Role of the mitogen-activated protein kinase pathway in the differential response of bovine monocytes to Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium

We compared the kinetics of activation and antimicrobial activities of MAPK-p38 and MAPK-ERK in bovine monocytes infected with Mycobacterium avium subsp. paratuberculosis (MAP) and Mycobacterium avium subsp. avium (Maa). Monocytes were incubated with MAP or Maa organisms with or without a specific inhibitor of the MAPK-p38 pathway (SB203580), and MAPK phosphorylation and antimicrobial functions of monocytes were evaluated. At early time points MAPK-p38 phosphorylation was greater in MAP-infected bovine monocytes than in Maa-infected monocytes. At later time points MAPK-p38 phosphorylation by both organisms was similar. MAPKp38 phosphorylation in MAP-infected monocytes was similar to negative control cells, whereas in Maa-infected this activation remained greater than negative control cells. Increase phosphorylation MAPK-ERK was similar at all time points for both organisms. Bovine monocytes had minimal capacity to kill MAP organisms, to acidify MAP-containing phagosomes, or to form phagolysosome. Alternatively, bovine monocytes were able to kill Maa organisms. Addition of SB203580 to monocyte cultures increased phagosome acidification, phagolysosome formation, and killing of MAP and Maa organisms. Taken together these data indicate that early transient activation of MAPK-p38 in bovine mononuclear phagocytes by MAP organisms may be a key mechanism involved in the capacity of MAP to survive in bovine monocytes.     

Differences in the physical properties of collagenases isolated from rheumatoid synovium and human skin

During purification of human collagenase from normal skin and rheumatoid synovium differences have been observed with regard to the size and charge properties of the two enzymes. Gel filtration experiments in Sephadex G-100 superfine have allowed molecular weights of approximately 63,000 and 32,000 daltons to be calculated for the skin and rheumatoid synovial enzyme respectively. Ion exchange chromatography using Sephadex QAE, A-50 has shown the rheumatoid synovial enzyme to possess charge properties more basic than that of the skin enzyme. Elution of collagenase activity from $\text{7}\text{1}\text{2}\text{\%}$ polyacrylamide gels has produced no evidence for a ‘fast-moving’ component of either enzyme.     

Cloning, expression, purification, and characterization of the acid alpha-mannosidase from Trypanosoma cruzi

The acid alpha-mannosidase of Trypanosoma cruzi is a broad-specificity hydrolase involved in the catabolism of glycoconjugates, presumably in the digestive vacuole. We have cloned the alpha-mannosidase gene from a T.cruzi epimastigote genomic library. The alpha-mannosidase gene was determined to be single copy by Southern analysis, and similar sequences were not detected in genomic digests of either Trypanosoma brucei or Leishmania donovani. The coding region was subcloned into the Pichia pastoris expression vector pPICZ, and alpha-mannosidase activity was detected in the medium of induced cultures. The recombinant alpha- mannosidase demonstrated a pH optimum, inhibition by swainsonine, Km, and substrate specificity consistent with the characteristics of the alpha-mannosidase previously purified from T.cruzi epimastigotes. The recombinant enzyme was purified 103-fold from the culture medium of Pichia pastoris and had a native molecular mass of 359 kDa by gel filtration. A combination of SDS-PAGE, deglycosylation with endo H, and NH2-terminal sequencing indicates that the enzyme is originally synthesized as a homodimeric polypeptide that is subsequently cleaved to form a heterotetramer composed of 57 and 46 kDa subunits. A polyclonal antibody raised to the recombinant enzyme was shown to immunoprecipitate the alpha-mannosidase from T.cruzi cell extracts and will be used in future immunolocalization studies.      

Effects of age,replicative lifespan and growth rate of human nucleus pulposus cells on selecting age range for cell-based biological therapies for degenerative disc diseases

Autologous disc cell implantation, growth factors and gene therapy appear to be promising therapies for disc regeneration. Unfortunately, the replicative lifespan and growth kinetics of human nucleus pulposus (NP) cells related to host age are unclear. We investigated the potential relations among age, replicative lifespan and growth rate of NP cells, and determined the age range that is suitable for cell-based biological therapies for degenerative disc diseases. We used NP tissues classified by decade into five age groups: 30s, 40s, 50s, 60s and 70s. The mean cumulative population doubling level (PDL) and population doubling rate (PDR) of NP cells were assessed by decade. We also investigated correlations between cumulative PDL and age, and between PDR and age. The mean cumulative PDL and PDR decreased significantly in patients in their 60s. The mean cumulative PDL and PDR in the younger groups (30s, 40s and 50s) were significantly higher than those in the older groups (60s and 70s). There also were significant negative correlations between cumulative PDL and age, and between PDR and age. We found that the replicative lifespan and growth rate of human NP cells decreased with age. The replicative potential of NP cells decreased significantly in patients 60 years old and older. Young individuals less than 60 years old may be suitable candidates for NP cell-based biological therapies for treating degenerative disc diseases.     

Combined effects of an anthropogenic (forest harvesting) and natural (extreme rainfall event) disturbance on headwater streams in New Zealand

1. Although extreme hydrological events are a natural component of river ecosystem disturbance regimes, their frequency is predicted to increase with climate change. Anthropogenic activities have the potential to exacerbate the impact of such disturbances but there are few studies on the combined effects of both anthropogenic and extreme hydrological disturbances on stream ecosystems.
2. We investigated the recovery of stream ecosystems over a 5-year period following the impact of an anthropogenic (forest clear-cut harvesting) and an extreme rainfall disturbance (estimated one-in-100 year average return interval) that generated debris flows in three headwater streams in New Zealand.
3. Initially, most of the riparian vegetation was eliminated and showed little recovery 1 year later. Subsequent riparian recovery was led by wind-borne, light-demanding, pioneering exotic weed species, lengthening and altering the long-term successional and recovery trajectories to a pre-disturbance composition of indigenous shrubs.
4. Stream shade, water temperature, and habitat had largely recovered after 5 years. However, the contribution of large wood to channel morphology and in-stream habitat was compromised due to diminished wood supplies in the stream channel and a hiatus in up-slope wood inputs until the riparian vegetation re-establishes and the next crop of trees matures.
5. After an initial decline, most indigenous fish taxa thrived in the post-disturbance conditions, with significant increases in densities and biomass. The more sensitive fish taxa were scarce or absent, particularly those taxa that prefer pools with overhead and in-stream cover provided by riparian vegetation and wood. Recovery of these taxa was outside the time frame of this study. Riffle dwelling fish communities were more resilient than pool dwelling fish communities.
6. Invertebrate densities showed a similar response to fish. Post-event invertebrate community composition differed from that typically found in post-harvest headwater streams, comprising comparatively lower proportions of Chironomidae, Oligochaetes, and Mollusca taxa, and higher proportions of Trichoptera taxa. Progression toward pre-event community composition was evident 5 years after the event.
7. The compounding effect of forest removal from harvesting, along with riparian vegetation and wood removal by debris flows, lengthened the recovery of riparian vegetation and wood supplies with cascading effects on in-stream habitat and biological communities.

     

Ultrastructural features of ethanol-induced cardiomyopathy in turkey poults

Alcoholic cardiomyopathy, characterized by cardiac hypertrophy, was induced in young turkey poults with 5% ethanol. Ultrastructural features included accumulation of glycogen, swollen mitochondria, myofibrillar lysis, increased number of lysosomes, dilated sarcoplasmic reticulum and dense myofibers. Similarity of these alterations to those described in human alcoholic cardiomyopathy confirms the usefulness of the turkey poult as an animal model for this disease syndrome.