Loss of miR-203 regulates cell shape and matrix adhesion through ROBO1/Rac/FAK in response to stiffness

Breast tumor progression is accompanied by changes in the surrounding extracellular matrix (ECM) that increase stiffness of the microenvironment. Mammary epithelial cells engage regulatory pathways that permit dynamic responses to mechanical cues from the ECM. Here, we identify a SLIT2/ROBO1 signaling circuit as a key regulatory mechanism by which cells sense and respond to ECM stiffness to preserve tensional homeostasis. We observed that Robo1 ablation in the developing mammary gland compromised actin stress fiber assembly and inhibited cell contractility to perturb tissue morphogenesis, whereas SLIT2 treatment stimulated Rac and increased focal adhesion kinase activity to enhance cell tension by maintaining cell shape and matrix adhesion. Further investigation revealed that a stiff ECM increased Robo1 levels by down-regulating miR-203. Consistently, patients whose tumor expressed a low miR-203/high Robo1 expression pattern exhibited a better overall survival prognosis. These studies show that cells subjected to stiffened environments up-regulate Robo1 as a protective mechanism that maintains cell shape and facilitates ECM adherence.     

NMR Model of PrgI–SipD Interaction and Its Implications in the Needle-Tip Assembly of the Salmonella Type III Secretion System

Salmonella and other pathogenic bacteria use the type III secretion system (T3SS) to inject virulence proteins into human cells to initiate infections. The structural component of the T3SS contains a needle and a needle tip. The needle is assembled from PrgI needle protomers and the needle tip is capped with several copies of the SipD tip protein. How a tip protein docks on the needle is unclear. A crystal structure of a PrgI–SipD fusion protein docked on the PrgI needle results in steric clash of SipD at the needle tip when modeled on the recent atomic structure of the needle. Thus, there is currently no good model of how SipD is docked on the PrgI needle tip. Previously, we showed by NMR paramagnetic relaxation enhancement (PRE) methods that a specific region in the SipD coiled coil is the binding site for PrgI. Others have hypothesized that a domain of the tip protein—the N-terminal α-helical hairpin—has to swing away during the assembly of the needle apparatus. Here, we show by PRE methods that a truncated form of SipD lacking the α-helical hairpin domain binds more tightly to PrgI. Further, PRE-based structure calculations revealed multiple PrgI binding sites on the SipD coiled coil. Our PRE results together with the recent NMR-derived atomic structure of the Salmonella needle suggest a possible model of how SipD might dock at the PrgI needle tip. SipD and PrgI are conserved in other bacterial T3SSs; thus, our results have wider implication in understanding other needle-tip complexes.     

Purification and characterization of a low molecular weight multifunctional cytotoxic phospholipase A2 from Russell's viper venom

A basic toxin from Russell's viper venom of 7.2 kDa (RVV-7) has been purified to homogeneity after partial unfolding by 4 M urea followed by filtration through Centricon-30 membrane. Its N-terminal sequence showed strong homology with snake venom cytotoxins. Cytotoxic activity of RVV-7 has been demonstrated with B16F10 melanoma cells. PLA2 activity was observed in cytotoxin (CX3) from Naja kauthia bearing sequence homology with RVV-7. Phospholipase A2 and trypsin inhibitory activities were also observed with RVV-7. Chemical modification and inhibition studies suggested independent functional sites for these activities. A qualitative assessment of tumor growth inhibition by RVV-7 has been made.     

Drop-in, drop-out allele-specific PCR

Allelotyping large numbers of samples by allele-specific polymerase chain reaction (PCR) can be problematic if the DNA samples to be tested are of highly variable concentration. On the one hand, analysis of dilute DNA samples often requires nested PCR to produce a product of sufficient yield to be detectable on ethidium bromide-stained agarose gels. Such two-step assays require additional reagents, are labor-intensive, and have a higher risk of contaiminations. On the other hand, the specificity of allele-specific PCR assays can be lost at high input DNA concentrations. Large population-based genetic studies using DNA from varied sources would benefit from one-tube assays that could detect mutations in samples over a wide range of concentration. We describe a one-tube nested allele-specific PCR-based assay, in which the input DNA concentration has little effect on the assay’s yield or specificity. An assay using this method is highly sensitive and specific, and was used to type several thousand DNA samples, obtained from various sources, for a G to A transition at human transthyretin codon 122. Similar assays could be readily adapted to any high-throughput allelotype assay where input DNA is of highly variable concentration.     

Recognition and long-range interactions of a minimal nanos RNA localization signal element

Localization of nanos (nos) mRNA to the germ plasm at the posterior pole of the Drosophila embryo is essential to activate nos translation and thereby generate abdominal segments. nos RNA localization is mediated by a large cis-acting localization signal composed of multiple, partially redundant elements within the nos 3' untranslated region. We identify a protein of approximately 75 kDa (p75) that interacts specifically with the nos +2' localization signal element. We show that the function of this element can be delimited to a 41 nucleotide domain that is conserved between D. melanogaster and D. virilis, and confers near wild-type localization when present in three copies. Two small mutations within this domain eliminate both +2' element localization function and p75 binding, consistent with a role for p75 in nos RNA localization. In the intact localization signal, the +2' element collaborates with adjacent localization elements. We show that different +2' element mutations not only abolish collaboration between the +2' and adjacent +1 element but also produce long-range deleterious effects on localization signal function. Our results suggest that higher order structural interactions within the localization signal, which requires factors such as p75, are necessary for association of nos mRNA with the germ plasm.     

Low-level X-ray exposures on rat skin

Integumentary structures naturally are more exposed to solar radiation than other body tissues, so there is a big question regarding the biological threshold of skin in respect to low-dose ionizing radiation. In this study, adult male albino rats were exposed chronically to low-dose (0.015 cGy/sec) X-rays for 9 and 18 mo, with total X-ray doses of 2.025 and 4.050 cGy, respectively. In both the dose groups, hyperkeratinization was noted in skin by transmission electron microscopic (TEM) study. Atomic absorption spectrometric (AAS) study revealed decreased zinc concentration (p<0.01), increased iron concentration (p<0.001), and status quo cadmium concentration. Moreover, the ratio between zinc and iron became highly depleted in both the irradiated groups. Hence it may be stated that chronic low-level X-rays induce redistribution of biometals in the skin. Nevertheless, specific concentrations of biometals indicate the risk-prone status of irradiated skin.