Polyanion-induced self-association of complement factor H.

Factor H is the primary soluble regulator of activation of the alternative pathway of complement. It prevents activation of complement on host cells and tissues upon association with C3b and surface polyanions such as sialic acids, heparin, and other glycosaminoglycans. Here we show that interaction with polyanions causes self-association forming tetramers of the 155,000 Da glycosylated protein. Monomeric human factor H is an extended flexible protein that exhibits an apparent size of 330,000 Da, relative to globular standards, during gel filtration chromatography in the absence of polyanions. In the presence of dextran sulfate (5000 Da) or heparin an intermediate species of apparent m.w. 700,000 and a limit species of m.w. 1,400,000 were observed by gel filtration. Sedimentation equilibrium analysis by analytical ultracentrifugation indicated a monomer Mr of 163,000 in the absence of polyanions and a Mr of 607,000, corresponding to a tetramer, in the presence of less than a 2-fold molar excess of dextran sulfate. Increasing concentrations of dextran sulfate increased binding of factor H to zymosan-C3b 4.5-fold. This result was accompanied by an increase in both the decay accelerating and cofactor activity of factor H on these cells. An expressed fragment encompassing the C-terminal polyanion binding site (complement control protein domains 18-20) also exhibited polyanion-induced self-association, suggesting that the C-terminal ends of factor H mediate self-association. The results suggest that recognition of polyanionic markers on host cells and tissues by factor H, and the resulting regulation of complement activation, may involve formation of dimers and tetramers of factor H.

Enzyme kinetics and characterization of mouse pancreatic elastase.

In the present study we have purified and characterized murine pancreatic elastase. The enzyme was extracted from acetone powders of mouse pancreas, fractionally precipitated with ammonium sulfate, and further purified by ion exchange chromatography to a single band on SDS-PAGE. The mouse enzyme exists in a proform, which was activated by removing a signal peptide by tryptic cleavage. The active form of mouse pancreatic elastase was shown by ultracentrifugation to have a molecular weight of 25.9 kDa and a frictional ratio of 1.26. The pH optimum for proteolytic activity was 8.0. Kinetic measurements were made with a variety of substrates and inhibitors and compared with elastases from other sources. The enzymatic properties and kinetic profiles for mouse pancreatic elastase were similar to other known serine elastases.

Measurements of asbestos burden in tissues.

Asbestos inhaled into the lung is recognized as a potential causal agent for the development of diseases in man. The diseases induced by asbestos include lung cancer, fibrosis of the lung (asbestosis), and extrapulmonary tumors including mesothelioma (a tumor of the serosal membrane), as well as fibrosis and other changes in the pleura linings. The cause of these diseases can often be more specifically linked to asbestos exposure once tissue burden of asbestos is established. The asbestos burden in tissue can be defined as the number of asbestos bodies and/or the numbers and types of asbestos fibers found in the tissue. In either of these cases the quality of information is directly dependent on the preparative techniques and instrumentation used in the analysis. The present article will discuss the significance of findings of tissue burden based on both these variables.

Stability of ferruginous bodies in human lung tissue following death, embalmment, and burial.

The identification of asbestos bodies in tissue sections is an indicator of past exposure to longer asbestos fibers. These structures are formed in lung tissue as a consequence of interactions with pulmonary macrophages resulting in the deposition of a ferroprotein (ferruginous) coating on the fiber. While the process of ferruginous body formation is known to take months in animal tissue, there is no published information on the stability of ferruginous bodies in tissue following death. The material assessed in the present study was obtained from lung material collected from an exhumed body approximately 8(1/2) mo after death, embalmment, and burial. Tissue sections were reviewed for the presence of asbestos bodies. Additional pieces of lung tissue were digested, with the digestate being evaluated by light microscopy for ferruginous bodies and by electron microscopy for uncoated asbestos fibers and core analysis of asbestos bodies. Classical ferruginous (asbestos) bodies were found in abundance in the tissue sections including in areas with fibrosis. The levels of uncoated asbestos fibers and classical appearing ferruginous bodies (asbestos bodies) were consistent with occupational levels of tissue burden. The data from this study indicate that ferruginous bodies remain morphologically stable within the tissue for months following death, embalmment, and burial. Thus the lung tissue from this exhumed individual was usable not only to pathologically confirm asbestosis but also to provide quantitative data of occupational exposure to asbestos.

Mutations in the GTP-binding and synergy loop domains of Mycobacterium tuberculosis ftsZ compromise its function in vitro and in vivo.

The Mycobacterium tuberculosis FtsZ (FtsZ(TB)), unlike other eubacterial FtsZ proteins, shows slow GTP-dependent polymerization and weak GTP hydrolysis activities [E.L. White, L.J. Ross, R.C. Reynolds, L.E. Seitz, G.D. Moore, D.W. Borhani, Slow polymerization of Mycobacterium tuberculosis FtsZ, J. Bacteriol. 182 (2000) 4028-4034]. In an attempt to understand the biological significance of these findings, we created mutations in the GTP-binding (FtsZ(G103S)) and GTP hydrolysis (FtsZ(D210G)) domains of FtsZ and characterized the activities of the mutant proteins in vitro and in vivo. We show that FtsZ(G103S) is defective for binding to GTP and polymerization activities, and exhibited reduced GTPase activity whereas FtsZ(D210G) protein is proficient in binding to GTP, showing reduced polymerization activity but did not show any measurable GTPase activity. Visualization of FtsZ-GFP structures in ftsZ merodiploid strains by fluorescent microscopy revealed that FtsZ(D210G) is proficient in associating with Z-ring structures whereas FtsZ(G103S) is not. Finally, we show that Mycobacterium smegmatis ftsZ mutant strains producing corresponding mutant FtsZ proteins are non-viable indicating that mutant FtsZ proteins cannot function as the sole source for FtsZ, a result distinctly different from that reported for Escherichia coli. Together, our results indicate that optimal GTPase and polymerization activities of FtsZ are required to sustain cell division in mycobacteria and that the same conserved mutations in different bacterial species have distinct phenotypes.

Evaluation of the size and type of free particulates collected from unused asbestos-containing brake components as related to potential for respirability.

BACKGROUND: Chrysotile asbestos has found multiple applications in the production of friction products. At one point it comprised 40-50% of the composition of brake linings thus generating the potential for the development of asbestos related diseases in millions of workers involved in vehicle repairs. While some attention has been given to the health status of workers involved in the handling of worn components, little has been given to the potential for exposure during the handling and fitting of new (unused) components as replacement parts. METHODS: Unused brake linings or brake shoes with attached linings from four different sources were gently rinsed with prefiltered water that was then collected on filters for analysis by Analytical Transmission Electron Microscope. RESULTS: Large numbers of chrysotile asbestos containing structures, the majority of respirable size, were present in each sample. CONCLUSIONS: We conclude that any manipulation of new asbestos containing brake components would be expected to yield free dust containing chrysotile asbestos of respirable size. That the vast majority of these fibers and particulates would not be counted as regulated fibers rendering assessment of potential exposures in the work place, based on data of Permissible Exposure Limits or Regulated Fibers, questionable.

Asbestos fiber length as related to potential pathogenicity: a critical review.

BACKGROUND: Asbestos inhalation is recognized as an exposure that increases the risk for the development of lung disease. It is unique among dusts in that it is both a carcinogen and capable of inducing extrapulmonary responses such as pleural thickening and fibrosis as well as malignancy. One feature of asbestos suggested as crucial in its pathological activity is its fibrous morphology. Long fibers that have been inhaled are cleared less readily and are thus more persistent in the body. Furthermore certain experimental models link fiber length to levels of risks for development of certain diseases. The present review will survey the data on this subject. METHODS: The review considers experimental models that have been used to assess the response to various lengths of fibers in animal models in addition to data obtained from studies of human materials. The review also emphasizes the importance in defining the method by which a sample is categorized. RESULTS: Data are offered which support the potential for longer fibers as well as shorter fibers to contribute to pathological responses. CONCLUSIONS: The data presented argue that asbestos fibers of all lengths induce pathological responses and that caution should be exerted when attempting to exclude any population of inhaled fibers, based on their length, from being contributors to the potential for development of asbestos-related diseases.

Conditional expression of Mycobacterium smegmatis ftsZ, an essential cell division gene.

To understand the role of Mycobacterium smegmatis ftsZ (ftsZ(smeg)) in the cell division process, the ftsZ gene was characterized at the genetic level. This study shows that ftsZ(smeg) is an essential gene in that it can only be disrupted in a merodiploid background carrying another functional copy. Expression of ftsZ(smeg) in M. smegmatis from a constitutively active mycobacterial promoter resulted in lethality whereas that from a chemically inducible acetamidase (ami) promoter led to FtsZ accumulation, filamentation and cell lysis. To further understand the roles of ftsZ in cell division a conditionally complementing ftsZ(smeg) mutant strain was constructed in which ftsZ expression is controlled by acetamide. Growth in the presence of 0.2 % acetamide increased FtsZ levels approximately 1.4-fold, but did not decrease viability or change cell length. Withdrawal of acetamide reduced FtsZ levels, decreased viability, increased cell length and eventually lysed the cells. Finally, it is shown that ftsZ(smeg) function in M. smegmatis can be replaced with the Mycobacterium tuberculosis counterpart, indicating that heterologous FtsZ(tb) can independently initiate the formation of Z-rings and catalyse the septation process. It is concluded that optimal levels of M. smegmatis FtsZ are required to sustain cell division and that the cell division initiation mechanisms are similar in mycobacteria.

Physiological consequences associated with overproduction of Mycobacterium tuberculosis FtsZ in mycobacterial hosts.

The ftsZ gene of Mycobacterium tuberculosis H37Rv has been characterized as the first step in determining the molecular events involved in the cell division process in mycobacteria. Western analysis revealed that intracellular levels of FtsZ are growth phase dependent in both M. tuberculosis and Mycobacterium smegmatis. Unregulated expression of M. tuberculosis ftsZ from constitutive hsp60 and dnaA promoters in M. tuberculosis hosts resulted in lethality whereas expression from only the hsp60 promoter was toxic in M. smegmatis hosts. Expression of ftsZ from the dnaA promoter in M. smegmatis resulted in approximately sixfold overproduction and the merodiploids exhibited slow growth, an increased tendency to clump and filament, and in some cases produced buds and branches. Many of the cells also contained abnormal and multiple septa. Expression of ftsZ from the chemically inducible acetamidase promoter in M. smegmatis hosts resulted in approximately 22-fold overproduction of FtsZ and produced filamentous cells, many of which lacked any visible septa. Visualization of the M. tuberculosis FtsZ tagged with green fluorescent protein in M. smegmatis by fluorescence microscopy revealed multiple fluorescent FtsZ foci, suggesting that steps subsequent to the formation of organized FtsZ structures but prior to septum formation are blocked in FtsZ-overproducing cells. Together these results suggest that the intracellular concentration of FtsZ protein is critical for productive septum formation in mycobacteria.

Purification, characterization and cloning of tensilin, the collagen-fibril binding and tissue-stiffening factor from Cucumaria frondosa dermis.

The inner dermis of the sea cucumber, Cucumaria frondosa, is a mutable collagenous tissue characterized by rapid and reversible changes in its mechanical properties regulated by one or more protein effectors that are released from neurosecretory cells. One such effector, tensilin, is a collagen-fibril binding protein, named for its ability to induce dermis stiffening. Tensilin was purified using an affinity column constructed from C. frondosa collagen-fibrils. The protein migrates as a single band on SDS-PAGE (Mr approximately 33 kDa) and has an isoelectric point of 5.8. Equilibrium sedimentation experiments suggest a molecular mass of approximately 28.5-29.4 kDa. Carbohydrate analysis of tensilin revealed no measurable sugar content. The molar amount of tensilin was determined to be 0.38% that of collagen and 47% that of stiparin, a constitutive matrix glycoprotein. A full-length cDNA clone for tensilin was obtained from a C. frondosa inner dermis cDNA expression library. Predicted properties derived from the deduced peptide sequence were in agreement with those of the native protein. A noted feature of tensilin's deduced peptide sequence, particularly in its N-terminal domain, is its homology to tissue inhibitor of metalloproteinases. Tensilin's C-terminal tail has no known homology to other proteins but contains a putative collagen-fibril binding site.