Use of moving aeration membrane bioreactor for the efficient production of tissue type plasminogen activator in serum free medium

A moving aeration-membrane (MAM) bioreactor was employed for the production of 2 μg/mL of tissue type Plasminogen Activator (tPA) in serum free medium from normal human fibroblast cells. This system could maintain high cell density for long periods of steady state conditions in perfusion cultivation. Under normal operating conditions, shear stress was as low as 0.65 dynes/cm² at the agitation speed of 80 rpm. Even though cell density gradually decreased with increasing agitation speed, tPA production increased linearly with increasing shear stress within a moderate range. This culture system allowed production of 2 μg tPA/mL while maintaining a high cell density of 1.0×10⁷ viable cells/mL.     

Ferrous Iron Induces Nrf2 Expression in Mouse Brain Astrocytes to Prevent Neurotoxicity

Free radical damage caused by ferrous iron is involved in the pathogenesis of secondary brain injury after intracerebral hemorrhage (ICH). NF‐E2‐related factor 2 (Nrf2), a major phase II gene regulator that binds to antioxidant response element, represents an important cellular cytoprotective mechanism against oxidative damage. We hypothesized that Nrf2 might protect astrocytes from damage by Fe²⁺. Therefore, we examined cytotoxicity in primary astrocytes induced by iron overload and evaluated the effects of Fe²⁺ on Nrf2 expression. The results demonstrated that 24‐h Fe²⁺ exposure exerted time‐ and concentration‐dependent cytotoxicity in astrocytes. Furthermore, Fe²⁺ exposure in astrocytes resulted in time‐ and concentration‐dependent increases in Nrf2 expression, which preceded Fe²⁺ toxicity. Nrf2‐specific siRNA further knocked down Nrf2 levels, resulting in greater Fe²⁺‐induced astrocyte cytotoxicity. These data indicate that induction of Nrf2 expression could serve as an adaptive self‐defense mechanism, although it is insufficient to completely protect primary astrocytes from Fe²⁺‐induced neurotoxicity.     

Extracellular expression, purification, and characterization of a winter flounder antifreeze polypeptide from Escherichia coli

HPLC6 is the major component of liver-type antifreeze polypeptides (AFPs) from the winter flounder, Pleuronectes americanus. To facilitate mutagenesis studies of this protein, a gene encoding the 37-amino acid mature polypeptide was chemically synthesized and cloned into the Tac cassette immediately after the bacterial ompA leader sequence for direct excretion of the AFP into the culture medium. Escherichia coli transformant with the construct placIQpar8AF was cultured in M9 medium. The recombinant AFP (rAFP) was detected by a competitive enzyme-linked immunosorbent assay (ELISA). After IPTG induction, a biologically active rAFP was expressed. The majority of the rAFP was excreted into the culture medium with only trace amounts trapped in the periplasmic space and cytoplasm. After 18 h of induction, the accumulated rAFP in the culture medium amounted to about 16 mg/L. The excreted AFP was purified from the culture medium by a single-step reverse-phase HPLC. Mass spectrometric and amino acid composition analyses confirmed the identity of the purified product. The rAFP, which lacked amidation at the C-terminal, was about 70% active when compared to the amidated wild-type protein, thus confirming the importance of C-terminal cap structure in protein stability and function.     

Nutrient-dependent requirement for SOD1 in lifespan extension by protein restriction in Drosophila melanogaster

Reactive oxygen species (ROS) modulate aging and aging-related diseases. Dietary composition is critical in modulating lifespan. However, how ROS modulate dietary effects on lifespan remains poorly understood. Superoxide dismutase 1 (SOD1) is a major cytosolic enzyme responsible for scavenging superoxides. Here we investigated the role of SOD1 in lifespan modulation by diet in Drosophila. We found that a high sugar-low protein (HS-LP) diet or low-calorie diet with low-sugar content, representing protein restriction, increased lifespan but not resistance to acute oxidative stress in wild-type flies, relative to a standard base diet. A low sugar-high protein diet had an opposite effect. Our genetic analysis indicated that SOD1 overexpression or dfoxo deletion did not alter lifespan patterns of flies responding to diets. However, sod1 reduction blunted lifespan extension by the HS-LP diet but not the low-calorie diet. HS-LP and low-calorie diets both reduced target of rapamycin (TOR) signaling and only the HS-LP diet increased oxidative damage. sod1 knockdown did not affect phosphorylation of S6 kinase, suggesting that SOD1 acts in parallel with or downstream of TOR signaling. Surprisingly, rapamycin decreased lifespan in sod1 mutant but not wild-type males fed the standard, HS-LP, and low-calorie diets, whereas antioxidant N-acetylcysteine only increased lifespan in sod1 mutant males fed the HS-LP diet, when compared to diet-matched controls. Our findings suggest that SOD1 is required for lifespan extension by protein restriction only when dietary sugar is high and support the context-dependent role of ROS in aging and caution the use of rapamycin and antioxidants in aging interventions.     

Characterization of phytoplankton assemblages in a tropical coastal environment using Kohonen self‐organizing map

This study was aimed at understanding the main abiotic environmental factors controlling the distribution patterns of abundance and composition of phytoplankton (size less than 10 μm) assemblages in the coastal waters of south‐eastern Côte d'Ivoire. Data were collected during two cruises, in January (low‐water period) and October (high‐water period) of 2014. A total of 67 species were identified and assigned to Bacillariophyceae (49%), Cyanophyceae (21%), Chlorophyceae (13%), Euglenophyceae (10%), Dinophyceae (4%) and Chrysophyceae (3%). Three biotic zones (I, IIA and IIB) were distinguishable on a Kohonen self‐organizing map after an unsupervised learning process. The diatom genera Eunotia sp., Navicula sp. and Actinoptychus senarius are significantly associated with I, IIA and IIB biotic zones, respectively. A clear seasonal cum salinity trend was apparent in phytoplankton distribution patterns. Turbidity and nitrate levels were the main abiotic factors controlling phytoplankton distribution in I, the upland tidal regions of the lagoon. In regions along the lagoon–sea continuum, phosphate and turbidity exert the most control during the low‐water season (IIA), while total dissolved solids control phytoplankton distribution during the high‐water season (IIB). These are climate‐sensitive parameters whose concentrations depend on prevailing hydroclimatic processes. Therefore, seasonality can have important consequences on phytoplankton community and inadvertently the productivity of these systems.     

Epidemiological Characteristics of Strongyloidiasis in Inhabitants of Indigenous Communities in Borneo Island,Malaysia

Epidemiological study on strongyloidiasis in humans is currently lacking in Malaysia. Thus, a cross-sectional study was carried out to determine the prevalence of Strongyloides stercoralis infection among the inhabitants of longhouse indigenous communities in Sarawak. A single stool and blood sample were collected from each participant and subjected to microscopy, serological and molecular techniques. Five species of intestinal parasites were identified by stool microscopy. None of the stool samples were positive for S. stercoralis. However, 11% of 236 serum samples were seropositive for strongyloidiasis. Further confirmation using molecular technique on stool samples of the seropositive individuals successfully amplified 5 samples, suggesting current active infections. The prevalence was significantly higher in adult males and tended to increase with age. S. stercoralis should no longer be neglected in any intestinal parasitic survey. Combination of more than 1 diagnostic technique is necessary to increase the likelihood of estimating the ‘true’ prevalence of S. stercoralis.     

The protein-tyrosine kinase Syk interacts with the C-terminal region of tensin2

Syk is a 72-kDa protein-tyrosine kinase that regulates signaling through multiple cell surface receptors including those for antigens, immunoglobulins and proteins of the extracellular matrix. As part of its function, Syk binds a variety of downstream effectors through interactions that are often mediated by motifs that recognize phosphotyrosines. In a search for novel Syk-interacting proteins by yeast two-hybrid analysis, we identified tensin2 as a Syk-binding protein. Syk interacts with a fragment of tensin2 located near the C-terminus that contains SH2 and PTB domains. In epithelial cells, tensin2 localizes both to focal adhesions and to large cytoplasmic puncta. It is within these punctuate structures that Syk and tensin2 are co-localized. The clustering of Syk within these structures leads to its phosphorylation on tyrosine.