Vibrio damsela infection in a stranded leatherback turtle (Dermochelys coriacea)

Necropsy of a stranded adult leatherback turtle (Dermochelys coriacea) determined that the animal died as a result of valvular endocarditis and septicemia. Vibrio damsela was isolated from the endocardial thrombus. The route of entry for infection probably was through the gastrointestinal tract.     

Culture of human corpus cavernosum endothelium

Summary A method for culturing endothelial cells (HCC-EC) from surgical specimens of human corpus cavernosum has been developed. The approach involves selective endothelial outgrowth from explants and may be generally applicable to tissue whose endothelium is not amenable to isolation by routine mechanical or enzymatic methods. The tissue is minced into pieces which are placed onto gelatin-or fibronectin-coated tissue culture plastic, and grown in medium suitable for microvascular endothelial cell growth (Carson and Haudenschild, In Vitro 22:344–354, 1986). By Days 5 to 7 EC colonies are found. Within a day or two after the appearance of the EC colonies, a non-EC cell type appears and, if undisturbed, quickly overgrows the EC. An exploitable temporal separation between the emergence of EC and non-EC is obtained when both conditioned medium (from bovine aortic endothelium) and retinal extract are present during the outgrowth period. Explants are removed by pipetting at the first sign of the emergence of the non-EC cell type. Once isolated, HCC-EC do not require conditioned medium but do require either retinal extract or acidic fibroblast growth factor for survival and growth. Approximately 60% of the first passage cultures are at least 80% EC as judged by DiI-Ac-LDL labeling. One corpus (0.3×0.3×0.5 cm) usually produces 120 cm² of primary culture within 2 wk. These EC form contact-inhibited monolayers and stain positively for Factor VIII. They have a doubling time at 6th passage of 48 h and a plateau density of 5 to 7×10⁴ cells/cm². The availability of such cultures should facilitate the study of endothelium-mediated responses which play an important role in the erectile function of human penile corpus cavernosum. Supported by NIH R01 HL23567-09, DK-39080-01, DK40025-01, DK40487-01.     

When the tail can't wag the dog: the implications of CNS-intrinsic initiation of neuroinflammation

The CNS (central nervous system) is unquestionably the central organ that regulates directly or indirectly all physiological systems in the mammalian body. Yet, when considering the defence of the CNS from pathogens, the CNS has often been considered passive and subservient to the pro-inflammatory responses of the immune system. In this view, neuroinflammatory disorders are examples of when the tail (the immune system) wags the dog (the CNS) to the detriment of an individual''s function and survival.     

Simple and sensitive procedure for the assay of serotonin and catecholamines in brain by high-performance liquid chromatography using fluorescence detection

A simple, rapid and specific method for the determination of serotonin and catecholamines in brain is described. After tissue homogenisation, catecholamines are isolated by adsorption onto alumina and elution with perchloric acid. Serotonin is isolated by extraction into n-heptanol and back-extraction into acid. High-performance liquid chromatography of the acid extracts is performed with a C₁₈ reversed-phase column and simple mobile phases. Detection is by the intrinsic fluorescence of the amines on excitation at 200 nm. Detection limits are 100 pg for norepinephrine, 300 pg for dopamine and 20 pg for serotonin. The results are found to correlate well with a catechol O-methyl transferase radioenzymatic assay for catecholamines and a ninhydrin derivatisation procedure for serotonin.     

Biomarkers affected by impact velocity and maximum strain of cartilage during injury

Osteoarthritis is one of the most common, debilitating, musculoskeletal diseases; 12% associated with traumatic injury resulting in post-traumatic osteoarthritis (PTOA). Our objective was to develop a single impact model with cartilage “injury level” defined in terms of controlled combinations of strain rate to a maximum strain (both independent of cartilage load resistance) to study their sensitivity to articular cartilage cell viability and potential PTOA biomarkers. A servo-hydraulic test machine was used to measure canine humeral head cartilage explant thickness under repeatable pressure, then subject it (except sham and controls) to a single impact having controlled constant velocity V=1 or 100 mm/s (strain rate 1.82 or 182/s) to maximum strain ε=10%, 30%, or 50%. Thereafter, explants were cultured in media for twelve days, with media changed at day 1, 2, 3, 6, 9, 12. Explant thickness was measured at day 0 (pre-injury), 6 and 12 (post-injury). Cell viability, and tissue collagen and glycosaminoglycan (GAG) were analyzed immediately post-injury and day 12. Culture media were tested for biomarkers: GAG, collagen II, chondroitin sulfate-846, nitric oxide, and prostaglandin E₂ (PGE₂). Detrimental effects on cell viability, and release of GAG and PGE₂ to the media were primarily strain-dependent, (PGE₂ being more prolonged and sensitive at lower strains). The cartilage injury model appears to be useful (possibly superior) for investigating the relationship between impact severity of injury and the onset of PTOA, specifically for discovery of biomarkers to evaluate the risk of developing clinical PTOA, and to compare effective treatments for arthritis prevention.     

Large, rapidly evolving intergenic spacers in the mitochondrial DNA of the salamander family Ambystomatidae (Amphibia: Caudata)

We report the presence, in the mitochondrial DNA (mtDNA) of all of the sexual species of the salamander family Ambystomatidae, of a shared 240- bp intergenic spacer between tRNAThr and tRNAPro. We place the intergenic spacer in context by presenting the sequence of 1,746 bp of mtDNA from Ambystoma tigrinum tigrinum, describe the nucleotide composition of the intergenic spacer in all of the species of Ambystomatidae, and compare it to other coding and noncoding regions of Ambystoma and several other vertebrate mtDNAs. The nucleotide substitution rate of the intergenic spacer is approximately three times faster than the substitution rate of the control region, as shown by comparisons among six Ambystoma macrodactylum sequences and eight members of the Ambystoma tigrinum complex. We also found additional inserts within the intergenic spacers of five species that varied from 87-444 bp in length. The presence of the intergenic spacer in all sexual species of Ambystomatidae suggests that it arose at least 20 MYA and has been a stable component of the ambystomatid mtDNA ever since. As such, it represents one of the few examples of a large and persistent intergenic spacer in the mtDNA of any vertebrate clade.      

IgG rheumatoid factors isolated by the surface-displaying phage library technique

 Our analysis of IgG rheumatoid factors (RFs) from three patients with rheumatoid arthritis (RA) revealed that most contained significant numbers of skewed mutations per V region, suggesting that these RFs arose from antigen-driven responses. To further study IgG RFs in RA, we used pComb3 vector to construct an IgG1,λ combinatorial antibody library from a synovial fluid sample. After panning against human IgG, Fab fragments from 71/96 phage clones bound to Fc-coated wells. Sequence analysis of 20 randomly chosen Fc-binders showed that 17 (85%) clones had identical heavy (H) and light (L) chain V regions, represented by Humha311 and Humla211, respectively. Of the remaining three clones, two had the same Humla211 L chain, but each with a different H chain V region. All the putative germline V genes for these RFs also encode RF in RA patients. However, none of these RF V regions are similar to those of the two IgG RFs derived by the hybridoma technique from the same synovial fluid. The Humha311 H chain has two frameshifts: a one-base insertion upstream of the JH region and a four-base deletion near the end of the CH1 region, resulting in a mainly unconventional amino acid sequence in the CH1 region. In the future, it will be important to study the presence of IgG molecules with such unconventional CH1 amino acid sequences, and the effects of these amino acid sequences on the structures and immunological properties of the IgG molecules. Received: 4 September 1996 / Revised: 22 October 1996