Precultivation technique for studies of microorganisms exhibiting overflow metabolism

A precultivation technique for microorganisms exhibiting overflow metabolism is presented. It is based on a low initial medium volume in the fermenter. The medium feed contains all the nutrients, but is diluted with respect to sugar, the concentration of which determines the biomass concentration at the end of the preculture. By this method, effects of varying activity of the inocula from shake flask cultures are minimised and the metabolic state, i.e. oxido-reductive, oxidative growth on sugar plus overflow metabolite or oxidative growth on sugar alone, can be controlled at the start of the main fermentation.     

An in vivo study of antifreeze protein adjuvant cryosurgery

Cryosurgery employs freezing to destroy undesirable tissue. However, under certain thermal conditions, frozen tissues survive. The survival of frozen undesirable tissue may lead to complications, such as recurrence of cancer. In a study of nude mice with subcutaneous metastatic prostate tumors, we showed that the preoperative injection of a phosphate-buffered saline solution with 10 mg/ml antifreeze protein of type I into the tumor prior to freezing enhances destruction under thermal conditions which normally yield cell survival. This suggests that the adjunctive use of antifreeze proteins in cryosurgery may reduce the complications from undesirable tissues that survive freezing.     

Multistep denaturation of Borrelia burgdorferi OspA, a protein containing a single-layer beta-sheet

Outer surface protein A (OspA) from the Lyme disease spirochete, Borrelia burgdorferi, is a dumbbell-shaped protein in which two globular domains are connected by a three-stranded beta-sheet segment that is solvent-exposed on both faces. Previous studies showed that the whole protein, including the single-layer beta-sheet, is highly rigid. To elucidate the folding mechanism and the role of the central beta-sheet in the formation of the rigid molecule, we investigated the equilibrium thermal denaturation reaction of OspA. We applied differential scanning calorimetry, heteronuclear NMR spectroscopy, and solution small-angle X-ray scattering (SAXS) to characterize the reaction in detail. All three techniques revealed that OspA denatures in two separable cooperative transitions. NMR measurements on OspA specifically 15N-labeled at Lys residues identified the locations of the two folding units and revealed that the C-terminal segment is less stable than the remaining N-terminal segment. The boundary between the two folding units is located within the central beta-sheet. The interconversion among the three folding states (fully folded, C-terminus unfolded, and fully denatured) is slow relative to chemical shift differences (<24 Hz), indicating that there are significant kinetic barriers in the denaturation reactions. SAXS measurements determined the radius of gyration of the native protein to be 25.0 +/- 0.3 A, which increases to 34.4 +/- 1.0 A in the first transition, and then to 56.1 +/- 1.6 A in the second transition. Thus, the intermediate state, in which the C-terminal folding unit is already denatured, is still compact. These results provide a basis for elucidating the folding mechanism of OspA.     

Poly(L-lysine)-mediated immobilisation of oligonucleotides on carboxy-rich polymer surfaces

The immobilisation efficiency of the complexes of oligonucleotide/poly(L-lysine) on two polymeric carboxy-rich surfaces, i.e. poly(styrene/maleic acid) (PSMA) and poly(styrene/maleic anhydride) (PSMAA), has been investigated using X-ray photoelectron spectroscopy, atomic force microscopy (AFM) and fluorescence-based measurements of DNA attachment. A molecularly thin layer of either electrostatically or covalently (via amide bond) bound poly(L-lysine) allows the 'switching' from COOH-based to NH(2)-based surface functionality. The results indicate that approximately 54-57% and 55-62% of the applied oligonucleotides bind to polymeric surfaces via the route of electrostatic adsorption of poly(L-lysine) and covalent bonding of poly(L-lysine), respectively. This system can be applied conveniently for the detection of nucleic acids in both disposable and reusable biosensors.     

Tumor-induced endothelial cell activation: role of vascular endothelial growth factor

Proangiogenic, proliferative effects of tumors have been extensively characterized in subconfluent endothelial cells (EC), but results in confluent, contact-inhibited EC are critically lacking. The present study examined the effect of tumor-conditioned medium (CM) of the malignant osteoblastic cell line MG63 on monolayer, quiescent bovine aorta EC. MG63-CM and MG63-CM + CoCl₂ significantly increased EC survival in serum-starved conditions, without inducing EC proliferation. Furthermore, MG63-CM and MG63-CM + CoCl₂, both containing high amounts of vascular endothelial growth factor (VEGF), induced relevant phenotypic changes in EC (all P < 0.01) involving increase of nucleoli/chromatin condensations, nucleus-to-cytosol ratio, capillary-like vacuolated structures, vessel-like acellular areas, migration through Matrigel, growth advantage in reseeding, and factor VIII content. All these actions were significantly inhibited by VEGF and VEGF receptor (VEGFR2) blockade. Of particular importance, a set of similar effects were detected in a human microvascular endothelial cell line (HMEC). With regard to gene expression, incubation with MG63-CM abolished endogenous VEGF mRNA and protein but induced a clear-cut increase in VEGFR2 mRNA expression in EC. In terms of mechanism, MG63-CM activates protein kinase B (PKB)/Akt, p44/p42-mitogen-activated protein kinase (MAPK)-mediated pathways, as suggested by both inhibition and phosphorylation experiments. In conclusion, tumor cells activate confluent, quiescent EC, promoting survival, phenotypic, and gene expression changes. Of importance, VEGF antagonism converts MG63-CM from protective to EC-damaging effects. vascular endothelial growth factor receptor 2; MG63-conditioned medium     

Bioconversion of waste office paper to L(+)-lactic acid by the filamentous fungus Rhizopus oryzae

L(+)-lactic acid production was investigated using an enzymatic hydrolysate of waste office automation (OA) paper in a culture of the filamentous fungus Rhizopus oryzae. In 4 d culture, 82.8 g/l glucose, 7 g/l xylose, and 3.4 g/l cellobiose contained in the hydrolysate were consumed to produce 49.1 g/l of lactic acid. The lactic acid yield and production rate were only 0.59 g/g and 16.3 g/l/d, respectively, only 75% and 61% of the results from the glucose medium. The low production rate from waste OA hydrolysate was elucidated by trials using xylose as the sole carbon source; in those trials, the lactic acid production rate was 7.3 g/l/d, only 28% that of glucose or cellobiose. The low lactic acid yield from waste OA hydrolysate was clarified by trials using artificial hydrolysates comprised of 7:2:1 or 7:1:2 ratios of glucose:cellobiose:xylose. For both, the lactic acid production rate of 17.4 g/l/d matched that of waste OA paper, while the lactic acid yield was similar to that of the glucose medium. This indicates that the production rate may be inhibited by xylose derived from hemicellulose, and the yield may be inhibited by unknown compounds derived from paper pulp.     

Extraction of fluorescent cell puncta by adaptive fuzzy segmentation

MOTIVATION: The discrimination and measurement of fluorescent-labeled vesicles using microscopic analysis of fixed cells presents a challenge for biologists interested in quantifying the abundance, size and distribution of such vesicles in normal and abnormal cellular situations. In the specific application reported here, we were interested in quantifying changes to the population of a major organelle, the peroxisome, in cells from normal control patients and from patients with a defect in peroxisome biogenesis. In the latter, peroxisomes are present as larger vesicular structures with a more restricted cytoplasmic distribution. Existing image processing methods for extracting fluorescent cell puncta do not provide useful results and therefore, there is a need to develop some new approaches for dealing with such a task effectively. RESULTS: We present an effective implementation of the fuzzy c-means algorithm for extracting puncta (spots), representing fluorescent-labeled peroxisomes, which are subject to low contrast. We make use of the quadtree partition to enhance the fuzzy c-means based segmentation and to disregard regions which contain no target objects (peroxisomes) in order to minimize considerable time taken by the iterative process of the fuzzy c-means algorithm. We finally isolate touching peroxisomes by an aspect-ratio criterion. The proposed approach has been applied to extract peroxisomes contained in several sets of color images and the results are superior to those obtained from a number of standard techniques for spot extraction. AVAILABILITY: Image data and computer codes written in Matlab are available upon request from the first author.     

Inhibition of constitutive NF-kappa B activation in mantle cell lymphoma B cells leads to induction of cell cycle arrest and apoptosis

Constitutive activation of the NF-kappaB has been documented to be involved in the pathogenesis of many human malignancies, including hemopoietic neoplasms. In this study, we examined the status of NF-kappaB in two non-Hodgkin's lymphoma cell lines derived from mantle cell lymphoma (MCL) samples and in patient MCL biopsy specimens by EMSA and confocal microscopic analysis. We observed that NF-kappaB is constitutively activated in both the MCL cell lines and in the MCL patient biopsy cells. Since NF-kappaB has been shown to play an important role in a variety of cellular processes, including cell cycle regulation and apoptosis, targeting the NF-kappaB pathways for therapy may represent a rational approach in this malignancy. In the MCL cell lines, inhibition of constitutive NF-kappaB by the proteasome inhibitor PS-341 or a specific pIkappaBalpha inhibitor, BAY 11-7082, led to cell cycle arrest in G(1) and rapid induction of apoptosis. Apoptosis was associated with the down-regulation of bcl-2 family members bcl-x(L) and bfl/A1, and the activation of caspase 3, that mediates bcl-2 cleavage, resulting in the release of cytochrome c from the mitochondria. PS-341or BAY 11-induced G(1) cell cycle arrest was associated with the inhibition of cyclin D1 expression, a molecular genetic marker of MCL. These studies suggest that constitutive NF-kappaB expression plays a key role in the growth and survival of MCL cells, and that PS-341 and BAY 11 may be useful therapeutic agents for MCL, a lymphoma that is refractory to most current chemotherapy regimens.