Demonstration of chondroitin sulphate and glycoproteins in articular cartilage matrix using periodic acid-Schiff (PAS) method

Summary Staining of articular cartilage by the periodic acid-Schiff (PAS) method was measured using microspectrophotometry. Standard PAS technique with 2 h oxidation produced a distinct Schiff reaction in the cartilage sections. The staining increased with depth of the articular cartilage demonstrating distribution of the glycoproteins. The modified PAS method included a second, longer periodic acid treatment, which made the uronic acid of glycosaminoglycans PAS-positive. The modified PAS method proved to be highly specific for chondroitin sulphate, which was determined from the samples with gas chromatography. A statistically significant correlation between the Schiff reactivity and galactosamine content of the sections was observed. It is concluded that for articular cartilage standard and modified PAS methods are useful procedures for demonstrating local changes of glycoproteins and chondroitin sulphate, respectively.     

Oligovalent Fab Display on M13 Phage Improved by Directed Evolution

Efficient display of antibody on filamentous phage M13 coat is crucial for successful biopanning selections. We applied a directed evolution strategy to improve the oligovalent display of a poorly behaving Fab fragment fused to phage gene-3 for minor coat protein (g3p). The Fab displaying clones were enriched from a randomly mutated Fab gene library with polyclonal anti-mouse IgG antibodies. Contribution of each mutation to the improved phenotype of one selected mutant was studied. It was found out that two point mutations had significant contribution to the display efficiency of Fab clones superinfected with hyperphage. The most dramatic effect was connected to a start codon mutation, from AUG to GUG, of the PelB signal sequence preceding the heavy chain. The clone carrying this mutation, FabM_GUG, displayed Fab 19-fold better and yielded twofold higher phage titers than the original Fab.     

Endostatin inhibits human tongue carcinoma cell invasion and intravasation and blocks the activation of matrix metalloprotease-2, -9, and -13

Endostatin, a 20-kDa collagen XVIII fragment, inhibits angiogenesis and tumor growth in vivo, but the mechanisms are still unclear. Matrix metalloproteases (MMPs), a family of extracellular and membrane-associated endopeptidases, collectively digest almost all extracellular matrix and basement membrane components, and thus play an important role in tumor progression. We studied the effects of recombinant human endostatin on human MMP-2, -9, -8, and -13. We found that endostatin inhibited the activation and catalytic activity of pro-MMP-9 and -13 as well as recombinant pro-MMP-2. It prevented the fragmentation of pro-MMP-2 that was associated with reduction of catalytic activity. Endostatin had no effect on MMP-8 as shown by collagenase activity assays. An in vitro migration assay and an in vivo chicken chorioallantoic membrane intravasation assay with the human tongue squamous cell carcinoma cell line HSC-3 revealed the biphasic nature of endostatin; low endostatin concentrations inhibited intravasation and migration of these cells in a dose-dependent manner, but at increased concentrations, the inhibitory effect was far less efficient. The results show that endostatin blocks the activation and activities of certain tumor-associated pro-MMPs, such as pro-MMP-2, -9, and -13, which may explain, at least in part, the antitumor effect of endostatin. Our results also suggest that endostatin inhibits tumor progression by directly affecting the tumor cells and not just acting via endothelial cells and blockage of angiogenesis.     

Comparative chemotherapeutic efficacy of balhimycin, desgluco-balhimycin against experimental MSSA and MRSA infection in mice

Balhimycin and desglucobalhimycin are glycopeptide antibiotics isolated from an Amycolatopsis spp during the search for novel antibacterials against MRSA from the natural product screening at the Research Centre of formerly Hoechst India Ltd. in Bombay, India. Both compounds show excellent in vitro activity against methicillin sensitive and resistant Staphylococcus aureus (MSSA, MRSA). Both compounds were also found to be active against a number of MRSA strain in the animal studies. The activities were comparable to that of the reference glycopeptides vancomycin and teicoplanin used in these studies. Teicoplanin displayed better in vivo efficacy against S. epidermidis 4929H and Streptococcus pyogenes A77 than either vancomycin or desgluco-balhimycin in the present study. Preliminary studies on pharmacokinetic and acute toxicity were done to get some idea at the early stage of the investigation about the promise of the compounds for development.     

Investigation of articular cartilage surface morphology with a semiquantitative scanning electron microscopic method

A semiquantitative scanning electron microscopic method for analysis of the articular cartilage surface morphology was developed. The method was based on a survey of large picture montages (ca. 70 X 100 cm) and classification of the cartilage surface changes at three levels. Computer technique was utilized in the analysis. The method ensured numerical expression and statistical treatment of the results. With this method we investigated the effects of physical exercise and immobilization on the articular cartilage of rabbit patella.     

Are biological classifications of headwater streams concordant across multiple taxonomic groups?

1. Studies assessing human impacts on freshwater ecosystems are typically based on a single taxonomic group, often macroinvertebrates or fish. Unfortunately, the degree to which such macroinvertebrate or fish‐based surveys can be generalised across other taxonomic groups remains largely unknown. A prerequisite for useful generalisations is that different taxonomic groups exhibit concordant patterns of community structure across sites. 2. We examined the concordance among fish, benthic macroinvertebrates and bryophytes in 32 streams in a boreal catchment in Finland. Our goal was to test how consistently different taxonomic groups classify stream sites; for example, can site groupings based on macroinvertebrates be used as a surrogate for bryophyte or fish assemblage classification? 3. Our results show that community classifications in headwater streams are not concordant across taxonomic groups, at least not at the within‐river system scale. The lack of concordance reflected the fact that all three groups responded to different environmental factors. Macroinvertebrate community structure was mainly correlated with stream size and pH, whereas bryophytes were related to water colour, nutrient content and in‐stream habitat variability. Fish community structure was best described by stream depth, substrate size and water oxygen concentration. 4. Our results suggest that great care should be taken when typologies based on benthic macroinvertebrates, or any other taxonomic group, are extrapolated to other groups in creating typologies of lotic environments.     

Complex metacommunity structure for benthic invertebrates in a low‐diversity coastal system

The majority of studies in metacommunity ecology have focused on systems other than marine benthic ecosystems, thereby providing an impetus to broaden the focus of metacommunity research to comprise marine systems. These systems are more open than many other systems and may thus exhibit relatively less discrete patterns in community structure across space. Metacommunity structure of soft‐sediment benthic invertebrates was examined using a fine‐grained (285 sites) data set collected during one summer across a large spatial extent (1700 km²). We applied the elements of metacommunity structure (EMS) approach, allowing multiple hypothesis of variation in community structure to be tested. We demonstrated several patterns associated with environmental variation and associated processes that could simultaneously assemble species to occur at the sites. A quasi‐Clementsian pattern was observed frequently, suggesting interdependent ecological relationships among species or similar response to an underlying environmental gradient across sites. A quasi‐nested clumped species loss pattern was also observed, which suggests nested habitat specialization. Species richness declined with depth (from 0.5 to 44.8 m). We argue that sensitive species may survive in shallower water, which are more stable with regard to oxygen conditions and present greater habitat complexity, in contrast to deeper waters, which may experience periodic disturbance due to hypoxia. Future studies should better integrate disturbance in terms of temporal dynamics and dispersal rates in the EMS approach. We highlight that shallow water sites may act as sources of recruitment to deeper water sites that are relatively more prone to periodic disturbances due to hypoxia. However, these shallow sites are not currently monitored and should be better prioritized in future conservation strategies in marine systems.