Objective. To determine the cellular and matrix responses to experimental wounding of articular cartilage.Methods. Immature and mature bovine articular cartilage was used as an in vitro model system to study the cellular responses to cartilage wounding. Explant cultures were wounded centrally with a trephine and maintained for up to 10 days. TUNEL labeling together with ultrastructural analyses were used to assess the nature of the observed cell death. In vitro labeling with 3 H-thymidine was used to detect cell proliferation, and 2 antibodies (COL2-3/4M and BC-13) were used to detect changes in matrix turnover.Results. Cell death was observed as a response to wounding and was considered to be a combination of necrosis and apoptosis. In immature tissue, cell death was more pronounced, particularly in the articular surface region. Within the area of cell death, many cells that did not die subsequently underwent proliferation. The collagenous network showed evidence of denaturation in the area of the wound, but "aggrecanase" activity was not detected.Conclusion. There are 2 contrasting, but related, responses to cartilage wounding-apoptosis and proliferation. In order to improve cartilage repair, future studies need to elucidate the regulatory mechanisms that determine these responses.

This paper reports for the first time the presence in the anaerobic rumen ciliate Dasytricha ruminantium (Schuberg) of microbody-like organelles, about 0.5 micrometer diameter, with a granular matrix and an equilibrium density of approx. 1.18 g/ml. These organelles can be isolated in a fraction sedimented at 10(5) g-min that contains 67% of the total pyruvate synthase (EC 1.2.7.1), 66% of the hydrogenase (EC 1.18.3.1) and 20% of the lactate dehydrogenase (EC 1.1.1.27). Thus in several respects this fraction is enzymically similar to those containing hydrogenosomes in some other parasitic anaerobic protozoa (the trichomonads). However, in contrast with the hydrogenosomes of trichomonads, the oxygen-tolerant enzyme malate dehydrogenase (decarboxylating) (EC 1.1.1.40) is not particulate, but occurs only in the cytosol. These results enable the proposal of a scheme for the pathway of product formation (acetate, lactate, CO2 and H2) from carbohydrates.

Dishevelled family proteins are multidomain intracellular transducers of Wnt signals. Ectopically expressed mammalian Dishevelled 2 (Dvl-2) activates downstream signalling and localises to cytoplasmic puncta. It has been suggested that these Dvl-2-containing structures correspond to intracellular vesicles and may be involved in the Wnt signal transduction process. We report that cytoplasmic puncta are primarily formed in cells expressing Dvl-2 at high levels. Lower levels of expression can activate signalling without forming puncta. The structures do not localise with markers of the early or late endocytic pathway and time-lapse analysis demonstrates that Dvl-2 puncta move in a random fashion over short distances but do not originate from the plasma membrane. Based on our findings, we propose that Dvl-2 puncta are protein aggregates that are not required for signalling.

Transmission electron microscopy (TEM), scanning electron microscopy (SEM) and energy-dispersive analysis of X-ray (EDAX) have been used to examine chlorhexidine diacetate (CHA)-sensitive and -resistant isolates of Pseudomonas stutzeri and to determine the effects of CHA on the cells. Significant differences were observed in the structure, size and elemental composition of CHA-sensitive and -resistant cells. Treatment with CHA produced considerably greater changes in CHA-sensitive cells, with widespread peeling of the outer membrane, a substantial loss of cytoplasmic electron-dense material and extensive lysis. Cells from the resistant isolates showed no blebbing of the outer membrane and no structural damage. X-ray mapping confirmed the difference in CHA uptake between CHA-sensitive and CHA-resistant cells. It is proposed that changes in the outer membrane form a major mechanism of resistance to CHA in P. stutzeri.

These studies demonstrate the utility of silicon microneedles in cutaneous gene delivery. Further studies are required to elucidate fully the influence of the physicochemical characteristics of gene therapy vectors, e.g. particle diameter and surface charge, on their diffusion through microchannels and to quantify gene expression in vivo.

Since the early 1960s, axenic culture and the development of procedures for the induction of encystation have made Acanthamoeba spp. superb experimental systems for studies of cell biology and differentiation. More recently, since their roles as human pathogens causing keratitis and encephalitis have become widely recognized, it has become urgent to understand the parameters that determine differentiation, as cysts are much more resistant to biocides than are the trophozoites. Viability of trophozoites of the soil amoeba Acanthamoeba castellanii (Neff), is conveniently measured by its ability to form plaques on a lawn of Escherichia coli. Use of confocal laser scanning microscopy with Calcofluor white, Congo Red or the anionic oxonol dye, DiBAC4(3) or flow cytometry with propidium iodide diacetate and fluorescein or oxonol provides more rapid assessment. For cysts, the plaque method is still the best, because dye exclusion does not necessarily indicate viability and therefore the plate count method has been used to study the sequence of development of biocide resistance during the differentiation process. After two hours, resistance to HCl was apparent. Polyhexamethylene biguanide, benzalkonium chloride, propamidine isethionate, pentamidine isethionate, dibromopropamine isethionate, and H2O2 and moist heat, all lost effectiveness at between 14 and 24 h after trophozoites were inoculated into encystation media. Chlorhexidine diacetate resistance was observed at between 24 and 36 h. The molecular biology and biochemistry of the modifications that underlie these changes are now being investigated.

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An arc-lamp based flow cytometer was used to obtain high resolution measurements of the light scattering characteristics and DNA contents of eight different bacteria. Light scatter profiles of bacteria are a useful first step when flow cytometry is used to characterize organisms. Scanning and transmission electron microscopy of the bacterial samples demonstrate that the structural basis of the light scattering profiles is not always clear, i.e. some organisms appear to have anomalous light scattering characteristics. The use of a third measurement parameter, DNA content, allowed much better discrimination of the organisms. Flow cytometry shows great promise as a method for the rapid discrimination and identification of bacterial populations.