The Cold Box stem-loop proximal to the 5'-end of the Escherichia coli cspA gene stabilizes its mRNA at low temperature.

The 5'-end region of cspA mRNA contains a Cold Box sequence conserved among several cold-shock mRNAs. This region forms a stable stem-loop structure followed by an AU-rich sequence. Here we show that the Cold Box region is essential for the normal scale of cspA mRNA induction after cold shock because a deletion of the stem-loop significantly destabilizes the mRNA and reduces the cold shock-induced cspA mRNA amount by approximately 50%. The AU-rich track, however, slightly destabilizes the mRNA. The integrity of the stem is essential for the stabilizing function, whereas that of the loop sequence is less important. Overexpression of a mutant cspA mRNA devoid of both the AUG initiation codon and the coding sequence results in a severe growth inhibition at low temperature along with a derepression of the chromosomal cspA expression. Furthermore, the overexpressed RNA is stably associated with the 30 S and 70 S ribosomes. Our results demonstrate that the AUG initiation codon and the coding region containing the downstream box are not required for cspA mRNA to bind ribosomes and that the 5'-untranslated region by itself has a remarkable affinity to ribosomes at low temperature.     

Involvement of Kv1.3 and p38 MAPK signaling in HIV-1 glycoprotein 120-induced microglia neurotoxicity

Inflammatory responses mediated by activated microglia play a pivotal role in the pathogenesis of human immunodeficiency virus type 1 (HIV-1)-associated neurocognitive disorders. Studies on identification of specific targets to control microglia activation and resultant neurotoxic activity are imperative. Increasing evidence indicate that voltage-gated K⁺ (K_v) channels are involved in the regulation of microglia functionality. In this study, we investigated K_v1.3 channels in the regulation of neurotoxic activity mediated by HIV-1 glycoprotein 120 (gp120)-stimulated rat microglia. Our results showed treatment of microglia with gp120 increased the expression levels of K_v1.3 mRNA and protein. In parallel, whole-cell patch-clamp studies revealed that gp120 enhanced microglia K_v1.3 current, which was blocked by margatoxin, a K_v1.3 blocker. The association of gp120 enhancement of K_v1.3 current with microglia neurotoxicity was demonstrated by experimental results that blocking microglia K_v1.3 attenuated gp120-associated microglia production of neurotoxins and neurotoxicity. Knockdown of K_v1.3 gene by transfection of microglia with K_v1.3-siRNA abrogated gp120-associated microglia neurotoxic activity. Further investigation unraveled an involvement of p38 MAPK in gp120 enhancement of microglia K_v1.3 expression and resultant neurotoxic activity. These results suggest not only a role K_v1.3 may have in gp120-associated microglia neurotoxic activity, but also a potential target for the development of therapeutic strategies.     

Isoliquiritigenin prevents hyperglycemia-induced renal injuries by inhibiting inflammation and oxidative stress via SIRT1-dependent mechanism

Diabetic nephropathy (DN) as a global health concern is closely related to inflammation and oxidation. Isoliquiritigenin (ISL), a natural flavonoid compound, has been demonstrated to inhibit inflammation in macrophages. Herein, we investigated the effect of ISL in protecting against the injury in STZ-induced type 1 DN and in high glucose-induced NRK-52E cells. In this study, it was revealed that the administration of ISL not only ameliorated renal fibrosis and apoptosis, but also induced the deterioration of renal function in diabetic mice. Mediated by MAPKs and Nrf-2 signaling pathways, respectively, upstream inflammatory response and oxidative stress were neutralized by ISL in vitro and in vivo. Moreover, as further revealed by the results of molecular docking, sirtuin 1 (SIRT1) binds to ISL directly, and the involvement of SIRT1 in ISL-mediated renoprotective effects was confirmed by studies using in vitro models of SIRT1 overexpression and knockdown. In summary, by reducing inflammation and oxidative stress, ISL has a significant pharmacological effect on the deterioration of DN. The benefits of ISL are associated with the direct binding to SIRT1, the inhibition of MAPK activation, and the induction of Nrf-2 signaling, suggesting the potential of ISL for DN treatment.Subject terms: Pharmacology, Molecular biology     

Reliability of wavefront shaping based on coherent optical adaptive technique in deep tissue focusing

Wavefront shaping can compensate the wavefront distortions in deep tissue focusing, leading to an improved penetration depth. However, when using the backscattered signals as the feedback, unexpected compensation bias may be introduced, resulting in focusing position deviations or even no focus in the illumination focal plane. Here we investigated the reliability of wavefront shaping based on coherent optical adaptive technique in deep tissue focusing by measuring the position deviations between the foci in the illumination focal plane and the epi‐detection plane. The experimental results show that when the penetration depth reaches 150 μm in mouse brain tissue (with scattering coefficient ~22.42 mm^?1) using a 488 nm laser and an objective lens with 0.75 numerical aperture, the center of the real focus will deviate out of one radius range of the Airy disk while the optimized focus in the epi‐detection plane maintained basically at the center. With the penetration depth increases, the peak to background ratio of the focus in the illumination focal plane decreases faster than that in the epi‐detection plane. The results indicate that when the penetration depth reaches 150 μm, feedback based on backscattered signals will make wavefront shaping lose its reliability, which may provide a guidance for applications of non‐invasive precise optogenetics or deep tissue optical stimulation using wavefront shaping methods. A, Intensity distribution in the epi‐detection plane and the illumination focal plane before and after correction, corresponding to brain sections with 250 and 300 μm thickness, respectively. Scale bar is 2 μm. B, Averaged focusing deviations in the epi‐detection plane (optimized) and the illumination focal plane (monitored) after compensation. The unit of the ordinate is one Airy disk diameter. Black dashed line represents one Airy disk radius. Bars represent the SE of each measurement set.      

Clinical Utility of Insulin-Like Growth Factor 1 and 2; Determination by High Resolution Mass Spectrometry

Measurement of insulin-like growth factor-1 (IGF-I) has utility for the diagnosis and management of growth disorders, but inter-assay comparison of results has been complicated by a multitude of reference standards, antibodies, detection methods, and pre-analytical preparation strategies. We developed a quantitative LC-MS method for intact IGF-I, which has advantages in throughput and complexity when compared to mass spectrometric approaches that rely on stable isotope dilution analysis of tryptic peptides. Since the method makes use of full-scan data, the assay was easily extended to provide quantitative measurement of IGF-II using the same assay protocol. The validated LC-MS assay for IGF-I and IGF-II provides accurate results across the pediatric and adult reference range and is suitable for clinical use.     

Apical Scaffolding Protein NHERF2 Modulates the Localization of Alternatively Spliced Plasma Membrane Ca2+ Pump 2B Variants in Polarized Epithelial Cells

The membrane localization of the plasma membrane Ca²⁺-ATPase isoform 2 (PMCA2) in polarized cells is determined by alternative splicing; the PMCA2w/b splice variant shows apical localization, whereas the PMCA2z/b and PMCA2x/b variants are mostly basolateral. We previously reported that PMCA2b interacts with the PDZ protein Na⁺/H⁺ exchanger regulatory factor 2 (NHERF2), but the role of this interaction for the specific membrane localization of PMCA2 is not known. Here we show that co-expression of NHERF2 greatly enhanced the apical localization of GFP-tagged PMCA2w/b in polarized Madin-Darby canine kidney cells. GFP-PMCA2z/b was also redirected to the apical membrane by NHERF2, whereas GFP-PMCA2x/b remained exclusively basolateral. In the presence of NHERF2, GFP-PMCA2w/b co-localized with the actin-binding protein ezrin even after disruption of the actin cytoskeleton by cytochalasin D or latrunculin B. Surface biotinylation and fluorescence recovery after photobleaching experiments demonstrated that NHERF2-mediated anchorage to the actin cytoskeleton reduced internalization and lateral mobility of the pump. Our results show that the specific interaction with NHERF2 enhances the apical concentration of PMCA2w/b by anchoring the pump to the apical membrane cytoskeleton. The data also suggest that the x/b splice form of PMCA2 contains a dominant lateral targeting signal, whereas the targeting and localization of the z/b form are more flexible and not fully determined by intrinsic sequence features.