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.     

Induction of Apoptotic Cell Death on Human Cervix Cancer HeLa cells by Extract from <Emphasis Type="Italic">Loranthus yadoriki</Emphasis>

Loranthus yadoriki, one of the Korean mistletoe species, has been already known for anti-viral effects, but the molecular basis that it caused apoptosis in cancer cells was not definitely revealed yet. The aim of this study was to estimate the mechanisms of apoptotic cell death of the extract from Loranthus yadoriki (named as ELY) in human cervix HeLa cells. We identified that ELY prevented the proliferation of HeLa cells between 50 and 300 μg/mL which did not affect non-cancerous HaCaT cells. In addition, ELY induced a morphological change and nucleus disruption as well as an accumulation of sub-G1 phase in HeLa cells. The mechanism study, by using western blot analysis, showed that the phosphorylation of Fas-associated death domain (FADD), Bim and Bak was up-regulated by ELY treatment. Furthermore, the expression of cytochrome c and Apaf-1 was increased by ELY treatment. In immunofluorescence staining, the increased intensity of cleaved caspase-3 and cleaved PARP was also observed under ELY treatment. Sequentially, the caspase cascade was activated by ELY from caspase-8 to caspase-3 and from caspase-9 to caspase-3, in both extrinsic and intrinsic pathways. The results of this study demonstrate that ELY has anti-cancer effects on human cervix cancer HeLa cells via caspase cascade in apoptotic signaling pathways.     

The highly stable alcohol dehydrogenase of Thermomicrobium roseum: purification and molecular characterization

An alcohol dehydrogenase (ADH) was purified to electrophoretic homogeneity from an extremely thermophilic bacterium, Thermomicrobium roseum. The native enzyme was found to be a homo-dimer of 43-kDa subunits. The pI of the enzyme was determined to be 6.2, while its optimum pH is 10.0. The enzyme oxidized mainly primary aliphatic alcohols and exhibited high substrate specificity towards ethanol, n-propanol and crotyl alcohol. The highest reaction rate was observed when ethanol was used as substrate and the K(m) value of the enzyme for ethanol was 24.2 mM. Pyrazole notably inhibited the enzymatic activity. The enzyme had the optimal temperature of 70 degrees C and was highly stable against high temperature.     

EGCG, a green tea polyphenol, improves endothelial function and insulin sensitivity, reduces blood pressure, and protects against myocardial I/R injury in SHR

Epigallocatechin gallate (EGCG), a bioactive polyphenol in green tea, may augment metabolic and vascular actions of insulin. Therefore, we investigated effects of EGCG treatment to simultaneously improve cardiovascular and metabolic function in spontaneously hypertensive rats (SHR; model of metabolic syndrome with hypertension, insulin resistance, and overweight). In acute studies, EGCG (1-100 microM) elicited dose-dependent vasodilation in mesenteric vascular beds (MVB) isolated from SHR ex vivo that was inhibitable by N(omega)-nitro-L-arginine methyl ester (L-NAME; nitric oxide synthase antagonist) or wortmannin [phosphatidylinositol (PI) 3-kinase inhibitor]. In chronic studies, 9-wk-old SHR were treated by gavage for 3 wk with EGCG (200 mg.kg(-1).day(-1)), enalapril (30 mg.kg(-1).day(-1)), or vehicle. A separate group of SHR receiving L-NAME (80 mg/l in drinking water) was treated for 3 wk with either EGCG or vehicle. Vasodilator actions of insulin were significantly improved in MVB from EGCG- or enalapril-treated SHR (when compared with vehicle-treated SHR). Both EGCG and enalapril therapy significantly lowered systolic blood pressure (SBP) in SHR. EGCG therapy of SHR significantly reduced infarct size and improved cardiac function in Langendorff-perfused hearts exposed to ischemia-reperfusion (I/R) injury. In SHR given L-NAME, beneficial effects of EGCG on SBP and I/R were not observed. Both enalapril and EGCG treatment of SHR improved insulin sensitivity and raised plasma adiponectin levels. We conclude that acute actions of EGCG to stimulate production of nitric oxide from endothelium using PI 3-kinase-dependent pathways may explain, in part, beneficial effects of EGCG therapy to simultaneously improve metabolic and cardiovascular pathophysiology in SHR. These findings may be relevant to understanding potential benefits of green tea consumption in patients with the metabolic syndrome.     

A role of retinoic acid in the regulation of the morphology and the levels of intermediate filament proteins and mRNAs in PC12 cells.

An adrenal tumor-derived cell line (PC12W) cultured in the presence of nerve growth factor exhibited a spindle-shaped cell morphology resembling neuronal cells. The shape of these cells can be specifically changed in vitamin A-depleted medium supplemented with retinoic acid. Retinoic acid promoted an epithelial-like cell morphology except for occasional neuronal processes. These morphological results were correlated with differential expression of intermediate filaments at the mRNA and protein levels in these cells. Retinoic acid suppressed the synthesis of peripherin, an intermediate filament protein predominantly found in peripheral nerve cells, but a high level of simple keratins, normally found in simple epithelial cells, was present in retinoic acid-treated PC12 cells. The neurofilaments typically expressed in neurons remained virtually unaffected under the same conditions. In contrast, nerve growth factor induced the production of neurofilaments, but suppressed the synthesis of simple keratins. Since intermediate filament expression is known to be tissue-specific, these changes in expression together with the cell morphology changes are consistent with PC12 cells undergoing an epithelial-like differentiation in the presence of retinoic acid and a neuronal-like differentiation in the presence of nerve growth factor. These results suggest that retinoic acid and nerve growth factor are both effective regulators of PC12 cell differentiation but stimulate opposing pathways.     

Structure-based functional inference in structural genomics

The dramatically increasing number of new protein sequences arising from genomics 4 proteomics requires the need for methods to rapidly and reliably infer the molecular and cellular functions of these proteins. One such approach, structural genomics, aims to delineate the total repertoire of protein folds in nature, thereby providing three-dimensional folding patterns for all proteins and to infer molecular functions of the proteins based on the combined information of structures and sequences. The goal of obtaining protein structures on a genomic scale has motivated the development of high throughput technologies and protocols for macromolecular structure determination that have begun to produce structures at a greater rate than previously possible. These new structures have revealed many unexpected functional inferences and evolutionary relationships that were hidden at the sequence level. Here, we present samples of structures determined at Berkeley Structural Genomics Center and collaborators laboratories to illustrate how structural information provides and complements sequence information to deduce the functional inferences of proteins with unknown molecular functions.Two of the major premises of structural genomics are to discover a complete repertoire of protein folds in nature and to find molecular functions of the proteins whose functions are not predicted from sequence comparison alone. To achieve these objectives on a genomic scale, new methods, protocols, and technologies need to be developed by multi-institutional collaborations worldwide. As part of this effort, the Protein Structure Initiative has been launched in the United States (PSI; www.nigms.nih.gov/funding/psi.html). Although infrastructure building and technology development are still the main focus of structural genomics programs [1–6], a considerable number of protein structures have already been produced, some of them coming directly out of semi-automated structure determination pipelines [6–10]. The Berkeley Structural Genomics Center (BSGC) has focused on the proteins of Mycoplasma or their homologues from other organisms as its structural genomics targets because of the minimal genome size of the Mycoplasmas as well as their relevance to human and animal pathogenicity (http://www.strgen.org). Here we present several protein examples encompassing a spectrum of functional inferences obtainable from their three-dimensional structures in five situations, where the inferences are new and testable, and are not predictable from protein sequence information alone.     

Borrelia burgdorferi binds fibronectin through a tandem beta-zipper, a common mechanism of fibronectin binding in staphylococci, streptococci, and spirochetes

BBK32 is a fibronectin-binding protein from the Lyme disease-causing spirochete Borrelia burgdorferi. In this study, we show that BBK32 shares sequence similarity with fibronectin module-binding motifs previously identified in proteins from Streptococcus pyogenes and Staphylococcus aureus. Nuclear magnetic resonance spectroscopy and isothermal titration calorimetry are used to confirm the binding sites of BBK32 peptides within the N-terminal domain of fibronectin and to measure the affinities of the interactions. Comparison of chemical shift perturbations in fibronectin F1 modules on binding of peptides from BBK32, FnBPA from S. aureus, and SfbI from S. pyogenes provides further evidence for a shared mechanism of binding. Despite the different locations of the bacterial attachment sites in BBK32 compared with SfbI from S. pyogenes and FnBPA from S. aureus, an antiparallel orientation is observed for binding of the N-terminal domain of fibronectin to each of the pathogens. Thus, these phylogenetically and morphologically distinct bacterial pathogens have similar mechanisms for binding to human fibronectin.     

The effects of follicular fluid on in vitro maturation, oocyte fertilization and the development of bovine embryos

We determined the effects of follicular fluid in the maturation medium on bovine oocyte maturation, fertilization and subsequent development, as well as on the number of cells in blastocysts following culture. Fluid and oocytes from bovine follicles less than 5 mm in diameter were collected from the ovaries of slaughtered cows. For the maturation medium, follicular fluid at concentrations of 10, 30 or 60% (v/v) was added to Medium 199 with Earle's salts supplemented with 0.1 microg/ml estradiol-17 beta (E(2), Experiment 1) or 0.1 microg/ml E2 and 100 IU/ml hCG (Experiment 2). The control medium contained polyvinylpyrrolidone (PVP; 3 mg/ml) instead of follicular fluid. After maturation for 24 h, oocytes were fertilized in vitro with bull frozen-thawed spermatozoa and cultured on a monolayer of granulosa cells for 9 d. There were no differences in maturation or fertilization rates of oocytes. In Experiment 1, maturation medium containing 10% follicular fluid did not affect the developmental rate of the oocytes to > 2-cell, 8 to 16-cell, blastocyst and hatched blastocyst stage embryos, respectively; whereas 60% decreased embryonic development (P < 0.05) compared with the control. Blastocysts and hatched blastocysts developed from fertilized oocytes which had been matured in medium containing 10 and 30% follicular fluid/E(2) had more cells than the controls (P < 0.01). In Experiment 2, maturation medium containing 10 or 30% follicular fluid did not affect the development fertilized oocytes to the blastocyst stage compared with the control, but decreased at 60% (P < 0.01). There were no differences in the number of cells from Day 9 blastocysts and hatched blastocysts from fertilized oocytes matured in maturation medium containing follicular fluid and E(2) + hCG. The results of these experiments suggest that the addition of bovine follicular fluid to the maturation medium enhances the cell numbers in blastocysts from bovine follicular oocytes matured in vitro.