Molecular localization of a redox-modulated process regulating plant mitochondrial electron transport

Using in organellar assays, we found that significant tobacco alternative oxidase (AOX) activity is dependent on both reduction of a putative regulatory disulfide bond and the presence of pyruvate, which may interact with a Cys sulfhydryl. This redox modulation and pyruvate activation thus may be important in determining the partitioning of electrons to AOX in vivo. To investigate these regulatory mechanisms, we generated tobacco plants expressing mutated AOX proteins. Mutation of the most N-terminal Cys residue (Cys-126) to an Ala residue produced an AOX that could not be converted to the disulfide-linked form, thus identifying this Cys residue as being responsible for redox modulation. Although this mutation might be expected to produce an AOX with constitutive high activity in the presence of pyruvate, we found it to have minimal in organellar activity in the presence of pyruvate. Nonetheless, the Cys-126 mutation did not appear to have compromised the catalytic function of AOX, given that cells expressing the protein displayed high rates of cyanide-resistant respiration in vivo. The striking difference between in vivo and in organellar results suggests that an additional mechanism(s), as yet unidentified by in organellar assays, may promote activity in vivo. Mutation of the Cys residue nearest the presumptive active site (Cys-176) to an Ala residue did not prevent disulfide bond formation or affect the ability of AOX to be stimulated by pyruvate, indicating that this Cys residue is involved in neither redox modulation nor pyruvate activation.     

A major invasion of transposable elements accounts for the large size of the <Emphasis Type="Italic">Blumeria graminis</Emphasis> f.sp. <Emphasis Type="Italic">tritici</Emphasis> genome

Powdery mildew of wheat (Triticum aestivum L.) is caused by the ascomycete fungus Blumeria graminis f.sp. tritici. Genomic approaches open new ways to study the biology of this obligate biotrophic pathogen. We started the analysis of the Bg tritici genome with the low-pass sequencing of its genome using the 454 technology and the construction of the first genomic bacterial artificial chromosome (BAC) library for this fungus. High-coverage contigs were assembled with the 454 reads. They allowed the characterization of 56 transposable elements and the establishment of the Blumeria repeat database. The BAC library contains 12,288 clones with an average insert size of 115 kb, which represents a maximum of 7.5-fold genome coverage. Sequencing of the BAC ends generated 12.6 Mb of random sequence representative of the genome. Analysis of BAC-end sequences revealed a massive invasion of transposable elements accounting for at least 85% of the genome. This explains the unusually large size of this genome which we estimate to be at least 174 Mb, based on a large-scale physical map constructed through the fingerprinting of the BAC library. Our study represents a crucial step in the perspective of the determination and study of the whole Bg tritici genome sequence.     

The variability of the hepatitis B virus genome: statistical analysis and biological implications

A statistical analysis of the nucleotide sequence variability in 14 published hepatitis B virus (HBV) genomes was carried out using parametric and nonparametric methods. A parametric statistical model revealed that the different regions of the genome differed significantly in their variability. The conclusion was supported by a nonparametric kernel-density model of the HBV genome. Genes S, C, and P, region X, the precore region, and the pre-S2/pre-S1 regions were ranked in order of increasing variability. In many instances, conserved regions of the genome identified with sequences of known function in HBV biology. However, other characterized regions (such as pre-S) showed much variability despite the involvement of their encoded peptides in specific functions. Point mutations that may result in the formation of stop codons and amino acid changes may affect the clinical picture of HBV infection and may be reflected in atypical serological patterns.      

Pollen clumping and wind dispersal in an invasive angiosperm

Pollen dispersal is a fundamental aspect of plant reproductive biology that maintains connectivity between spatially separated populations. Pollen clumping, a characteristic feature of insect-pollinated plants, is generally assumed to be a detriment to wind pollination because clumps disperse shorter distances than do solitary pollen grains. Yet pollen clumps have been observed in dispersion studies of some widely distributed wind-pollinated species. We used Ambrosia artemisiifolia (common ragweed; Asteraceae), a successful invasive angiosperm, to investigate the effect of clumping on wind dispersal of pollen under natural conditions in a large field. Results of simultaneous measurements of clump size both in pollen shedding from male flowers and airborne pollen being dispersed in the atmosphere are combined with a transport model to show that rather than being detrimental, clumps may actually be advantageous for wind pollination. Initial clumps can pollinate the parent population, while smaller clumps that arise from breakup of larger clumps can cross-pollinate distant populations.     

Stomatal dimensions and resistance to diffusion

In the past, relations of diffusive resistance to stomatal geometry have concerned circular pores or pores that are replaced by equivalent circles of the same area. We calculated the resistance for general shapes that include the realistic slit. The resistance comprises two terms. The first is an outer resistance that depends only on ventilation and leaf geometry and is independent of stomata. The second is an inner resistance and is a function of stomatal interference and of stomatal geometry only. If interstomatal spacing is at least three times stomatal length, interstomatal interference is negligible. The inner resistance can then be calculated by adding the resistance of the two ends and the throat of each stoma. In the case of an elongated stoma, the part of the diffusive resistance per square centimeter determined by stomatal geometry is [Formula: see text] where a, b, d, and n are the semilength, semiwidth, depth, and density of the stomata, and D is the diffusivity. This is the familiar Brown and Escombe result applied to slits.     

STOMATAL MECHANICS

A model of stomatal movement due to changes in turgor is presented which systematically illustrates the role of certain anatomical features. During the expansion of paired guard cells, there are two physical constraints that cause the guard cells to bend and thus open the stomatal pore. The radial orientation of the micellae is shown to be the crucial feature which directly transmits the movement of the dorsal wall of the polar and central section to the stomatal slit. Furthermore, it is necessary that either the overall length of the entire stomatal apparatus or length of the common wall between the polar segments of the guard cells be constrained during the expansion of the guard cells. The model also shows that asymmetrically thickened guard cell walls are not necessary to cause bending of the guard cell. The ideas set forth in our model are consistent with the opening movements of both elliptical and grass-type stomata.     

Discovery of biclonal origin and a novel oncogene SLC12A5 in colon cancer by single-cell sequencing

Single-cell sequencing is a powerful tool for delineating clonal relationship and identifying key driver genes for personalized cancer management. Here we performed single-cell sequencing analysis of a case of colon cancer. Population genetics analyses identified two independent clones in tumor cell population. The major tumor clone harbored APC and TP53 mutations as early oncogenic events, whereas the minor clone contained preponderant CDC27 and PABPC1 mutations. The absence of APC and TP53 mutations in the minor clone supports that these two clones were derived from two cellular origins. Examination of somatic mutation allele frequency spectra of additional 21 whole-tissue exome-sequenced cases revealed the heterogeneity of clonal origins in colon cancer. Next, we identified a mutated gene SLC12A5 that showed a high frequency of mutation at the single-cell level but exhibited low prevalence at the population level. Functional characterization of mutant SLC12A5 revealed its potential oncogenic effect in colon cancer. Our study provides the first exome-wide evidence at single-cell level supporting that colon cancer could be of a biclonal origin, and suggests that low-prevalence mutations in a cohort may also play important protumorigenic roles at the individual level.     

Differential regulation of the expression of aquaporins 3 and 9 by Auphen and dbcAMP in the SMMC-7721 hepatocellular carcinoma cell line

Aquaglycero-aquaporins (agAQPs) are the structural foundation of rapid water transport and they appear to participate in cancer proliferation and malignancy. AQP3 expression is increased and AQP9 expression is decreased in hepatocellular carcinoma (HCC) compared to normal liver, which suggests their possible use as targets for cancer treatment. AQP-based modifiers, such as Auphen and dibutyryladenosine 3′, 5′-cyclic monophosphate (dbcAMP), might be used to treat several diseases and as chemical tools for assessing the functions of AQPs in biological systems. We investigated the effects of both Auphen on AQP3 and dbcAMP on AQP9 in SMMC-7721 cells. We used western blotting, real-time quantitative polymerase chain reaction (qPCR) and immunohistochemistry to evaluate changes in AQP3 and AQP9 expression in SMMC-7721 cells after culturing with Auphen and dbcAMP, respectively. We also determined the proliferation of SMMC-7721 cells. We found that compared to HL-7702 (L02) liver cells, Auphen increased AQP3 expression in tumor cells, whereas dbcAMP decreased expression of AQP9 in these cells. Also, high concentrations of Auphen and dbcAMP inhibited proliferation of SMMC-7721 cells in vitro. Auphen and dbcAMP may inhibit HCC development and could be considered targets for HCC diagnosis and therapy.