A critical period for functional vestibular development in zebrafish.

We have determined a critical period for vestibular development in zebrafish by using a bioreactor designed by NASA to simulate microgravity for cells in culture. A critical period is defined as the briefest period of time during development when stimulus deprivation results in long lasting or permanent sensory deficits. Zebrafish eggs were collected within 3 hours of being laid and fertilized. In experiment 1, eggs were placed in the bioreactor at 3, 24, 30, 36, 48, or 72 hours postfertilization (hPF) and maintained in the bioreactor until 96 hPF. In experiment 2, eggs were placed in the bioreactor immediately after they were collected and maintained in the bioreactor until 24, 36, 48, 60, 66, 72, or 96 hPF. Beginning at 96 hPF, all larvae had their vestibulo-ocular reflexes (VOR) evaluated once each day for 5 days. Only larvae that hatched from eggs that were placed in the bioreactor before 30 hPF in experiment 1 or removed from the bioreactor later than 66 hPF in experiment 2 had VOR deficits that persisted for at least 5 days. These data suggest a critical period for vestibular development in the zebrafish that begins before 30 hPF and ends after 66 hPF. To confirm this, zebrafish eggs were placed in the bioreactor at 24 hPF and removed at 72 hPF. VORs were evaluated in these larvae once each day for 5 days beginning at 96 hPF. These larvae had VOR deficits that persisted for at least 5 days. In addition, larvae that had been maintained in the bioreactor from 24 to 66 hPF or from 30 to 72 hPF, had only temporary VOR deficits. In a final experiment, zebrafish eggs were placed in the bioreactor at 3 hPF and removed at 96 hPF but the bioreactor was turned off from 24 hPF to 72 hPF. These larvae had normal VORs when they were removed from the bioreactor at 96 hPF. Taken as a whole, these data support the idea that there is a critical period for functional maturation of the zebrafish vestibular system. The developmental period identified includes the timeframe during which the vestibular primary afferent neurons are born, innervate their central and peripheral targets, and remodel their central projections.     

Synthesis and release of 5-fluorouracil from poly(N-vinylpyrrolidinone) bearing 5-fluorouracil derivatives.

1-beta-allyloxycarbonyloxymethyl-5-fluorouracil (4) and 1,3-bis(beta-allyloxycarbonyloxymethyl)-5-fluorouracil (5) were synthesised by reacting 5-fluorouracil with formaldehyde followed by treating the product with isopropenyl chloroformate. The monomers 4 and 5 were copolymerized separately with N-vinylpyrrolidinone to form linear copolymers and cross-linked polymer networks, respectively. The monomer reactivity ratios in the copolymerization of 4 with NVP were evaluated by both linear and non-linear methods and the effect of monomer feed composition on copolymer molecular weight was examined. The degradation of the polymer networks in phosphate buffer (pH 7.4) was investigated. The hydrolytic scission of the carbonate groups resulted in release of 5-fluorouracil and a decrease in cross-linking density. The time-dependent fractional release of the 5-FU could be fitted by a power relationship with exponents between 0.10 and 0.25.     

Cellular and molecular basis of age-related sarcopenia.

Sarcopenia, the decline in muscle bulk and performance associated with normal aging, is an important component of frailty in the elderly. The gradual loss of both motor nerves and muscle fibers during senescence appears to be the major problem. Atrophy (especially in fast-twitch fibers) and impaired function of the surviving cells also contribute to sarcopenia. Although skeletal muscle has the capacity to regenerate itself, this process is not activated by the gradual age-related loss of muscle fibers. The endocrine, autocrine, and paracrine environment in old muscle is less supportive of protein synthesis, reinnervation of muscle fibers, and satellite cell activation, proliferation, and differentiation. Lifelong exposure of DNA to free radical damage results in accumulation of somatic mutations in nerves and muscle fibers. Reduced protein synthesis leads to atrophy, and slower fractional protein turnover contributes to longer retention of proteins that may have been damaged by free radicals. Many genes are differentially expressed in young and old muscle, but additional research is needed to determine which of these genes have a significant role in the pathogenesis or adaptation to sarcopenia.     

A new immune-competent animal model of mucosally derived squamous cell carcinoma.

OBJECTIVES: To develop an immune-competent animal model for mucosally derived squamous cell carcinoma (SCCA). STUDY DESIGN: Fifteen Fischer 344 rats were inoculated with 1, 2, 5, 10, or 20 x 10(6) FAT7 cells in their flanks. The animals were observed for tumor growth and metastasis. RESULTS: All animals developed tumors that grew exponentially. Pulmonary metastases developed in all animals and 13% developed lymph node metastases. CONCLUSION: The FAT7 flank tumor in Fischer 344 rats is a new animal model that closely resembles the behavior of human mucosal head and neck cancer. SIGNIFICANCE: The existence of an immune-competent, mucosally derived, and reliable animal model of SCCA that somewhat resembles human head and neck SCCA gives the opportunity to perform immune-modulating experiments on head and neck cancer in these animals. EBM rating: B-3.     

Crosslinking of fibrinogen and fibronectin by free radicals: a possible initial step in adhesion formation in osteoarthritis of the temporomandibular joint.

PURPOSE: Adhesion formation in osteoarthritis (OA) of the temporomandibular joint (TMJ) typically results in a sustained limitation of joint movement. We propose the hypothesis that free-radical-mediated crosslinking of proteins underlies this adhesion formation in affected joints. Free radicals may cause oxidative modification of proteins, creating an opportunity for the formation of intramolecular and intermolecular crosslinks via covalent bonds. This may stabilize protein aggregates, rendering them more resistant to degradation. In this study, the free-radical-mediated crosslinking of model proteins (fibrinogen and fibronectin) was investigated to test our hypothesis that free radicals contribute to adhesion formation via this mechanism in OA of the TMJ. MATERIALS AND METHODS: Physiological clot formation of fibrinogen by thrombin and free-radical-induced crosslinking of fibrinogen and of fibronectin were analyzed using spectrophotometric turbidity measurements, light-scattering techniques, polyacrylamide gel electrophoresis (PAGE), and rotary shadowing. RESULTS: Fibrinogen was shown to aggregate after free radical treatment, as detected using turbidity measurements and light-scattering techniques. Using PAGE, fibrinogen as well as fibronectin was shown to degrade under low oxidative stress. Under high oxidative stress, however, fragments from both proteins were found to be covalently crosslinked, resulting in high-molecular-weight protein aggregates. The aggregation was shown to be at random with rotary shadowing. CONCLUSION: The study shows that high oxidative stress contributes to the formation of crosslinked proteins that may serve as an initial scaffolding for the development of adhesions frequently seen in OA of the TMJ.