Experimental osteoarthritic articular cartilage is enriched in guanidine soluble type VI collagen

Experimental osteoarthritis was surgically induced in the right knee joint of dogs; the left knee served as a control. Articular cartilage was extracted with 4 M guanidinium chloride, 0.05 M sodium acetate, pH 6.0, containing proteinase inhibitors and the proteins purified by associative CsCl density gradient centrifugation. Equal quantities of protein were electrophoresed in agarose-acrylamide gradient gels and the high molecular weight type VI collagen bands detected in immunoblots with a polyclonal antiserum. Type VI collagen bands between 185 and 220 kDa were evident in the pathological specimens of dogs sacrificed 3, 5, and 7 months after surgery and were either absent or only very weakly visible in the controls. These results demonstrate that experimental osteoarthritic cartilage is enriched in 4 M guanidine-soluble type VI collagen.     

Characterisation of Tat protein transport complexes carrying inactivating mutations

The Tat system functions to transport folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. Tat transport involves a high molecular weight TatBC-containing complex that transiently associates with TatA during protein translocation. Sedimentation equilibrium experiments were used to determine a protein-only molecular mass for the TatBC complex of 630+/-30kDa, suggesting that it contains approximately 13 copies of the TatB and TatC protomers. Point mutations that inactivate Tat transport have previously been identified in each of TatA, TatB, and TatC. Analysis of the TatBC complexes formed by these inactive variants demonstrates that the amino acid substitutions neither affect the composition of the TatBC complex nor cause accumulation of the assembled TatABC translocation site. In addition, the TatA protein is shown not to be required for the assembly or stability of the TatBC complex.     

Aerobic nitrate respiration in a nitrite-oxidising bioreactor

The ability of heterotrophic bacteria in a nitrite-oxidising bioreactor to respire with nitrate as an electron acceptor was examined. Approximately 70% of 1000 heterotrophic isolates were able to express a nitrate reductase. A detailed survey of 15 isolates showed that five expressed the azide-insensitive nitrate reductase encoded by the napA gene. A two-round PCR amplification of the napA gene using degenerate PCR primers and DNA sequence analysis of these products confirmed the presence of this gene in the positive isolates. Partial 16S rDNA products and napA products were amplified from the biomass in the bioreactor and denaturing gradient gel electrophoresis of these products identified 21 distinct ribotypes and 12 distinct napA sequences. The results show that the ability to respire with nitrate as an electron acceptor under aerobic conditions is widespread among the heterotrophic population of this bioreactor.     

The rel gene is essential for in vitro growth of Staphylococcus aureus

The stringent response in Staphylococcus aureus is mediated by the nucleotide guanosine pentaphosphate, whose synthesis is catalyzed by the product of the rel gene. We report here that the rel gene is essential for the in vitro growth of S. aureus, distinguishing it from all other bacteria tested for this requirement.     

A microbead array chemical sensor using capillary-based sample introduction: toward the development of an "electronic tongue"

The development of a micromachined fluidic structure for the introduction of liquid samples into a chip-based sensor array composed of individually addressable polymeric microbeads is presented. The micromachined structure consists of micromachined storage cavities combined with a covering glass layer that confines the microbeads and fluidic channels. In our sensor array transduction occurs via optical (colorimetric and fluorescence) changes to receptors and indicator molecules that are covalently attached to termination sites on the polymeric microbeads. Spectral data are acquired for each of the individual microbeads using a charged-coupled device (CCD) allowing for the near-real-time analysis of liquid sample. Hence the micromachined fluidic structure must allow for both optical access to the microbeads and fluid flow through the micromachined cavities that serve as the microreactors/analysis chambers. One of the key parts of the structure is a passive fluid introduction system driven only by capillary force. This simple means of fluid introduction realizes a compact device. The capillary flow on the inlet channel has been studied, and the responses of the microbeads (alizarin complexone) to a liquid sample have been characterized. The test results show that this system is useful in a micro-total-analysis-system (mu-TAS) and biomedical applications.     

Gene Expression Signatures of Extracellular Matrix and Growth Factors during Embryonic Stem Cell Differentiation

Pluripotent stem cells are uniquely capable of differentiating into somatic cell derivatives of all three germ lineages, therefore holding tremendous promise for developmental biology studies and regenerative medicine therapies. Although temporal patterns of phenotypic gene expression have been relatively well characterized during the course of differentiation, coincident patterns of endogenous extracellular matrix (ECM) and growth factor expression that accompany pluripotent stem cell differentiation remain much less well-defined. Thus, the objective of this study was to examine the global dynamic profiles of ECM and growth factor genes associated with early stages of pluripotent mouse embryonic stem cell (ESC) differentiation. Gene expression analysis of ECM and growth factors by ESCs differentiating as embryoid bodies for up to 14 days was assessed using PCR arrays (172 unique genes total), and the results were examined using a variety of data mining methods. As expected, decreases in the expression of genes regulating pluripotent stem cell fate preceded subsequent increases in morphogen expression associated with differentiation. Pathway analysis generated solely from ECM and growth factor gene expression highlighted morphogenic cell processes within the embryoid bodies, such as cell growth, migration, and intercellular signaling, that are required for primitive tissue and organ developmental events. In addition, systems analysis of ECM and growth factor gene expression alone identified intracellular molecules and signaling pathways involved in the progression of pluripotent stem cell differentiation that were not contained within the array data set. Overall, these studies represent a novel framework to dissect the complex, dynamic nature of the extracellular biochemical milieu of stem cell microenvironments that regulate pluripotent cell fate decisions and morphogenesis.     

Conversion of iris epithelial cells as a model of differentiation control

Abstract. It has been shown that, upon lentectomy or in culture, iris epithelial cells (IECs) of adult newts become converted into lens cells, and this conversion is the basic event of lens regeneration in newts. Whether in situ or in cell culture, the conversion requires the passage of a specific number of cell cycles. The progeny of IECs which fails to traverse this cell-cycle number redifferentiates as IECs in situ. The passage through cell cycles of IECs is associated with progressive alterations of cytoplasm and cell surface, during which the original state of differentiation disappears (dedifferentiation). It is speculated that the altered state of cells caused by proliferation leads to the appearance of factors which interact with the genome and switch the gene activation pattern to that of the lens cell. In this model, developmental controls are geared to the cell-cycle progression and not directly to the activation of lens-characteristic genes. A number of points are raised which speak against the long-held idea that a factor from neural retina induces lens differentiation in IECs. It is proposed that the retinal factor plays the role of growth factor which is essential in the conversion in situ, but not required in the conversion in cell culture. The proposed model is compared with reprogramming of differentiation of some cell lines by cytidine analogs and with ontogenic systems of differentiation control.