A PHABULOSA-Controlled Genetic Pathway Regulates Ground Tissue Patterning in the Arabidopsis Root

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Effect of Aqueous and Nonaqueous Fixatives on the Quantitative Estimation of Collagen-Proteoglycan Interaction in Tissue Sections

The present study was designed to assess the influence of aqueous and nonaqueous fixatives on the quantitative estimation of collagen-proteoglycan interaction in tissue sections. Tissues containing different collagen types and distinct sulfated proteoglycan classes were isolated from pig costal cartilage, human skin, and the inner muscular layer of dog small intestine and fixed using aqueous or nonaqueous methods. The results showed that the best fixation method was exposure to paraformaldehyde gas. When using aqueous fixatives, proteoglycans were lost to different degrees among the various tissues analyzed, reflecting differences in chemical properties of proteoglycan classes and/or in their interactions with other matrix components such as collagen.     

Using antibodies against P2Y and P2X receptors in purinergic signaling research

The broad expression pattern of the G protein-coupled P2Y receptors has demonstrated that these receptors are fundamental determinants in many physiological responses, including neuromodulation, vasodilation, inflammation, and cell migration. P2Y receptors couple either G_q or G_i upon activation, thereby activating different signaling pathways. Ionotropic ATP (P2X) receptors bind extracellular nucleotides, a signal which is transduced within the P2X protein complex into a cation channel opening, which usually leads to intracellular calcium concentration elevation. As such, this family of proteins initiates or shapes several cellular processes including synaptic transmission, gene expression, proliferation, migration, and apoptosis. The ever-growing range of applications for antibodies in the last 30 years attests to their major role in medicine and biological research. Antibodies have been used as therapeutic tools in cancer and inflammatory diseases, as diagnostic reagents (flow cytometry, ELISA, and immunohistochemistry, to name a few applications), and in widespread use in biological research, including Western blot, immunoprecipitation, and ELISPOT. In this article, we will showcase several of the advances that scientists around the world have achieved using the line of antibodies developed at Alomone Labs for P2Y and P2X receptors.

     

A Locus at 5q33.3 Confers Resistance to Tuberculosis in Highly Susceptible Individuals

Immunosuppression resulting from HIV infection increases the risk of progression to active tuberculosis (TB) both in individuals newly exposed to Mycobacterium tuberculosis (MTB) and in those with latent infections. We hypothesized that HIV-positive individuals who do not develop TB, despite living in areas where it is hyperendemic, provide a model of natural resistance. We performed a genome-wide association study of TB resistance by using 581 HIV-positive Ugandans and Tanzanians enrolled in prospective cohort studies of TB; 267 of these individuals developed active TB, and 314 did not. A common variant, rs4921437 at 5q33.3, was significantly associated with TB (odds ratio = 0.37, p = 2.11 × 10⁻⁸). This variant lies within a genomic region that includes IL12B and is embedded in an H3K27Ac histone mark. The locus also displays consistent patterns of linkage disequilibrium across African populations and has signals of strong selection in populations from equatorial Africa. Along with prior studies demonstrating that therapy with IL-12 (the cytokine encoded in part by IL12B, associated with longer survival following MTB infection in mice deficient in CD4 T cells), our results suggest that this pathway might be an excellent target for the development of new modalities for treating TB, especially for HIV-positive individuals. Our results also indicate that studying extreme disease resistance in the face of extensive exposure can increase the power to detect associations in complex infectious disease.     

The role of GDP-l-galactose phosphorylase in the control of ascorbate biosynthesis

The enzymes involved in l-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff–Wheeler [SW] pathway) are well established. Here, we analyzed their subcellular localizations and potential physical interactions and assessed their role in the control of ascorbate synthesis. Transient expression of C terminal-tagged fusions of SW genes in Nicotiana benthamiana and Arabidopsis thaliana mutants complemented with genomic constructs showed that while GDP-d-mannose epimerase is cytosolic, all the enzymes from GDP-d-mannose pyrophosphorylase (GMP) to l-galactose dehydrogenase (l-GalDH) show a dual cytosolic/nuclear localization. All transgenic lines expressing functional SW protein green fluorescent protein fusions driven by their endogenous promoters showed a high accumulation of the fusion proteins, with the exception of those lines expressing GDP-l-galactose phosphorylase (GGP) protein, which had very low abundance. Transient expression of individual or combinations of SW pathway enzymes in N. benthamiana only increased ascorbate concentration if GGP was included. Although we did not detect direct interaction between the different enzymes of the pathway using yeast-two hybrid analysis, consecutive SW enzymes, as well as the first and last enzymes (GMP and l-GalDH) associated in coimmunoprecipitation studies. This association was supported by gel filtration chromatography, showing the presence of SW proteins in high-molecular weight fractions. Finally, metabolic control analysis incorporating known kinetic characteristics showed that previously reported feedback repression at the GGP step, combined with its relatively low abundance, confers a high-flux control coefficient and rationalizes why manipulation of other enzymes has little effect on ascorbate concentration.

Metabolic engineering, genetic analysis, and functional mutant complementation identify GDP-l-galactose phosphorylase as the main control point in ascorbate biosynthesis in green tissues.     

Redox activation of ATM enhances GSNOR translation to sustain mitophagy and tolerance to oxidative stress

The denitrosylase S‐nitrosoglutathione reductase (GSNOR) has been suggested to sustain mitochondrial removal by autophagy (mitophagy), functionally linking S‐nitrosylation to cell senescence and aging. In this study, we provide evidence that GSNOR is induced at the translational level in response to hydrogen peroxide and mitochondrial ROS. The use of selective pharmacological inhibitors and siRNA demonstrates that GSNOR induction is an event downstream of the redox‐mediated activation of ATM, which in turn phosphorylates and activates CHK2 and p53 as intermediate players of this signaling cascade. The modulation of ATM/GSNOR axis, or the expression of a redox‐insensitive ATM mutant influences cell sensitivity to nitrosative and oxidative stress, impairs mitophagy and affects cell survival. Remarkably, this interplay modulates T‐cell activation, supporting the conclusion that GSNOR is a key molecular effector of the antioxidant function of ATM and providing new clues to comprehend the pleiotropic effects of ATM in the context of immune function.     

Pollen tube growth and self-incompatibility in three Ziziphus species (Rhamnaceae)

Self-incompatibility and synchronous protandrous dichogamy have previously been reported in Ziziphus species. In this work, we conducted a comparative analysis of fluorescence microscopy observations of pollen tube growth following controlled cross pollinations of emasculated flowers and self-pollinations of non-emasculated flowers in three Ziziphus species, Z. jujuba, Z. mauritiana and Z. spina-christi, with the aim to determine the type of the self-incompatibility system of each species. In addition, to test whether autonomous self-pollination, parthenocarpy or agamospermy occurs, flowers were emasculated (or not) and covered. Fruit set and seed viability were monitored. The presence of binucleate pollen grains and the cessation of pollen tube growth in the style suggest that the self-incompatibility system operating in the studied Ziziphus species is gametophytically controlled. Controlled self-pollination in Z. mauritiana resulted in fruits that dropped off before maturation, whereas in Z. spina-christi the flowers dropped off one or two days after pollination. Following controlled self-pollination, small fruits lacking viable seeds were obtained in Z. jujuba, probably due to the stimulus provided by pollination (stimulative parthenocarpy). In the cultivar Tamar of Z. jujuba, the relatively high percentage of seedless fruits obtained in emasculated bagged flowers without hand pollination suggests that this cultivar can set seedless fruits without any pollination stimulus.