Production and characterization of monoclonal antibodies to rabbit lymphocyte subpopulations. I. Tissue immunofluorescence and flow cytometric analysis

Nine monoclonal antibodies to rabbit T cells and B subpopulations have been generated from three separate fusions of spleen cells from mice immunized with fractionated populations of rabbit lymphocytes. These monoclonal antibodies, as well as a previously described rabbit T cell monoclonal antibody, 9AE10, have been analyzed by immunofluorescence staining on frozen tissue sections of rabbit thymus, spleen, and appendix. This screening method permits rapid identification of the lymphocyte subdomains in each tissue which is not possible by other screening methods. Each monoclonal antibody selected has a unique tissue staining pattern. Flow cytometric analysis of these monoclonal antibodies, using indirect immunofluorescence techniques on thymocytes, splenocytes, and PBL, revealed varying percentages of positive cells and individual mean fluorescence intensities indicating different epitope densities for each antigen. These monoclonal antibodies are now being used to characterize normal lymphocyte function and the role of specific lymphocyte subpopulations in experimental disease models in the rabbit.     

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.     

Cathepsin B stability,but not activity,is affected in cysteine:cystine redox buffers

In order to test the hypothesis that the lysosomal cysteine protease cathepsin B may be redox regulated in vivo, cathepsin B activity and stability were measured in cysteine- and/or cystine-containing buffers. Cathepsin B activity in cysteine-containing buffers was similar at pH 6.0 and pH 7.0, over all thiol concentrations tested. In contrast, the stability of the enzyme was greater at pH 6.0 than at pH 7.0. This suggests that the enzyme's operational pH in vivo may be < pH 7.0. The activity of the enzyme was depressed in glutathione-containing buffers. When assessed in cysteine:cystine redox buffers (pH 6.0-7.0) cathepsin B was active over a broad redox potential range, suggesting that cathepsin B activity may not be redox regulated. However, at pH 7.0, the stability of cathepsin B decreased with increasing reduction potential and ambient cystine concentration. This suggests that the stability of the enzyme at neutral pH is dependent on redox potential, and on the presence of oxidising agents.     

VSV transmembrane domain (TMD) peptide promotes PEG-mediated fusion of liposomes in a conformationally sensitive fashion

Helical instability induced by gly residues in the transmembrane domain (TMD) of G protein, the fusion protein of vesicular stomatitis virus (VSV), was speculated to aid in the later steps of the fusion process, because G protein with ala's substituted for the two TMD gly's was inactive (Cleverley, D. Z., and Lenard, J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3425-30). Here we examine the conformations of synthetic peptides corresponding to fusion-active (GGpep) and inactive (AApep; G's replaced by A's) TMDs by CD spectroscopy, and then their effects on the kinetics of poly (ethyleneglycol) (PEG)-mediated fusion of small unilamellar vesicles. GGpep and AApep both assumed history-dependent, non-interconvertible ordered structures. Both peptides were largely helical under all conditions if derived from trifluoroethanol solutions, and aggregated in a beta-sheet form if derived from acetonitrile solutions. In solvent, detergents or lipid bilayers, GGpep showed a greater range of secondary structural features than did AApep. The two peptides had large but different effects on PEG-mediated fusion. Both enhanced the rate but not the extent of lipid mixing. AApep significantly inhibited the extent of fusion pore formation while GGpep had no effect. The initial rate of fusion was enhanced 6-fold by GGpep and less than 2-fold by AApep. Addition of 5 mol % hexadecane overrode all peptide-induced effects. We suggest that both GGpep and hexadecane promote pore formation by stabilizing the nonlamellar structures in fusion intermediates or initial small pores. AApep, which had fewer nonhelical features in its CD spectrum than GGpep, actually inhibited fusion pore formation.     

Factors determining the efficacy of alpha-helical antimicrobial peptides

A database of alpha-helical antimicrobial peptides (AMP) was established and their minimum inhibitory concentrations (MIC) were compared with their physiochemical characteristics in an attempt to establish those features that determine efficacy. There is no significant difference in AMP sensitivity between Gram-positive and Gram-negative bacteria but fungi did require higher concentrations to achieve the same degree of growth inhibition. For antibacterial peptides there appears to be a positive correlation between MIC and hydrophobic arc size and a negative correlation between MIC and net charge.     

Epidemiology of osteoporotic fractures

Osteoporosis constitutes a major public health problem through its association with age-related fractures. These fractures typically occur at the hip, spine and distal forearm. It has been estimated that the lifetime risk of a hip fracture in white women is 17.5%, with a comparable risk in men of 6%. Hip fractures lead to an overall reduction in survival of about 15% (relative or observed/expected survival at 5 years of 0.83), and the majority of excess deaths occur within the first 6 months following the fracture. Such fractures are also associated with considerable morbidity. Although all vertebral deformities do not come to clinical attention, the lifetime risk of clinically diagnosed vertebral fractures is about 15% in white women. Vertebral fractures tend to be associated with back pain and kyphosis, and also with an impairment of survival, though this is likely to be due to clustering of comorbidity. About one-quarter of clinically diagnosed vertebral deformities result in hospitalization.     

Seagrasses of south–west Australia: A conceptual synthesis of the world's most diverse and extensive seagrass meadows

South–west Australia contains extensive seagrass meadows along 2,500 km of coastline from the shallow subtidal to 50+ m water depths, and in many of the 51 bar-built estuaries along the coast. There are geomorphological differences between the south and west coasts that result in different patterns of swell exposure influencing the processes that structure seagrass habitats. In this paper, ‘sheltered’, ‘exposed’ and ‘estuarine’ seagrass habitat types are defined for south–west Australia to synthesize processes influencing seagrass communities. Sheltered habitats in south–west Australia are characterized by high light, low to moderate water motion and sporadic disturbance from storms, making them ideal habitats for a diversity of seagrass assemblages. Exposed seagrass habitats are characterized by the presence of strong and consistent ocean swells (3–8 m), predominantly from the south or south–west and seagrasses exhibit a suite of adaptive traits to survive the effects of exposure to ocean swell and associated sand movement. These include morphological features such as heavy fiber reinforcement to strengthen the aboveground stems or leaves, deep vertical rhizomes and life history traits such as rapid growth and high seed set. Within estuarine habitats highly dynamic seagrass communities are the result of fluctuating annual cycles in temperature, light and salinity. Compared to global seagrass meadows, coastal south–west Australian seagrass habitats experience high light, low nutrients and high water movement. Despite these differences, similarities with other regions do exist and here we place the habitats of south–west Australia into a global context using comparative data. The wide array of morphology and life history traits displayed among seagrass species of south–west Australia are presented in a conceptual model including habitat type, physical stressors and seagrass responses. The combination of adaptations to the habitats and processes that define them make south–west Australia a region where there is an unusually high number of co-occurring seagrass species, the highest in the world for a temperate environment (19 species), and approaching the species diversity of many tropical environments. Linking aspects of seagrass habitat, physical aspects of the environment and seagrass life history provides a context for applying knowledge gained from seagrasses in south–west Australia to other coastal ecosystems throughout the world.