneum. The data definitively show that the stratum corneum acid mantle results from the presence of aqueous acidic pockets within the lipid-rich extracellular matrix.     

25-Hydroxyvitamin D-1alpha-hydroxylase activity is diminished in human prostate cancer cells and is enhanced by gene transfer

The hormone 1alpha,25-dihydroxyvitamin D (1alpha,25(OH)(2)D) inhibits growth and induces differentiation of prostate cells. The enzyme responsible for 1alpha,25(OH)(2)D synthesis, 25-hydroxyvitamin D (25(OH)D)-1alpha-hydroxylase (1alpha-OHase), has been demonstrated in human prostate cells. We compared the levels of 1alpha-OHase activity in prostate cancer cell lines, LNCaP, DU145 and PC-3 and in primary cultures of normal, cancerous and benign prostatic hyperplasia (BPH) prostate cells. We observed a marked decrease in 1alpha-OHase activity in prostate cancer cells, including an undetectable level of activity in LNCaP cells. Transient or stable transfection of 1alpha-OHase cDNA into LNCaP cells increased 1alpha-OHase activity from undetectable to 4.95pmole/mg+/-0.69pmole/mg and 5.8pmole/mg+/-0.7pmole/mg protein per hour, respectively. In response to 25(OH)D, the prohormone of 1alpha,25(OH)(2)D, the transfected LNCaP cells showed a significant inhibition of 3H-thymidine incorporation (37%+/-6% and 56%+/-4% at 10(-8)M for transiently and stably transfected cells, respectively). These findings support an important autocrine role for 1alpha,25(OH)(2)D in the prostate and suggest that the re-introduction of the 1alpha-OHase gene to prostate cancer cells, in conjunction with the systemic administration of 25(OH)D, constitutes an endocrine form of gene therapy that may be less toxic than the systemic administration of 1alpha,25(OH)(2)D.     

Normal greeble learning in a severe case of developmental prosopagnosia

A central question in cognitive neuroscience is whether mechanisms exist that are specialized for particular domains. One of the most commonly cited examples of a domain-specific competence is the human ability to recognize upright faces. However, according to a widely discussed alternative hypothesis, face recognition is instead performed by mechanisms specialized for processing any object class for which an individual has expertise. Faces, according to this domain-general hypothesis, are just one example of an expert class. Nonface object expertise has been intensively investigated using a training procedure involving an artificial stimulus class known as greebles. A key prediction of this hypothesis is that individuals with face recognition impairments will also have impairments with other categories that control subjects have expertise with. Our results show that a man with severe prosopagnosia performed normally throughout the standard greeble training procedure. These findings indicate that face recognition and greeble recognition rely on separate mechanisms.     

Quantitative analysis of indigo and indigo precursors in leaves of Isatis spp. and Polygonum tinctorium

Analysis of extracts from two woad species (Isatis tinctoria and Isatis indigotica) and Polygonum tinctorium revealed that only one indigo precursor (indican) was present in Polygonum, but two precursors were found in Isatis spp. This was done using high performance liquid chromatography (HPLC), coupled to an evaporative light scattering detector (ELSD). In Isatis spp., the indigo precursors indican and a fraction representing isatan B were identified. The proportion of indican and isatan B was different between the two Isatis spp. tested. For the first time, it was possible to quantify the precursors in woad plant species, and the results were found to be in good agreement with those made from total indigo quantification using two different spectrophotometric methods or a derivatization technique.     

Cre recombinase specificity defined by the tau locus

We generated a transgenic mouse line (tau::Cre) by targeting the Cre to the tau locus (Mapt). Based on previous reports on the expression of Tau during development, we expected the Cre recombinase to be expressed in a neuron-specific and pan-neuronal manner. However, intercrosses between the tau::Cre and the Cre-activatable reporter animals resulted in offspring with recombination either restricted to the nervous system or throughout the entire conceptus, indicating expression of Tau early in development. The percentage of neuron-specific excision was dependent on the Cre reporter used representing different Cre target sites in the mouse genome. In spite of the observed variability, our data suggest that the tau::Cre mouse line can be used for pan-neuronal recombination of floxed alleles when it is used with caution.