Molecular cloning of the Ecotin gene in Escherichia coli

AbstractThe nucleotide sequence of a 876 bp region in E. coli chromosome that encodes Ecotin was determined. The proposed coding sequence for Ecotin is 486 nucleotides long, which would encode a protein consisting of 162 amino acids with a calculated molecular weight of 18 192 Da. The deduced primary sequence of Ecotin includes a 20-residue signal sequence, cleavage of which would give rise to a mature protein with a molecular weight of 16 099 Da. Ecotin does not contain any consensus reactive site sequences of known serine protease inhibitor families, suggesting that Ecotin is a novel inhibitor

Repurposing hyperpolarization-activated cyclic nucleotide-gated channels as a novel therapy for breast cancer.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are members of the voltage-gated cation channel family known to be expressed in the heart and central nervous system. Ivabradine, a small molecule HCN channel-blocker, is FDA-approved for clinical use as a heart rate-reducing agent. We found that HCN2 and HCN3 are overexpressed in breast cancer cells compared with normal breast epithelia, and the high expression of HCN2 and HCN3 is associated with poorer survival in breast cancer patients. Inhibition of HCN by Ivabradine or by RNAi, aborted breast cancer cell proliferation in vitro and suppressed tumour growth in patient-derived tumour xenograft models established from triple-negative breast cancer (TNBC) tissues, with no evident side-effects on the mice. Transcriptome-wide analysis showed enrichment for cholesterol metabolism and biosynthesis as well as lipid metabolism pathways associated with ER-stress following Ivabradine treatment. Mechanistic studies confirmed that HCN inhibition leads to ER-stress, in part due to disturbed Ca2+ homeostasis, which subsequently triggered the apoptosis cascade. More importantly, we investigated the synergistic effect of Ivabradine and paclitaxel on TNBC and confirmed that both drugs acted synergistically in vitro through ER-stress to amplify signals for caspase activation. Combination therapy could suppress tumour growth of xenografts at much lower doses for both drugs. In summary, our study identified a new molecular target with potential for being developed into targeted therapy, providing scientific grounds for initiating clinical trials for a new treatment regimen of combining HCN inhibition with chemotherapy

Intra-host competition and interactions between Soybean mosaic virus (SMV) strains in mixed-infected soybean

Abstract Over the past two decades, the dominant Soybean mosaic virus (SMV) strain in South Korea has changed from G5 to G7H. To examine the dominance of G7H, intra-host competition between G7H and G5 was evaluated in soybean plants infected with a mixture of SMV strains. The distribution patterns of the two SMV strains in soybean plants inoculated with G7H, G5 and G7H/G5 were investigated at designated time points by RT-PCR/RFLP analysis, which enables the specific differentiation of low concentrations of SMV strains and the detection of mixed infection at any given time. When leaves of 'Kwangankong' and 'Tawonkong' were infected with both strains, the upper leaves had only the G7H strain in simultaneous infections. In sequential inoculations, the leaves exhibited mosaic symptoms caused by G7H, and the G5 strain was not detected in plants pre-inoculated with the G7H strain before inoculation with the G5 strain. In the reverse treatment, both G5 and G7H were present at every vegetative stage. In addition, interactions between the virulence and dominance of G7H, G5, and G1, a less virulent strain, were investigated. Three landrace soybeans were co-inoculated with G7H/G5, G7H/G1, G5/G1, and G7H/G5/G1 sets. There was no significant difference between virulence and dominance. These results demonstrate the dominance of G7H in mixed infections and could explain the prevalence of G7H in South Korea

Clinical Features and Outcomes of Idiopathic Pulmonary Alveolar Proteinosis in Korean Population

Idiopathic pulmonary alveolar proteinosis (PAP) is a rare disorder in which lipoproteinaceous material accumulates within alveoli. There were few reports on Asian populations with idiopathic PAP. We retrospectively reviewed 38 patients with idiopathic PAP in Korea. We assessed clinical features, therapeutic efficacy and outcomes of whole lung lavage in patients with idiopathic PAP. The mean age at diagnosis was 52 yr. Eighty six percent of patients were symptomatic at diagnosis. Dyspnea and cough were the most common symptoms. Crackles were the most common physical examination finding. On pulmonary function test, a mild restrictive ventilatory defect was common, with a predicted mean forced vital capacity (FVC) of 77% and forced expiratory volume in one second (FEV1) of 84.6%. Diffusing capacity was disproportionately reduced at 67.7%. Arterial blood gas analysis revealed hypoxemia with a decreased PaO2 of 69.0 mmHg and an increased D(A-a)O2 of 34.2 mmHg. After whole lung lavage, PaO2, D(A-a)O2 and DLCO were significantly improved, but FVC and total lung capacity (TLC) were not different. This is the first multicenter study to analyze 38 Korean patients with idiopathic PAP. The clinical features and pulmonary parameters of Korean patients with idiopathic PAP are consistent with reports in other published studies. Whole lung lavage appears to be the most effective form of treatment

Expression analysis and functional characterization of the monosaccharide transporters, OsTMTs, involving vacuolar sugar transport in rice (Oryza sativa)

In Arabidopsis, the compartmentation of sugars into vacuoles is known to be facilitated by sugar transporters. However, vacuolar sugar transporters have not been studied in detail in other plant species. To characterize the rice (Oryza sativa) tonoplast monosaccharide transporters, OsTMT1 and OsTMT2, we analysed their subcellular localization using green fluorescent protein (GFP) and expression patterns using reverse-transcription polymerase chain reaction (RT-PCR), performed histochemical beta-glucuronidase (GUS) assay and in situ hybridization analysis, and assessed sugar transport ability using isolated vacuoles. Expression of OsTMT-GFP fusion protein in rice and Arabidopsis revealed that the OsTMTs localize at the tonoplast. Analyses of OsTMT promoter-GUS transgenic rice indicated that OsTMT1 and OsTMT2 are highly expressed in bundle sheath cells, and in vascular parenchyma and companion cells in leaves, respectively. Both genes were found to be preferentially expressed in the vascular tissues of roots, the palea/lemma of spikelets, and in the main vascular tissues and nucellar projections on the dorsal side of the seed coats. Glucose uptake studies using vacuoles isolated from transgenic mutant Arabidopsis (tmt1-2-3) expressing OsTMT1 demonstrated that OsTMTs are capable of transporting glucose into vacuoles. Based on expression analysis and functional characterization, our present findings suggest that the OsTMTs play a role in vacuolar glucose storage in rice

Phosphorylation of Nicastrin by SGK1 Leads to Its Degradation through Lysosomal and Proteasomal Pathways

The gamma-secretase complex is involved in the intramembranous proteolysis of a variety of substrates, including the amyloid precursor protein and the Notch receptor. Nicastrin (NCT) is an essential component of the gamma-secretase complex and functions as a receptor for gamma-secretase substrates. In this study, we determined that serum- and glucocorticoid-induced protein kinase 1 (SGK1) markedly reduced the protein stability of NCT. The SGK1 kinase activity was decisive for NCT degradation and endogenous SGK1 inhibited gamma-secretase activity. SGK1 downregulates NCT protein levels via proteasomal and lysosomal pathways. Furthermore, SGK1 directly bound to and phosphorylated NCT on Ser437, thereby promoting protein degradation. Collectively, our findings indicate that SGK1 is a gamma-secretase regulator presumably effective through phosphorylation and degradation of NCT