T16Ainh - A01

Thermostable chitinase from Cohnella sp. A01: isolation and product optimization.[Pubmed:27528085]

Braz J Microbiol. 2016 Oct - Dec;47(4):931-940.

Twelve bacterial strains isolated from shrimp farming ponds were screened for their growth activity on chitin as the sole carbon source. The highly chitinolytic bacterial strain was detected by qualitative cup plate assay and tentatively identified to be Cohnella sp. A01 based on 16S rDNA sequencing and by matching the key morphological, physiological, and biochemical characteristics. The cultivation of Cohnella sp. A01 in the suitable liquid medium resulted in the production of high levels of enzyme. The colloidal chitin, peptone, and K2HPO4 represented the best carbon, nitrogen, and phosphorus sources, respectively. Enzyme production by Cohnella sp. A01 was optimized by the Taguchi method. Our results demonstrated that inoculation amount and temperature of incubation were the most significant factors influencing chitinase production. From the tested values, the best pH/temperature was obtained at pH 5 and 70 degrees C, with Km and Vmax values of chitinase to be 5.6mg/mL and 0.87mumol/min, respectively. Ag(+), Co(2+), iodoacetamide, and iodoacetic acid inhibited the enzyme activity, whereas Mn(2+), Cu(2+), Tweens (20 and 80), Triton X-100, and EDTA increased the same. In addition, the study of the morphological alteration of chitin treated by enzyme by SEM revealed cracks and pores on the chitin surface, indicating a potential application of this enzyme in several industries.

Anoctamin-1 Cl(-) channels in nociception: activation by an N-aroylaminothiazole and capsaicin and inhibition by T16A[inh]-A01.[Pubmed:26364309]

Mol Pain. 2015 Sep 12;11:55.

BACKGROUND: Anoctamin 1 (ANO1 or TMEM16A) Ca(2+)-gated Cl(-) channels of nociceptor neurons are emerging as important molecular components of peripheral pain transduction. At physiological intracellular Cl(-) concentrations ([Cl(-)]i) sensory neuronal Cl(-) channels are excitatory. The ability of sensory neuronal ANO1 to trigger action potentials and subsequent nocifensive (pain) responses were examined by direct activation with an N-aroylaminothiazole. ANO1 channels are also activated by intracellular Ca(2+) ([Ca(2+)]i) from sensory neuronal TRPV1 (transient-receptor-potential vallinoid 1) ion channels and other noxicant receptors. Thus, sensory neuronal ANO1 can facilitate TRPV1 triggering of action potentials, resulting in enhanced nociception. This was investigated by reducing ANO1 facilitation of TRPV1 effects with: (1) T16A[inh]-A01 ANO1-inhibitor reagent at physiological [Cl(-)]i and (2) by lowering sensory neuronal [Cl(-)]i to switch ANO1 to be inhibitory. RESULTS: ANO1 effects on action potential firing of mouse dorsal root ganglia (DRG) neurons in vitro and mouse nocifensive behaviors in vivo were examined with an N-aroylaminothiazole ANO1-activator (E-act), a TRPV1-activator (capsaicin) and an ANO1-inhibitor (T16A[inh]-A01). At physiological [Cl(-)]i (40 mM), E-act (10 microM) increased current sizes (in voltage-clamp) and action potential firing (in current-clamp) recorded in DRG neurons using whole-cell electrophysiology. To not disrupt TRPV1 carried-Ca(2+) activation of ANO1 in DRG neurons, ANO1 modulation of capsaicin-induced action potentials was measured by perforated-patch (Amphotericin-B) current-clamp technique. Subsequently, at physiological [Cl(-)]i, capsaicin (15 microM)-induced action potential firing was diminished by co-application with T16A[inh]-A01 (20 microM). Under conditions of low [Cl(-)]i (10 mM), ANO1 actions were reversed. Specifically, E-act did not trigger action potentials; however, capsaicin-induced action potential firing was inhibited by co-application of E-act, but was unaffected by co-application of T16A[inh]-A01. Nocifensive responses of mice hind paws were dramatically induced by subcutaneous injections of E-act (5 mM) or capsaicin (50 microM). The nocifensive responses were attenuated by co-injection with T16A[inh]-A01 (1.3 mM). CONCLUSIONS: An ANO1-activator (E-act) induced [Cl(-)]i-dependent sensory neuronal action potentials and mouse nocifensive behaviors; thus, direct ANO1 activation can induce pain perception. ANO1-inhibition attenuated capsaicin-triggering of action potentials and capsaicin-induced nocifensive behaviors. These results indicate ANO1 channels are involved with TRPV1 actions in sensory neurons and inhibition of ANO1 could be a novel means of inducing analgesia.

Complete genome sequence of Streptococcus thermophilus MN-BM-A01, a strain with high exopolysaccharides production.[Pubmed:26956372]

J Biotechnol. 2016 Apr 20;224:45-6.

Streptococcus thermophilus MN-BM-A01 (ST MN-BM-A01) (CGMCC No. 11383) was a strain isolated from Yogurt Block in Gansu, China. The yogurt fermented with this strain has good flavor, acidity, and viscosity. Moreover, ST MN-BM-A01 could produce a high level of EPS which can confer the yogurt with improved rheological properties. We reported the complete genome sequence of ST MN-BM-A01 that contains 1,876,516bp encoding 1704 coding sequences (CDSs), 67 tRNA genes and 6 rRNA operons. The genomic sequence indicated that this strain included a 35.3-kb gene cluster involved in EPS biosynthesis.

TMEM16A induces MAPK and contributes directly to tumorigenesis and cancer progression.[Pubmed:22564524]

Cancer Res. 2012 Jul 1;72(13):3270-81.

Frequent gene amplification of the receptor-activated calcium-dependent chloride channel TMEM16A (TAOS2 or ANO1) has been reported in several malignancies. However, its involvement in human tumorigenesis has not been previously studied. Here, we show a functional role for TMEM16A in tumor growth. We found TMEM16A overexpression in 80% of head and neck squamous cell carcinoma (SCCHN), which correlated with decreased overall survival in patients with SCCHN. TMEM16A overexpression significantly promoted anchorage-independent growth in vitro, and loss of TMEM16A resulted in inhibition of tumor growth both in vitro and in vivo. Mechanistically, TMEM16A-induced cancer cell proliferation and tumor growth were accompanied by an increase in extracellular signal-regulated kinase (ERK)1/2 activation and cyclin D1 induction. Pharmacologic inhibition of MEK/ERK and genetic inactivation of ERK1/2 (using siRNA and dominant-negative constructs) abrogated the growth effect of TMEM16A, indicating a role for mitogen-activated protein kinase (MAPK) activation in TMEM16A-mediated proliferation. In addition, a developmental small-molecule inhibitor of TMEM16A, T16A-inh01 (A01), abrogated tumor cell proliferation in vitro. Together, our findings provide a mechanistic analysis of the tumorigenic properties of TMEM16A, which represents a potentially novel therapeutic target. The development of small-molecule inhibitors against TMEM16A may be clinically relevant for treatment of human cancers, including SCCHN.

Epidermal growth factor chronically upregulates Ca(2+)-dependent Cl(-) conductance and TMEM16A expression in intestinal epithelial cells.[Pubmed:22351639]

J Physiol. 2012 Apr 15;590(8):1907-20.

Dysregulated epithelial fluid and electrolyte transport is a common feature of many intestinal disorders. However, molecular mechanisms that regulate epithelial transport processes are still poorly understood, thereby limiting development of new therapeutics. Previously, we showed that epidermal growth factor (EGF) chronically enhances intestinal epithelial secretory function. Here, we investigated a potential role for altered expression or activity of apical Cl(-) channels in mediating the effects of EGF. Cl(-) secretion across monolayers of T(84) colonic epithelia was measured as changes in short-circuit current. Protein expression/phosphorylation was measured by RT-PCR and Western blotting. Under conditions that specifically isolate apical Ca(2+)-activated Cl(-) channel (CaCC) currents, EGF pretreatment (100 ng ml(-1) for 15 min) potentiated carbachol (CCh)-induced responses to 173 +/- 25% of those in control cells, when measured 24 h later (n = 26; P < 0.01). EGF-induced increases in CaCC currents were abolished by the transmembrane protein 16A (TMEM16A) inhibitor, T16A(inh)-A01 (10 mum). Furthermore, TMEM16A mRNA and protein expression was increased by EGF to 256 +/- 38% (n = 7; P < 0.01) and 297 +/- 46% (n = 9, P < 0.001) of control levels, respectively. In contrast, EGF did not alter CFTR expression or activity. EGF-induced increases in Cl(-) secretion, CaCC currents and TMEM16A expression were attenuated by a PKCdelta inhibitor, rottlerin (20 mum), and a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY290042 (25 mum). Finally, LY290042 inhibited EGF-induced phosphorylation of PKCdelta. We conclude that EGF chronically upregulates Ca(2+)-dependent Cl(-) conductances and TMEM16A expression in intestinal epithelia by a mechanism involving sequential activation of PI3K and PKCdelta. Therapeutic targeting of EGF receptor-dependent signalling pathways may provide new approaches for treatment of epithelial transport disorders.

TMEM16A inhibitors reveal TMEM16A as a minor component of calcium-activated chloride channel conductance in airway and intestinal epithelial cells.[Pubmed:21084298]

J Biol Chem. 2011 Jan 21;286(3):2365-74.

TMEM16A (ANO1) functions as a calcium-activated chloride channel (CaCC). We developed pharmacological tools to investigate the contribution of TMEM16A to CaCC conductance in human airway and intestinal epithelial cells. A screen of approximately 110,000 compounds revealed four novel chemical classes of small molecule TMEM16A inhibitors that fully blocked TMEM16A chloride current with an IC(50) < 10 muM, without interfering with calcium signaling. Following structure-activity analysis, the most potent inhibitor, an aminophenylthiazole (T16A(inh)-A01), had an IC(50) of approximately 1 muM. Two distinct types of inhibitors were identified. Some compounds, such as tannic acid and the arylaminothiophene CaCC(inh)-A01, fully inhibited CaCC current in human bronchial and intestinal cells. Other compounds, including T16A(inh)-A01 and digallic acid, inhibited total CaCC current in these cells poorly, but blocked mainly an initial, agonist-stimulated transient chloride current. TMEM16A RNAi knockdown also inhibited mainly the transient chloride current. In contrast to the airway and intestinal cells, all TMEM16A inhibitors fully blocked CaCC current in salivary gland cells. We conclude that TMEM16A carries nearly all CaCC current in salivary gland epithelium, but is a minor contributor to total CaCC current in airway and intestinal epithelia. The small molecule inhibitors identified here permit pharmacological dissection of TMEM16A/CaCC function and are potential development candidates for drug therapy of hypertension, pain, diarrhea, and excessive mucus production.

T16Ainh-A01, an aminophenylthiazole, is a potent transmembrane protein 16A (TMEM16A) inhibitor, inhibiting TMEM16A-mediated chloride currents with an IC50 value of ~1 µM. TMEM16A (ANO1) functions as a calcium-activated chloride channel (CaCC).

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