Molecular genetic manipulation of Pichia pastoris SEC4 governs cell growth and glucoamylase secretion

We have previously engineered a recombinant Pichia pastoris GS115 transformant, MSPGA-7, harboring seven copies of glucoamylase (GA) fused with modified signal peptide. High yield secretion of GA was achieved as an extra copy of SEC4 was integrated to the transformant. To elucidate the physiological role of SEC4, a dominant-negative mutant of SEC4, SEC4(S28N), was overexpressed under the control of alchohol oxidase 1 (AOX1) promoter in P. pastoris strain MSPGA-7 as well as a set of host cells harboring multi-copy of wild type SEC4. We found that SEC4(S28N) mutation in the key guanine nucleotide binding domain reduced guanine nucleotide binding affinity, hence it blocked the transport of vesicles required for targeting and fusion to the plasma membrane. The inhibitory levels of cell growth and GA secretion were correlated with the dosage of SEC4(S28N) gene. In addition, overexpression of SEC4 driven by AOX1 promoter in MSPGA-7 improved the secretory production of GA, but demonstrated the delay of cell growth by increased gene dosage of SEC4. Interestingly, a limited level of Sec4p did not disturb the cell growth. It was because expression of only one copy of SEC4 resulted in delay of cell growth at an early stage while still maintaining high level Sec4p at long-term incubation. Accordingly, as glyceraldehyde-3-phosphate dehydrogenase promoter was used to substitute AOX1 promoter to drive the SEC4 expression, enhanced GA secretion but not inhibition of cell growth was achieved. Taken together, our results demonstrate that SEC4 is essential for P. pastoris in regulating cell growth and heterologous protein secretion in a dosage-dependent manner.     

Enhanced nuclear factor-kappa B-associated Wnt-1 expression in hepatitis B- and C-related hepatocarcinogenesis: identification by functional proteomics

Summary Chronic infections with hepatitis B and C viruses (HBV and HCV) are etiologically linked to hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Both viruses may induce activation of nuclear factor-kappa B (NF-κB) in hepatocytes that plays a crucial role in the regulation of cell growth and apoptosis. Functional proteomics analysis of proteins associated with NF-κB signaling complexes in both viruses-related HCC tumor and non-tumor tissues may disclose possible common mechanisms in hepatocarcinogenesis. By functional proteomics, we analyzed proteins associated with NF-κB-signaling complexes in four-paired human HCC tumor and non-tumor tissues from HBV- and HCV-infected patients, respectively, and in one-paired tissue with dual viral infection. The quantity of NF-κB-associated proteins was semi-quantitatively measured by protein spot intensity on the gels of two-dimensional polyacrylamide gel electrophoresis. The results showed that overexpression of NF-κB-associated Wnt-1 protein in tumor part was detected in the␣majority of HBV- and HCV-infected HCC samples. These data suggest that enhanced expression of NF-κB-associated Wnt-1 protein might be a mechanism of hepatocarcinogenesis common to HBV- and HCV-infected patients. NF-κB signaling pathway and Wnt-1 protein could be potential targets for designing highly effective therapeutic agents in treating HCC and for chemoprevention of hepatocarcinogenesis.     

TIMP2 deficient mice develop accelerated osteoarthritis via promotion of angiogenesis upon destabilization of the medial meniscus

Vascular invasion into the normally avascular articular surface is a hallmark of advanced osteoarthritis (OA). In this study, we demonstrated that the expression of tissue inhibitor of metalloproteinases-2 (TIMP2), an anti-angiogenic factor, was present at high levels in normal articular chondrocytes, and was drastically decreased shortly after destabilization of the medial meniscus (DMM). We also investigated the anti-angiogenic properties of TIMP2 via knockout. We hypothesized that the loss of TIMP2 could accelerate osteoarthritis development via promotion of angiogenesis. Loss of TIMP2 led to increased periarticular vascular formation 1 month post DMM, compared to wild-type mice, and did so without altering the expression pattern of matrix metalloproteinases and vascular endothelial growth factors. The increased vascularization eventually resulted in a severe degeneration of the articular surface by 4 months post DMM. Our findings suggest that reduction of TIMP2 levels and increased angiogenesis are possible primary events in OA progression. Inhibiting or delaying angiogenesis by TIMP2 expression or other anti-angiogenic therapies could improve OA prevention and treatment.     

P wave dispersion and maximum p wave duration are independently associated with rapid renal function decline

The P wave parameters measured by 12-lead electrocardiogram (ECG) are commonly used as noninvasive tools to assess for left atrial enlargement. There are limited studies to evaluate whether P wave parameters are independently associated with decline in renal function. Accordingly, the aim of this study is to assess whether P wave parameters are independently associated with progression to renal end point of ≥25% decline in estimated glomerular filtration rate (eGFR). This longitudinal study included 166 patients. The renal end point was defined as ≥25% decline in eGFR. We measured two ECG P wave parameters corrected by heart rate, i.e. corrected P wave dispersion (PWdisperC) and corrected P wave maximum duration (PWdurMaxC). Heart function and structure were measured from echocardiography. Clinical data, P wave parameters, and echocardiographic measurements were compared and analyzed. Forty-three patients (25.9%) reached renal end point. Kaplan-Meier curves for renal end point-free survival showed PWdisperC > median (63.0 ms) (log-rank P = 0.004) and PWdurMaxC > median (117.9 ms) (log-rank P<0.001) were associated with progression to renal end point. Multivariate forward Cox-regression analysis identified increased PWdisperC (hazard ratio [HR], 1.024; P = 0.001) and PWdurMaxC (HR, 1.029; P = 0.001) were independently associated with progression to renal end point. Our results demonstrate that increased PWdisperC and PWdurMaxC were independently associated with progression to renal end point. Screening patients by means of PWdisperC and PWdurMaxC on 12 lead ECG may help identify a high risk group of rapid renal function decline.     

Correlation between body composition and risk factors for cardiovascular disease and metabolic syndrome

Body mass index (BMI) is an important diagnostic tool for determining obesity; however, while BMI reflects the influence of body height over body weight, it does not reveal body fat percentage (BF%). We explored whether BF% correlated with risk factors for cardiovascular disease and metabolic syndrome and whether metabolically obese, normal weight people were at risk for these diseases. A total of 2,867 healthy volunteers participated in this study. Blood pressure, height, weight, waist circumference, BMI, BF%, lipid profile, fasting glucose, uric acid, and lifestyle factors were collected from healthy subjects during their annual health examinations. In both males and females, BF% correlated positively with BMI and waist circumference. Participants were divided into three groups according to BF% and data were compared between groups. The results suggest that BF% correlates with risk factors for cardiovascular disease and metabolic syndrome for both men and women, and that BF% may be a useful predictor of risk, particularly in metabolically obese, normal weight individuals. ? 2012 International Union of Biochemistry and Molecular Biology, Inc.     

Peptidoglycan-induced IL-6 production in RAW 264.7 macrophages is mediated by cyclooxygenase-2, PGE2/PGE4 receptors, protein kinase A, I kappa B kinase, and NF-kappa B

In this study, we investigated the signaling pathway involved in IL-6 production caused by peptidoglycan (PGN), a cell wall component of the Gram-positive bacterium, Staphylococcus aureus, in RAW 264.7 macrophages. PGN caused concentration- and time-dependent increases in IL-6, PGE(2), and cAMP production. PGN-mediated IL-6 production was inhibited by a nonselective cyclooxygenase (COX) inhibitor (indomethacin), a selective COX-2 inhibitor (NS398), a PGE(2) (EP2) antagonist (AH6809), a PGE(4) (EP4) antagonist (AH23848), and a protein kinase A (PKA) inhibitor (KT5720), but not by a nonselective NO synthase inhibitor (N(G)-nitro-l-arginine methyl ester). Furthermore, PGE(2), an EP2 agonist (butaprost), an EP2/PGE(3) (EP3)/EP4 agonist (misoprostol), and misoprostol in the presence of AH6809 all induced IL-6 production, whereas an EP1/EP3 agonist (sulprostone) did not. PGN caused time-dependent activations of IkappaB kinase alphabeta (IKKdbeta) and p65 phosphorylation at Ser(276), and these effects were inhibited by NS398 and KT5720. Both PGE(2) and 8-bromo-cAMP also caused IKKdbeta kinase alphabeta phosphorylation. PGN resulted in two waves of the formation of NF-kappaB-specific DNA-protein complexes. The first wave of NF-kappaB activation occurred at 10-60 min of treatment, whereas the later wave occurred at 2-12 h of treatment. The PGN-induced increase in kappaB luciferase activity was inhibited by NS398, AH6809, AH23848, KT5720, a protein kinase C inhibitor (Ro31-8220), and a p38 MAPK inhibitor (SB203580). These results suggest that PGN-induced IL-6 production involves COX-2-generated PGE(2), activation of the EP2 and EP4 receptors, cAMP formation, and the activation of PKA, protein kinase C, p38 MAPK, IKKdbeta, kinase alphabeta, p65 phosphorylation, and NF-kappaB. However, PGN-induced NO release is not involved in the signaling pathway of PGN-induced IL-6 production.     

Temperature sensitive nitrogen fixation mutants of Azotobacter vinelandii

Summary Temperature-sensitive nitrogen fixation mutants of Azotobacter vinelandii were obtained by nitrosoguanidine mutagenesis and penicillin selection. The mutants were unable to grow on N₂ at 39° but grew normally at 30° on N₂ and at both temperatures in the presence of metabolizable nitrogen compounds. Growth experiments and assays of whole cells for nitrogenase activity separated the mutants into two classes: 1. mutants in which the nitrogenase activity present in cells grown at 30° was unaffected by a shift to 39°, and 2. mutants which lost their nitrogen fixation activity after such a temperature shift. Assays of cell-free extracts of the second class of mutants showed that in all cases tested the enzymatic activity of the nitrogenase complex itself was not affected by the mutation. These mutants might therefore contain some other temperature-sensitive proteins specifically involved in nitrogen fixation.