Cloning and expression of a zebrafish SCN1B ortholog and identification of a species-specific splice variant

Abstract Background Voltage-gated Na+ channel β1 (Scn1b) subunits are multi-functional proteins that play roles in current modulation, channel cell surface expression, cell adhesion, cell migration, and neurite outgrowth. We have shown previously that β1 modulates electrical excitability in vivo using a mouse model. Scn1b null mice exhibit spontaneous seizures and ataxia, slowed action potential conduction, decreased numbers of nodes of Ranvier in myelinated axons, alterations in nodal architecture, and differences in Na+ channel α subunit localization. The early death of these mice at postnatal day 19, however, make them a challenging model system to study. As a first step toward development of an alternative model to investigate the physiological roles of β1 subunits in vivo we cloned two β1-like subunit cDNAs from D. rerio. Results Two β1-like subunit mRNAs from zebrafish, scn1ba_tv1 and scn1ba_tv2, arise from alternative splicing of scn1ba. The deduced amino acid sequences of Scn1ba_tv1 and Scn1ba_tv2 are identical except for their C-terminal domains. The C-terminus of Scn1ba_tv1 contains a tyrosine residue similar to that found to be critical for ankyrin association and Na+ channel modulation in mammalian β1. In contrast, Scn1ba_tv2 contains a unique, species-specific C-terminal domain that does not contain a tyrosine. Immunohistochemical analysis shows that, while the expression patterns of Scn1ba_tv1 and Scn1ba_tv2 overlap in some areas of the brain, retina, spinal cord, and skeletal muscle, only Scn1ba_tv1 is expressed in optic nerve where its staining pattern suggests nodal expression. Both scn1ba splice forms modulate Na+ currents expressed by zebrafish scn8aa, resulting in shifts in channel gating mode, increased current amplitude, negative shifts in the voltage dependence of current activation and inactivation, and increases in the rate of recovery from inactivation, similar to the function of mammalian β1 subunits. In contrast to mammalian β1, however, neither zebrafish subunit produces a complete shift to the fast gating mode and neither subunit produces complete channel inactivation or recovery from inactivation. Conclusion These data add to our understanding of structure-function relationships in Na+ channel β1 subunits and establish zebrafish as an ideal system in which to determine the contribution of scn1ba to electrical excitability in vivo.http://deepblue.lib.umich.edu/bitstream/2027.42/112585/1/12864_2007_Article_939.pd

The Intratubular and Intracrine Renin-Angiotensin System in the Proximal Tubules of the Kidney and Its Roles in Angiotensin II-Induced Hypertension

The kidney plays a fundamental role in the physiological regulation of basal blood pressure and the development of hypertension. Although the mechanisms underlying hypertension are very complex, the renin-angiotensin system (RAS) in the kidney, especially intratubular and intracellular RAS, undoubtedly plays a critical role in maintaining basal blood pressure homeostasis and the development of angiotensin II (ANG II)-dependent hypertension. In the proximal tubules, ANG II activates two G protein-coupled receptors, AT1 and AT2, to exert powerful effects to regulate proximal tubular sodium and fluid reabsorption by activating cell surface as well as intracellular AT1 receptors. Increased production and actions of ANG II in the proximal tubules may cause salt and fluid retention, impair the pressure-natriuresis response, and consequently increase blood pressure in hypertension. The objectives of this chapter are to critically review and discuss our current understanding of intratubular and intracellular RAS in the kidney, and their contributions to basal blood pressure homeostasis and the development of ANG II-dependent hypertension. The new knowledge will likely help uncover novel renal mechanisms of hypertension, and develop kidney- or proximal tubule-specific strategies or drugs to prevent and treat hypertension in humans

Scn1b deletion in adult mice results in seizures and SUDEP

Pathogenic lossâ ofâ function variants in SCN1B are linked to Dravet syndrome (DS). Previous work suggested that neuronal pathfinding defects underlie epileptogenesis and SUDEP in the Scn1b null mouse model of DS. We tested this hypothesis by inducing Scn1b deletion in adult mice that had developed normally. Epilepsy and SUDEP, which occur by postnatal day 21 in Scn1b null animals, were observed within 20 days of induced Scn1b deletion in adult mice, suggesting that epileptogenesis in SCN1Bâ DS does not result from defective brain development. Thus, the developmental brain defects observed previously in Scn1b null mice may model other coâ morbidities of DS.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149575/1/acn3785.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149575/2/acn3785_am.pd

Spectrochemical determination of unique bacterial responses following long-term low-level exposure to antimicrobials

Agents arising from engineering or pharmaceutical industries may have significant environmental impacts. Particularly, antimicrobials not only act as efficient eliminators of certain microbes but also facilitate the propagation of organisms with antimicrobial resistance, giving rise to critical health issues, e.g., the bloom of multidrug-resistant bacteria. Although many investigations have examined microbial responses to antimicrobials and characterized relevant mechanisms, they have focused mainly on high-level and short-term exposures, instead of simulating real-world scenarios in which the antimicrobial exposure is at a low-level for long periods. Herein, we developed a spectrochemical tool, attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy, as a high-throughput and nondestructive approach to interrogate the long-term effects of low-level antimicrobial exposure in bacterial cells. Post-exposure to nanoparticulate silver (AgNP), tetracycline or their mixtures for 12 days, Gram-positive (Mycobacterium vanbaalenii PYR-1) and Gram-negative (Pseudomonas fluorescens) bacteria exhibited distinct IR spectral alterations. Multivariate analysis coupled with multivariate regression trees (MRT) indicates nutrient depletion and exposure time as the primary factors in bacterial behaviour, followed by exposure category and bacterial type. Nutrient depletion and starvation during long-term exposure drives bacterial cells into a dormant state or to exhibit additional cellular components (e.g., fatty acids) in response to antimicrobials, consequently causing a broader range of spectral alterations compared to short-term exposure. This work is the first report highlighting the more important roles of exposure duration and nutrient depletion, instead of treatment regimens of antimicrobials, in microbial responses to low-level and prolonged environmental exposures

Spectrochemical analyses of growth phase-related bacterial responses to low (environmentally-relevant) concentrations of tetracycline and nanoparticulate silver

Exposure to environmental insults generally occurs at low levels, making it challenging to measure bacterial responses to such interactions. Additionally, microbial behaviour and phenotype varies in differing bacterial types or growth phases, likely giving rise to growth- or species-specific responses to environmental stimuli. The present study applied a spectrochemical tool, infrared (IR) spectral interrogation coupled with multivariate analysis, to investigate the growth- and species-specific responses of two bacterial strains, Gram-negative Pseudomonas fluorescens and Gram-positive Mycobacterium vanbaalenii, to low concentrations of tetracycline, nanoparticulate silver (AgNP) or mixtures thereof. Results indicate the tendency for tetracycline-induced biospectral alterations to occur in outer-cellular components, e.g., phospholipids or proteins, while AgNPs-induced changes are mainly associated with proteins (∼964 cm−1, ∼1485 cm−1, ∼1550 cm−1, ∼1650 cm−1). The primary altered targets are correlated with bacterial membranes or outer-cellular components. Furthermore, significant lipid changes at 1705–1750 cm−1 were only present in P. fluorescens cells compared to M. vanbaalenii, owing to differences in cell wall structure between Gram-positive and -negative bacteria. This study also found distinct biospectral alterations in non-log phase compared to log phase, confirming bacterial growth-dependent responses to environmental exposures. It implies that previous studies on log phase only may underestimate the impacts from exposures of interest in situ, where bacteria stay in different growth stages. Our work proves the feasibility of biospectroscopy in determining bacterial responses to low-level environmental exposures in a fast and efficient manner, revealing sufficient biochemical information continuously through growth phases. As a nondestructive approach, biospectroscopy may provide deeper insights into the actual and in situ interactions between microbes and environmental stimuli, regardless of the exposure level, growth phase, or bacterial types

The Transcription Factor GLI1 Mediates TGFb1 Driven EMT in Hepatocellular Carcinoma via a SNAI1-Dependent Mechanism

The role of the epithelial-to-mesenchymal transition (EMT) during hepatocellular carcinoma (HCC) progression is well established, however the regulatory mechanisms modulating this phenomenon remain unclear. Here, we demonstrate that transcription factor glioma-associated oncogene 1 (GLI1) modulates EMT through direct up-regulation of SNAI1 and serves as a downstream effector of the transforming growth factor-b1 (TGFb1) pathway, a well-known regulator of EMT in cancer cells. Overexpression of GLI1 increased proliferation, viability, migration, invasion, and colony formation by HCC cells. Conversely, GLI1 knockdown led to a decrease in all the above-mentioned cancer-associated phenotypes in HCC cells. Further analysis of GLI1 regulated cellular functions showed that this transcription factor is able to induce EMT and identified SNAI1 as a transcriptional target of GLI1 mediating this cellular effect in HCC cells. Moreover, we demonstrated that an intact GLI1-SNAI1 axis is required by TGFb1 to induce EMT in these cells. Together, these findings define a novel cellular mechanism regulated by GLI1, which controls the growth and EMT phenotype in HCC.National Institutes of Health Grants CA100882 and CA128633 (to LRR) and CA165076; the Mayo Clinic Center for Cell Signaling in Gastroenterology (NIDDK P30DK084567) (to MEFZ); the Mayo Clinic Cancer Center (CA15083), the Mayo Clinic Center for Translational Science Activities (NIH/NCRR CTSA Grant Number KL2 RR024151), and an American Gastroenterological Association Foundation for Digestive Health and Nutrition Bridging Grant (to LRR)

4-Nonylphenol effects on rat testis and sertoli cells determined by spectrochemical techniques coupled with chemometric analysis

Herein, vibrational spectroscopy has been applied for qualitative identification of biomolecular alterations that occur in cells and tissues following chemical treatment. Towards this end, we combined attenuated total reflection Fourier-transform infrared (ATR-FTIR) and Raman spectroscopy to assess testicular toxicology after 4-nonylphenol (NP) exposure, an estrogenic endocrine disruptor affecting testicular function in rats and other species. Rats aged 21, 35 or 50 days received NP at intra-peritoneal doses of 0, 25, 50 or 100 mg/kg for 20 consecutive days. Primary Sertoli cells (SCs) were treated with NP at various concentrations (0, 2.5, 5, 10 or 20 μM) for 12 h. Post-exposure, testicular cells, interstitial tissue and SCs were interrogated respectively using spectrochemical techniques coupled with multivariate analysis. Distinct biomolecular segregation between the NP-exposed samples vs. control were observed based on infrared (IR) spectral regions of 3200–2800 cm−1 and 1800-900 cm−1, and the Raman spectral region of 1800–900 cm−1. For in vivo experiments, the main wavenumbers responsible for segregation varied significantly among the three age classes. The main IR and Raman band differences between NP-exposed and control groups were observed for Amide (proteins), lipids and DNA/RNA. An interesting finding was that the peptide aggregation level, Amide Ӏ-to-Amide II ratio, and phosphate-to-carbohydrate ratio were considerably reduced in ex vivo NP-exposed testicular cells or SCs in vitro. This study demonstrates that ATR-FTIR and Raman spectroscopy techniques can be applied towards analysing NP-induced testicular biomolecular alterations

Exploring Off-Targets and Off-Systems for Adverse Drug Reactions via Chemical-Protein Interactome — Clozapine-Induced Agranulocytosis as a Case Study

In the era of personalized medical practice, understanding the genetic basis of patient-specific adverse drug reaction (ADR) is a major challenge. Clozapine provides effective treatments for schizophrenia but its usage is limited because of life-threatening agranulocytosis. A recent high impact study showed the necessity of moving clozapine to a first line drug, thus identifying the biomarkers for drug-induced agranulocytosis has become important. Here we report a methodology termed as antithesis chemical-protein interactome (CPI), which utilizes the docking method to mimic the differences in the drug-protein interactions across a panel of human proteins. Using this method, we identified HSPA1A, a known susceptibility gene for CIA, to be the off-target of clozapine. Furthermore, the mRNA expression of HSPA1A-related genes (off-target associated systems) was also found to be differentially expressed in clozapine treated leukemia cell line. Apart from identifying the CIA causal genes we identified several novel candidate genes which could be responsible for agranulocytosis. Proteins related to reactive oxygen clearance system, such as oxidoreductases and glutathione metabolite enzymes, were significantly enriched in the antithesis CPI. This methodology conducted a multi-dimensional analysis of drugs' perturbation to the biological system, investigating both the off-targets and the associated off-systems to explore the molecular basis of an adverse event or the new uses for old drugs

Direct Interaction between Two Viral Proteins, the Nonstructural Protein 2CATPase and the Capsid Protein VP3, Is Required for Enterovirus Morphogenesis

In spite of decades-long studies, the mechanism of morphogenesis of plus-stranded RNA viruses belonging to the genus Enterovirus of Picornaviridae, including poliovirus (PV), is not understood. Numerous attempts to identify an RNA encapsidation signal have failed. Genetic studies, however, have implicated a role of the non-structural protein 2CATPase in the formation of poliovirus particles. Here we report a novel mechanism in which protein-protein interaction is sufficient to explain the specificity in PV encapsidation. Making use of a novel “reporter virus”, we show that a quasi-infectious chimera consisting of the capsid precursor of C-cluster coxsackie virus 20 (C-CAV20) and the nonstructural proteins of the closely related PV translated and replicated its genome with wild type kinetics, whereas encapsidation was blocked. On blind passages, encapsidation of the chimera was rescued by a single mutation either in capsid protein VP3 of CAV20 or in 2CATPase of PV. Whereas each of the single-mutation variants expressed severe proliferation phenotypes, engineering both mutations into the chimera yielded a virus encapsidating with wild type kinetics. Biochemical analyses provided strong evidence for a direct interaction between 2CATPase and VP3 of PV and CAV20. Chimeras of other C-CAVs (CAV20/CAV21 or CAV18/CAV20) were blocked in encapsidation (no virus after blind passages) but could be rescued if the capsid and 2CATPase coding regions originated from the same virus. Our novel mechanism explains the specificity of encapsidation without apparent involvement of an RNA signal by considering that (i) genome replication is known to be stringently linked to translation, (ii) morphogenesis is known to be stringently linked to genome replication, (iii) newly synthesized 2CATPase is an essential component of the replication complex, and (iv) 2CATPase has specific affinity to capsid protein(s). These conditions lead to morphogenesis at the site where newly synthesized genomes emerge from the replication complex