Genomic comparison of Mycobacterium avium subsp. paratuberculosis sheep and cattle strains by microarray hybridization

Microarray-based comparisons of three Mycobacterium avium subsp. paratuberculosis isolates, including one sheep strain and two cattle strains, identified three large genomic deletions in the sheep strain, totaling 29,208 bp and involving 24 open reading frames. These deletions may help explain some of the differences in pathogenicity and host specificity observed between the cattle and sheep strains of Mycobacterium avium subsp. paratuberculosis.     

Inhibition kinetics of lactate dehydrogenase isoenzymes by gossypol acetic acid

The effect of gossypol acetic acid, a potent male sterilent was studied on LDH from goat liver (LDH-A4), heart (LDH-B4) and testis (LDH-C4) in vitro. All the preparations of LDH were inhibited by gossypol when the reaction was carried out in pyruvate-lactate (direct) or lactate to pyruvate (reverse) directions. The IC50 of gossypol for the pyruvate oxidation by LDH isozymes varied between 16 and 42 microM in presence of 0.27 mM pyruvate and 0.15 mM NADH at 25 degrees C and pH 7.4 whereas for the lactate oxidation, IC50 was 125 microM in a system containing 3.3 mM lactic acid and 1.8 mM NAD at 25 degrees C and pH 9.0. Reciprocal plots due to Lineweaver-Burk showed that these isozymes are inhibited in a non-competitive manner with respect to pyruvate and lactate, and in a competitive fashion when NAD and NADH were varied as substrates. Ki values of LDH-A4, -B4 and -C4 isozymes in presence of gossypol were 20, 34 and 29 microM against pyruvate; 33, 43 and 45 microM against NADH; 85, 85 and 125 microM against lactate and 94, 108 and 83 microM against NAD respectively.     

Andrographolide inhibits prostate cancer by targeting cell cycle regulators,CXCR3 and CXCR7 chemokine receptors

Despite state of the art cancer diagnostics and therapies offered in clinic, prostate cancer (PCa) remains the second leading cause of cancer-related deaths. Hence, more robust therapeutic/preventive regimes are required to combat this lethal disease. In the current study, we have tested the efficacy of Andrographolide (AG), a bioactive diterpenoid isolated from Andrographis paniculata, against PCa. This natural agent selectively affects PCa cell viability in a dose and time-dependent manner, without affecting primary prostate epithelial cells. Furthermore, AG showed differential effect on cell cycle phases in LNCaP, C4-2b and PC3 cells compared to retinoblastoma protein (RB^?/?) and CDKN2A lacking DU-145 cells. G2/M transition was blocked in LNCaP, C4-2b and PC3 after AG treatment whereas DU-145 cells failed to transit G1/S phase. This difference was primarily due to differential activation of cell cycle regulators in these cell lines. Levels of cyclin A2 after AG treatment increased in all PCa cells line. Cyclin B1 levels increased in LNCaP and PC3, decreased in C4-2b and showed no difference in DU-145 cells after AG treatment. AG decreased cyclin E2 levels only in PC3 and DU-145 cells. It also altered Rb, H3, Wee1 and CDC2 phosphorylation in PCa cells. Intriguingly, AG reduced cell viability and the ability of PCa cells to migrate via modulating CXCL11 and CXCR3 and CXCR7 expression. The significant impact of AG on cellular and molecular processes involved in PCa progression suggests its potential use as a therapeutic and/or preventive agent for PCa.     

Development and use of a partial Mycobacterium avium subspecies paratuberculosis protein array

As an initial step toward systematically characterizing all antigenic proteins produced by a significant veterinary pathogen, 43 recombinant Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) expression clones were constructed, cataloged, and stored. NC filters were spotted with purified proteins from each clone along with a whole cell lysate of M. paratuberculosis. Spots on the resulting dot array consisted of hypothetical proteins (13), metabolic proteins (3), cell envelope proteins (7), known antigens (4), and unique proteins with no similarity in public sequence databases (16). Dot blot arrays were used to profile antibody responses in a rabbit and mouse exposed to M. paratuberculosis as well as in cattle showing clinical signs of Johne's disease. The M. paratuberculosis heat shock protein DnaK, encoded by ORF MAP3840 and a membrane protein (MAP2121c), were identified as the most strongly immunoreactive in both the mouse and rabbit hosts, respectively. MAP3155c, which encodes a hypothetical protein, was most strongly immunoreactive in sera from Johne's disease cattle. This study has enabled direct comparisons of antibody reactivity for an entire panel of over 40 proteins and has laid the foundation for future high throughput production and arraying of M. paratuberculosis surface proteins for immune profiling experiments in cattle.     

Calcium tips the balance: a microtubule plus end to lattice binding switch operates in the carboxyl terminus of BPAG1n4

Microtubules (MTs) are integral to numerous cellular functions, such as cell adhesion, differentiation and intracellular transport. Their dynamics are largely controlled by diverse MT‐interacting proteins, but the signalling mechanisms that regulate these interactions remain elusive. In this report, we identify a rapid, calcium‐regulated switch between MT plus end interaction and lattice binding within the carboxyl terminus of BPAG1n4. This switch is EF‐hand dependent, and mutations of the EF‐hands abolish this dynamic behaviour. Our study thus uncovers a new, calcium‐dependent regulatory mechanism for a spectraplakin, BPAG1n4, at the MT plus end.     

Application of Gene Network Analysis Techniques Identifies AXIN1/PDIA2 and Endoglin Haplotypes Associated with Bicuspid Aortic Valve

Bicuspid Aortic Valve (BAV) is a highly heritable congenital heart defect. The low frequency of BAV (1% of general population) limits our ability to perform genome-wide association studies. We present the application of four a priori SNP selection techniques, reducing the multiple-testing penalty by restricting analysis to SNPs relevant to BAV in a genome-wide SNP dataset from a cohort of 68 BAV probands and 830 control subjects. Two knowledge-based approaches, CANDID and STRING, were used to systematically identify BAV genes, and their SNPs, from the published literature, microarray expression studies and a genome scan. We additionally tested Functionally Interpolating SNPs (fitSNPs) present on the array; the fourth consisted of SNPs selected by Random Forests, a machine learning approach. These approaches reduced the multiple testing penalty by lowering the fraction of the genome probed to 0.19% of the total, while increasing the likelihood of studying SNPs within relevant BAV genes and pathways. Three loci were identified by CANDID, STRING, and fitSNPS. A haplotype within the AXIN1-PDIA2 locus (p-value of 2.926×10⁻⁰⁶) and a haplotype within the Endoglin gene (p-value of 5.881×10⁻⁰⁴) were found to be strongly associated with BAV. The Random Forests approach identified a SNP on chromosome 3 in association with BAV (p-value 5.061×10⁻⁰⁶). The results presented here support an important role for genetic variants in BAV and provide support for additional studies in well-powered cohorts. Further, these studies demonstrate that leveraging existing expression and genomic data in the context of GWAS studies can identify biologically relevant genes and pathways associated with a congenital heart defect.     

A case of true hermaphroditism reveals an unusual mechanism of twinning

Traditionally twins are classified as dizygous or fraternal and monozygous or identical (Hall Twinning, 362, 2003 and 735-743). We report a rare case of 46,XX/46,XY twins: Twin A presented with ambiguous genitalia and Twin B was a phenotypically normal male. These twins demonstrate a third, previously unreported mechanism for twinning. The twins underwent initial investigation with 17-hydroxyprogesterone and testosterone levels, pelvic ultrasound and diagnostic laparoscopy. Cytogenetic analysis was performed on peripheral blood cells and skin fibroblasts. Histological examination and Fluorescence in situ hybridization studies on touch imprints were performed on gonadal biopsies. DNA analysis using more than 6,000 DNA markers was performed on skin fibroblast samples from the twins and on peripheral blood samples from both parents. Twin A was determined to be a true hermaphrodite and Twin B an apparently normal male. Both twins had a 46,XX/46,XY chromosome complement in peripheral lymphocytes, skin fibroblasts, and gonadal biopsies. The proportion of XX to XY cells varied between the twins and the tissues evaluated. Most significantly the twins shared 100% of maternal alleles and approximately 50% of paternal alleles in DNA analysis of skin fibroblasts. The twins are chimeric and share a single genetic contribution from their mother but have two genetic contributions from their father thus supporting the existence of a third, previously unreported type of twinning.     

Reliability of detecting rRNA sequences of Chlamydia trachomatis with fluorescence in situ hybridization without amplification

OBJECTIVE: To use fluorescence in situ hybridization (FISH) using ribosomal RNA (rRNA) oligonucleotide probes as the target nucleic acid for the detection of Chlamydia trachomatis. STUDY DESIGN: Suitable sequences selected from the rRNA sequence of C trachomatis were labeled with a fluorescent dye and used in FISH for detecting chlamydial inclusion bodies and/ or elementary bodies in paraformaldehyde-fixed urogenital swab samples. The sensitivity and specificity of the FISH assay were compared with those of the polymerase chain reaction (PCR) using plasmid primers. Positive known C trachomatis-infected McCoy cells were used as positive controls. Urogenital swab specimens that were C trachomatis negative on culture and PCR were used as negative controls. RESULT: Among the 128 samples included in the study, FISH was positive in 28 (21.8%) and PCR in 33 (25.7%). A significant correlation was found between the 2 detection methods. Results of PCR and FISH were consistent in 115 of the 128 samples (R = 0.89). Thirteen samples showed discordant results. Of these, 9 FISH negative samples were PCR positive and 4 FISH positive samples were PCR negative. CONCLUSION: FISH was a highly specific and fairly sensitive technique for detecting C trachomatis. Signal amplification techniques and use of different fluorophores may further increase the sensitivity of this technique.     

UV radiation reduces epidermal cell expansion in Arabidopsis thaliana leaves without altering cellular microtubule organization

Upon chronic UV treatment pavement cell expansion in Arabidopsis leaves is reduced, implying alterations in symplastic and apoplastic properties of the epidermal cells. In this study, the effect of UV radiation on microtubule patterning is analysed, as microtubules are thought to serve as guiding rails for the cellulose synthase complexes depositing cellulose microfibrils. Together with hemicelluloses, these microfibrils are regarded as the load-bearing components of the cell wall. Leaves of transgenic plants with fluorescently tagged microtubules (GFP-TUA6) were as responsive to UV as wild type plants. Despite the UV-induced reduction in cell elongation, confocal microscopy revealed that cellular microtubule arrangements were seemingly not affected by the UV treatments. This indicates an unaltered deposition of cellulose microfibrils in the presence of UV radiation. Therefore, we surmise that the reduction in cell expansion in UV-treated leaves is most probably due to changes in cell wall loosening and/or turgor pressure.Key words: arabidopsis, cell expansion, GFP-TUA6, leaf development, microtubule cytoskeleton, UV radiationPhotosynthetic functions such as solar light capture and carbon fixation are highly evolved features of plant leaves. To fulfil these functions in an optimal way, leaf development needs to be tuned to environmental conditions. Leaves are continuously exposed and subjected to environmental influences, which serve as co-regulators of leaf and plant development.¹ This ability of plants to adapt, secures the plant''s survival, even under non-optimal conditions. An example of a regulatory environmental parameter is solar light, indispensable for photosynthesis but potentially causing photoinhibition and/or UV-radiation stress. The highly energetic ultraviolet B (UV-B) rays of short wavelengths (280–315 nm) can both cause damage, as well as induce a range of specific metabolic and morphogenic plant responses. It was reported before that exposure to low dose UV radiation reduces Arabidopsis leaf size due to a decreased cell size.² Expansion of leaf epidermal cells of Arabidopsis thaliana is the combined action of promotion and restriction of growth, resulting in the typical irregular sinuous pavement cells. It has been postulated that cellulose microfibrils are responsible for generating a force opposing isotropic expansion by creating neck regions in between outgrowing lobes.³ As the microtubule cytoskeleton is believed to serve as guiding rails for the cellulose synthase complexes (CESAs),⁴ the deposition of the cellulose fibrils is intimately linked to the cortical microtubule arrangement. We have studied the UV-effect on microtubule organisation in leaf epidermal cells whose expansion had decreased upon this UV radiation. Microtubules in the adaxial pavement cells of the fourth leaf were monitored on several successive days in a transgenic line containing GFP fused to tubulin A6.⁵ The chronic UV treatment was started on day 0 when the plants were 2 weeks old, using UV exposure conditions as described in reference ². First the responsiveness of the GFP-TUA6 plants to UV radiation was evaluated. Similar to wild type (WT) plants,² the GFP-TUA6 plants had smaller leaves following 8 days of UV treatment (t-test, p < 0.01) (Fig. 1). This was caused by a significant reduction in the generalized cell area average of all measured cells, irrespective of the location within the leaf (Fig. 1; t-test, p < 0.01). In more detail, the average cell area within the base, middle and top zones of the GFP-TUA6 leaf was systematically lower in UV-treated leaves from 8 days after the treatment started onwards (data not shown).Open in a separate windowFigure 1Effect of UV radiation on leaf and cell area after different days of UV radiation. Open asterisks indicate a statistically significant difference in leaf area between UV-treated and control plants, black asterisks indicate statistically significant difference in cell area (t-test, *p < 0.05, **p < 0.01, ***p < 0.001). Error bars indicate the standard error for five different leaves at all measured time-points and 600, 170 and 180 cells at day 0, 8 and 12 respectively.As GFP-TUA6 leaves were as responsive to UV radiation as wild type leaves, confocal microscopy was used to visualize the organisation of the cortical microtubules facing the outer periclinal wall of the adaxial epidermis. No clear difference in microtubule (re)organization could be detected during the development of pavement cells, and throughout the UV treatment period. As shown in Figure 2 at day 2, pavement cells with comparable areas are similarly shaped in control and UV-irradiated plants and contain similar microtubule arrangements (Fig. 2 and marked cells). This means that microtubule organization is not directly affected by the UV exposure and that shape development proceeds in an analoguous manner as under control conditions. This lack of alteration in the microtubule arrangement can be observed for cells at the leaf tip, which were already in the process of lobe formation at the start of the exposure period, as well as for cells at the leaf base. Under our growth conditions, and in the monitored leaf number 4, cell proliferation still took place in this part of the leaf and lobes only started to appear on the cell surface. As microtubules are linked to the deposition of cellulose microfibrils, it can be assumed that no alterations in cellulose deposition occur upon UV treatment either. We can therefore conclude that the process of lobe formation and microtubule patterning is not impeded and that only the extent of cell expansion is restricted upon UV exposure.Open in a separate windowFigure 2Microtubule pattern in control and UV-exposed leaves visualized using GFP-TUA6 and confocal microscopy. Both images are from cells at the mid zone of the fourth leaf at day 2. Microtubules are similarly arranged in equally shaped and sized cells of control and UV-exposed leaves. The marked cells show a pattern whereby the tubules are centred in the neck regions between two outgrowing lobes.According to the Lockhart equation,⁶ cell (wall) growth is modulated by wall biomechanics and turgor pressure. Concerning turgor pressure, no clear differences in this factor between UV-exposed and control plants of Lactuca sativa L.⁷ and Pisum sativum⁸ could be observed, reinforcing the idea that especially the modulation of cell wall properties is the main factor causing the observed UV-induced reduction in cell expansion. Some reports indicate differential expression of wall loosening enzymes like expansins or xyloglucan endotransglycosylase/hydrolases (XTHs),^9,10 or cell wall strengthening enzymes as particular peroxidases⁷ after UV exposure. Another key event could involve UV-mediated changes in the phenylpropanoid pathway, which may cause changes in the lignin biosynthesis. As shown by the literature^11–14 lignin may well be an important modulator of cell wall architecture in Arabidopsis and therefore alterations in lignin synthesis could form the basis for morphological modifications. Further research on the cell wall properties of UV-treated plants may resolve this uncertainty.As a general conclusion we can state that the patterning of microtubules is not altered, but that alterations in cell wall composition or arrangements are the most plausible candidates for the observed reduction in pavement cell expansion upon chronic UV treatment.