Expression of xylanase enzymes from thermophilic microorganisms in fungal hosts

Bulk production of xylanases from thermophilic microorganisms is a prerequisite for their use in industrial processes. As effective secretors of gene products, fungal expression systems provide a promising, industrially relevant alternative to bacteria for heterologous enzyme production. We are currently developing the yeast Kluyveromyces lactis and the filamentous fungus Trichoderma reesei for the extracellular production of thermophilic enzymes for the pulp and paper industry. The K. lactis system has been tested with two thermophilic xylanases and secretes gram amounts of largely pure xylanase A from Dictyoglomus thermophilum in chemostat culture. The T. reesei expression system involves the use of the cellobiohydrolase I (CBHI) promoter and gene fusions for the secretion of heterologous thermostable xylanases of both bacterial and fungal origin. We have reconstructed the AT-rich xynB gene of Dictyoglomus thermophilum according to Trichoderma codon preferences and demonstrated a dramatic increase in expression. A heterologous fungal gene, Humicola grisea xyn2, could be expressed without codon modification. Initial amounts of the XYN2 protein were of a gram per liter range in shake-flask cultivations, and the gene product was correctly processed by the heterologous host. Comparison of the expression of three thermophilic heterologous microbial xylanases in T. reesei demonstrates the need for addressing each case individually.     

Mapping of antibody responses to the protective antigen of Bacillus anthracis by flow cytometric analysis

BACKGROUND: Different plant species vary as to the ratio of nucleotide base pairs of genomic DNA. A correlation between genome size and base pair ratio has been claimed. Base composition can be analyzed by base-specific dyes. METHODS: Genome size is determined by flow cytometry of suspensions of nuclei stained by the base independent dye, PI. For estimation of the AT frequency, the AT-specific dyes 4,6-diamidino-2-phenylindole, dihydrochloride (DAPI) and Hoechst 33342 (HO) were used. We define a dye factor (DF) as the ratio of the two estimates (peak ratios) of nuclear fluorescence intensities of sample relative to reference plant nuclei using a given dye and an intercalating fluorochrome. RESULTS: No significant correlation between genome size and the DF for DAPI was found when 54 plant species were investigated. However, similarities within and differences among the plant families were shown. The comparison of DAPI and HO DFs gave no consistent differences as would be predicted from the model of different binding site length of dyes. This result may be explained by the nonrandom distribution of base pairs. CONCLUSIONS: There is no general correlation between genome size and AT/GC ratio in higher plants. Similar AT/GC ratios within a plant family result from the general similarity of the DNA sequences within a family. The fluorescence of base-specific dyes is influenced by the nonrandom distribution of bases in the DNA molecule.     

Recombinant carbazole-degrading strains for enhanced petroleum processing

Biotechnological upgrading of fossil fuels is of increasing interest as remaining stocks of petroleum show increasing levels of contaminants such as heavy metals, sulfur and nitrogen-containing heteroaromatic compounds. Carbazole is of particular interest as a major petroleum component known to reduce refining yields through catalyst poisoning. In this study, the biotransformation of carbazole was successfully demonstrated in a liquid two-phase system, when solubilized in either 1-methylnaphthalene or in diesel fuel. The effects of solvent toxicity were investigated by expressing the carbazole-transformation genes from MB1332, a rifampicin-resistant derivative of Pseudomonas sp. LD2, in a solvent-resistant heterologous host, P. putida Idaho [1]. This solvent-resistant strain successfully degraded carbazole solubilized in 1-methylnaphthalene and in the presence of 10 vol% xylenes similar to the non-recombinant strain Pseudomonas sp. LD2. Identification of a suitable recombinant host, however, was essential for further investigations of partial pathway transformations. Recombinant P. putida Idaho expressing only the initial dioxygenase enzymes transformed carbazole to an intermediate well retained in the oil phase. Partial carbazole transformation converts carbazole to non-aromatic species; their effect is unknown on refinery catalyst poisoning, but would allow almost complete retention of carbon content and fuel value. Electronic Publication     

Radiation-induced cyclooxygenase 2 up-regulation is dependent on redox status in prostate cancer cells

Cyclooxygenase 2 (COX2) is the inducible isozyme of COX, a key enzyme in arachidonate metabolism and the conversion of arachidonic acid (AA) to prostaglandins (PGs) and other eicosanoids. Previous studies have demonstrated that the COX2 protein is up-regulated in prostate cancer cells after irradiation and that this results in elevated levels of PGE(2). In the present study, we further investigated whether radiation-induced COX2 up-regulation is dependent on the redox status of cells from the prostate cancer cell line PC-3. l-Buthionine sulfoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (gammaGCS), and the antioxidants alpha-lipoic acid and N-acetyl-l-cysteine (NAC) were used to modulate the cellular redox status. BSO decreased the cellular GSH level and increased cellular reactive oxygen species (ROS) in PC-3 cells, whereas alpha-lipoic acid and NAC increased the GSH level and decreased cellular ROS. Both radiation and the oxidant H(2)O(2) had similar effects on COX2 up-regulation and PGE(2) production in PC-3 cells, suggesting that radiation-induced COX2 up-regulation is secondary to the production of ROS. The relative increases in COX2 expression and PGE(2) production induced by radiation and H(2)O(2) were even greater when PC-3 cells were pretreated with BSO. When the cells were pretreated with alpha-lipoic acid or NAC for 24 h, both radiation- and H(2)O(2)-induced COX2 up-regulation and PGE(2) production were markedly inhibited. These results demonstrate that radiation-induced COX2 up-regulation in prostate cancer cells is modulated by the cellular redox status. Radiation-induced increases in ROS levels contribute to the adaptive response of PC-3 cells, resulting in elevated levels of COX2.     

Concatenation cDNA sequencing for transcriptome analysis

We describe a high-throughput cDNA sequencing pipeline (http://www.hgsc.bcm.tmc.edu/projects/cdna) built in response to the emerging need for rapid sequencing of large cDNA collections. Using this strategy cDNA inserts are purified and joined through concatenation into large molecules. These 'pseudo-BACs' are subjected to random shotgun sequencing whereby the majority of cDNA inserts in the pool are sequenced. Using this concatenation cDNA sequencing platform, we have contributed more than 13000 full-length cDNA sequences from human and mouse to the Mammalian Gene Collection (MGC).     

In vitro development and characterization of a manatee bronchial cell line

In the present study, culture conditions that promote the growth and differentiation of manatee respiratory tract epithelial cells toward a mucociliary phenotype were determined. Characterization of a manatee-specific cell line enables investigators to conduct in vitro testing where live-animal experimentation is not possible. Cell cultures were established from both explants and enzymatically dissociated cells that were isolated from manatee bronchial tissue. To modulate their differentiation, bronchial epithelial cells were grown on Transwell collagen membranes either submerged or at an air-liquid interface. Growth on a collagen membrane at an air-liquid interface and medium supplemented with retinoic acid was required to promote a mucociliary phenotype. When cells were grown in submerged cultures without retinoic acid, they appeared more squamous and were not ciliated. Intracellular keratin proteins were detected in both submerged and interface cultures. Cultured manatee bronchial epithelial cells will facilitate future studies to investigate their potential role in pulmonary disease associated with brevetoxicosis after exposure to the red-tide organism, Karenia brevis.     

Some observations on the effect of Daflon (micronized purified flavonoid fraction of Rutaceae aurantiae) in bancroftian filarial lymphoedema

BACKGROUND: Morbidity management is a core component of the global programme for the elimination of lymphatic filariasis. In a double-blind clinical trial, the tolerability and efficacy of Daflon (500 mg) + DEC (25 mg) or DEC (25 mg) alone, twice daily for 90 days, was studied in 26 patients with bancroftian filarial lymphoedema. RESULTS: None of the patients in either drug group reported any adverse reaction throughout the treatment period (90 days). Haematological and biochemical parameters were within normal limits and there was no significant difference between the pre-treatment (day 0) and post-treatment (day 90) values. The group receiving Daflon showed significant reduction in oedema volume from day 90 (140.6 PlusMinus; 18.8 ml) to day 360 (71.8 PlusMinus; 20.7 ml) compared to the pre-treatment (day 0, 198.4 PlusMinus; 16.5 ml) value. This accounted for a 63.8% reduction in oedema volume by day 360 (considering the pre-treatment (day 0) as 100%). In the DEC group, the changes in oedema volume (between day 1 and day 360) were not significant when compared to the pre-treatment (day 0) value. The percentage reduction at day 360 was only 9%, which was not significant (P > 0.05). CONCLUSION: This study has shown that Daflon (500 mg, twice a day for 90 days) is both safe and efficacious in reducing oedema volume in bancroftian filarial lymphoedema. Further clinical trials are essential for strengthening the evidence base on the role of this drug in the morbidity management of lymphatic filariasis.     

Regulation of neuronal proliferation and differentiation by nitric oxide

Many studies have revealed the free radical nitric oxide (NO) to be an important modulator of vascular and neuronal physiology. It also plays a developmental role in regulating synapse formation and patterning. Recent studies suggest that NO may also mediate the switch from proliferation to differentiation during neurogenesis. Many mechanisms of this response are conserved between neuronal precursor cells and the cells of the vascular system, where NO can inhibit the proliferative response of endothelial and smooth-muscle cells to injury. In cultured neuroblastoma cells, NO synthase (NOS) expression is increased in the presence of various growth factors and mitogens. Subsequent production of NO leads to cessation of cell division and the acquisition of a differentiated phenotype. The inhibitory action of NO on neuroblast proliferation has also been demonstrated in vivo for vertebrate and invertebrate nervous systems, as well as in the adult brain. Potential downstream effectors of NO include the second messenger cyclic GMP, activation of the tumor-suppressor genes p53 and Rb, and the cyclin-dependent kinase inhibitor p21. These studies highlight a new role for NO in the nervous system, as a coordinator of proliferation and patterning during neural development and adult neurogenesis.     

Glucose metabolism in perfused mouse hearts overexpressing human GLUT-4 glucose transporter

Glucose and fatty acid metabolism was assessed in isolated working hearts from control C57BL/KsJ-m+/+db mice and transgenic mice overexpressing the human GLUT-4 glucose transporter (db/+-hGLUT-4). Heart rate, coronary flow, cardiac output, and cardiac power did not differ between control hearts and hearts overexpressing GLUT-4. Hearts overexpressing GLUT-4 had significantly higher rates of glucose uptake and glycolysis and higher levels of glycogen after perfusion than control hearts, but rates of glucose and palmitate oxidation were not different. Insulin (1 mU/ml) significantly increased glycogen levels in both groups. Insulin increased glycolysis in control hearts but not in GLUT-4 hearts, whereas glucose oxidation was increased by insulin in both groups. Therefore, GLUT-4 overexpression increases glycolysis, but not glucose oxidation, in the heart. Although control hearts responded to insulin with increased rates of glycolysis, the enhanced entry of glucose in the GLUT-4 hearts was already sufficient to maximally activate glycolysis under basal conditions such that insulin could not further stimulate the glycolytic rate.     

Altered metabolism causes cardiac dysfunction in perfused hearts from diabetic (db/db) mice

Contractile function and substrate metabolism were characterized in perfused hearts from genetically diabetic C57BL/KsJ-lepr(db)/lepr(db) (db/db) mice and their non-diabetic lean littermates. Contractility was assessed in working hearts by measuring left ventricular pressures and cardiac power. Rates of glycolysis, glucose oxidation, and fatty acid oxidation were measured using radiolabeled substrates ([5-(3)H]glucose, [U-(14)C]glucose, and [9,10-(3)H]palmitate) in the perfusate. Contractile dysfunction in db/db hearts was evident, with increased left ventricular end diastolic pressure and decreased left ventricular developed pressure, cardiac output, and cardiac power. The rate of glycolysis from exogenous glucose in diabetic hearts was 48% of control, whereas glucose oxidation was depressed to only 16% of control. In contrast, palmitate oxidation was increased twofold in db/db hearts. The hypothesis that altered metabolism plays a causative role in diabetes-induced contractile dysfunction was tested using perfused hearts from transgenic db/db mice that overexpress GLUT-4 glucose transporters. Both glucose metabolism and palmitate metabolism were normalized in hearts from db/db-human insulin-regulatable glucose transporter (hGLUT-4) hearts, as was contractile function. These findings strongly support a causative role of impaired metabolism in the cardiomyopathy observed in db/db diabetic hearts.