Hypertrophy and physiological death of equine chondrocytes in vitro

REASON FOR PERFORMING STUDY: Equine osteochondrosis results from a failure of endochondral ossification during skeletal growth. Endochondral ossification involves chondrocyte proliferation, hypertrophy and death. Until recently no culture system was available to study these processes in equine chondrocytes. OBJECTIVE: To optimise an in vitro model in which equine chondrocytes can be induced to undergo hypertrophy and physiological death as seen in vivo. METHODS: Chondrocytes isolated from fetal or older (neonatal, growing and mature) horses were cultured as pellets in 10% fetal calf serum (FCS) or 10% horse serum (HS). The pellets were examined by light and electron microscopy. Total RNA was extracted from the pellets, and quantitative PCR carried out to investigate changes in expression of a number of genes regulating endochondral ossification. RESULTS: Chondrocytes from fetal foals, grown as pellets, underwent hypertrophy and died by a process morphologically similar to that seen in vivo. Chondrocytes from horses age >5 months did not undergo hypertrophy in pellet culture. They formed intramembranous inclusion bodies and the cultures included cells of osteoblastic appearance. Pellets from neonatal foals cultured in FCS resembled pellets from older horses, however pellets grown in HS underwent hypertrophy but contained inclusion bodies. Chondrocytes from fetal foals formed a typical cartilage-like tissue grossly and histologically, and expressed the cartilage markers collagen type II and aggrecan mRNA. Expression of Sox9, collagen type II, Runx2, matrix metalloproteinase-13 and connective tissue growth factor mRNA increased at different times in culture. Expression of fibroblast growth factor receptor-3 and vascular endothelial growth factor mRNA decreased with time in culture. CONCLUSIONS: Freshly isolated cells from fetal growth cartilage cultured as pellets provide optimal conditions for studying hypertrophy and death of equine chondrocytes. POTENTIAL RELEVANCE: This culture system should greatly assist laboratory studies aimed at elucidating the pathogenesis of osteochondrosis.     

Primitive neuroectodermal tumor in the spinal cord of a brahman crossbred calf

A variety of embryonal tumors of the central nervous system, typically malignant and occurring in young individuals, are recognized in humans and animals. This report describes an invasive subdural but predominantly extramedullary primitive neuroectodermal tumor developing at the lumbosacral junction in a 6-month-old Brahman crossbred calf. The tumor was composed of spindloid embryonal cells organized in interlacing fascicles. The cells had oval to elongate or round hyperchromic nuclei, single to double nucleoli, and scant discernible cytoplasm. Immunohistochemical staining for neuron-specific enolase, synaptophysin, and S-100 protein and formation of pseudorosettes suggested neuronal and possibly ependymal differentiation.     

Granulomatous lymphadenitis and splenitis associated with Monocillium indicum infection in a dog.

This report describes severe generalized granulomatous lymphadenitis and splenitis in a 5-year-old, spayed female, Rottweiler dog with anorexia and diarrhea. There was replacement or effacement of much of the parenchyma of the lymph nodes and spleen by sheets of macrophages, multinucleated giant cells, and myriad nonpigmented fungal organisms, most of which appeared to be intracellular. These organisms were very pleomorphic, including large chlamydospore-like cells, small round yeast-like cells, and septate hyphae. A fungus identified as Monocillium indicum was isolated from lymph node tissue. To the authors' knowledge, this is the first report of infection with Monocillium in either humans or other animals.     

Metagenomic discovery of biomass-degrading genes and genomes from cow rumen

The paucity of enzymes that efficiently deconstruct plant polysaccharides represents a major bottleneck for industrial-scale conversion of cellulosic biomass into biofuels. Cow rumen microbes specialize in degradation of cellulosic plant material, but most members of this complex community resist cultivation. To characterize biomass-degrading genes and genomes, we sequenced and analyzed 268 gigabases of metagenomic DNA from microbes adherent to plant fiber incubated in cow rumen. From these data, we identified 27,755 putative carbohydrate-active genes and expressed 90 candidate proteins, of which 57% were enzymatically active against cellulosic substrates. We also assembled 15 uncultured microbial genomes, which were validated by complementary methods including single-cell genome sequencing. These data sets provide a substantially expanded catalog of genes and genomes participating in the deconstruction of cellulosic biomass.     

Identification of fish species after cooking by SDS-PAGE and urea IEF: a collaborative study

A collaborative study, to validate the use of SDS-PAGE and urea IEF, for the identification of fish species after cooking has been performed by nine laboratories. By following optimized standard operation procedures, 10 commercially important species (Atlantic salmon, sea trout, rainbow trout, turbot, Alaska pollock, pollack, pink salmon, Arctic char, chum salmon, and New Zealand hake) had to be identified by comparison with 22 reference samples. Some differences in the recoveries of proteins from cooked fish flesh were noted between the urea and the SDS extraction procedures used. Generally, the urea extraction procedure appears to be less efficient than the SDS extraction for protein solubilization. Except for some species belonging to the Salmonidae family (Salmo, Oncorhynchus), both of the analytical techniques tested (urea IEF, SDS-PAGE) enabled identification of the species of the samples to be established. With urea IEF, two laboratories could not differentiate Salmo salar from Salmo trutta. The same difficulties were noted for differentiation between Oncorhynchus gorbuscha and Oncorhynchus keta samples. With SDS-PAGE, three laboratories had some difficulties in identifying the S. trutta samples. However, in the contrast with the previous technique, SDS-PAGE allows the characterization of most of the Oncorhynchus species tested. Only Oncorhynchus mykiss was not clearly recognized by one laboratory. Therefore, SDS-PAGE (Excel gel homogeneous 15%) appears to be better for the identification, after cooking, of fish such as the tuna and salmon species which are characterized by neutral and basic protein bands, and urea IEF (CleanGel) is better for the gadoid species, which are characterized by acid protein bands (parvalbumins). Nevertheless, in contentious cases it is preferable to use both analytical methods.     

Fecal microbiota of calves in the clinical setting: effect of penicillin treatment

The effect of parenteral penicillin treatment on the intestinal microbiota was determined by monitoring the phenotypic antimicrobial resistance among Escherichia coli in 19 calves (15 calves received treatment and four calves were healthy controls) and by examining changes in the fecal microbial community structure using molecular fingerprinting techniques in a subset of eight calves (five treated calves and three control calves). After five days of penicillin treatment an increased resistance to multiple unrelated antimicrobial agents, including non-β-lactams, was seen in E. coli from treated calves, and this was not seen in the controls. Automated ribosomal intergenic spacer analysis (ARISA) and terminal restriction fragment length polymorphism (TRFLP) revealed that penicillin treatment causes a significant variation in the microbial structure within an individual calf. The study shows that parenteral administration of penicillin has an impact on the composition of the fecal microbiota in calves, and on the antimicrobial resistance pattern of their fecal E. coli.