Effect of chilling and cryopreservation on expression of Pax genes in zebrafish (Danio rerio) embryos and blastomeres

Cryopreservation is now common practice in the fields of aquaculture, conservation and biomedicine. However, there is a lack of information on the effect of chilling and cryopreservation at the molecular level. In the present study, we used real-time RT-PCR analysis to determine the effect of chilling and cryopreservation on expression of Pax2a, Pax2b, Pax5 and Pax8 which constitute one subgroup of the Pax gene family. As intact embryos of zebrafish have not yet been successfully cryopreserved, we have used two alternatives: chilling of intact embryos and cryopreservation of isolated blastomeres. Cryopreservation was found to affect the normal pattern of gene expression in zebrafish embryonic blastomeres. The trends, profile changes, in expression of Pax2a and Pax5 occurred to a lesser extent in frozen-thawed blastomeres than in fresh blastomeres whilst the opposite was true for Pax8. The trends in expression of Pax2b were delayed in frozen-thawed blastomeres compared to fresh blastomeres. Cryopreservation can therefore disrupt normal gene expression patterns in zebrafish embryonic blastomeres which could have a detrimental effect on embryo development.     

Wendell L. Hughes' life and contributions to plastic surgery

Wendell L. Hughes was a pioneer in ophthalmic plastic surgery and best known for the "Hughes flap," a tarsoconjunctival flap used for lower eyelid reconstruction. In 1937, Wendell L. Hughes sought to achieve the criterion standard of replacing "like with like" in his development of the tarsoconjunctival flap for lower lid reconstruction. This work was published in his ground-breaking thesis, Reconstructive Surgery of the Eyelids, the most comprehensive book on ophthalmic plastic surgery of its time. Although this flap has undergone many modifications, it has stood the test of time and is still used today. In addition, Dr. Hughes was heavily involved in surgical education, a founding member of the American Board of Plastic Surgery, and a leader in the development of sutures and microneedles. More importantly, he was a gracious humanitarian and inspiring mentor loved by peers and patients alike. Other authors have reviewed the intricacies of the Hughes flap; however, little attention has been given to the contributions of its creator.     

Cytoskeleton proteins F-actin and tubulin distribution and interaction with mitochondria in the granulosa cells surrounding stage III zebrafish (Danio rerio) oocytes

The distributional arrangement of mitochondria in the granulosa cells surrounding stage III zebrafish oocyte has been reported as a contiguous aggregation of mitochondria at the margin of the each granulosa cell. The aim of the present study was to further investigate the mitochondrial distribution in the granulosa cell layer in stage III ovarian follicles and the interaction between mitochondria and cytoskeleton elements actin and tubulin. To determine mitochondrial distribution/transport, immunocytochemistry analysis of tubulin and mitochondrial COX-I was carried out along with phalloidin staining of polymerised F-actin. The follicles were also exposed to a range of conditions that are known to affect mitochondria and the cytoskeleton proteins actin and tubulin. The mitochondrial inhibitor FCCP, the anti-mitotic drug nocodazole, and actin polymerisation inhibitor cytochalasin B were used. Levels of ATP, mtDNA copy number, and viability assessed by Trypan blue were also studied after exposure to inhibitors in order to determine the relationship between mitochondrial distribution/activity and ATP production. F-actin showed a hexagonal-polygonal distribution surrounding the mitochondria in granulosa cells, with the F-actin network adjacent to the plasma membrane of each granulosa cell. Tubulin structure presented a less organised distribution than F-actin, it was sparse in the cytosol. Interaction between mitochondria and tubulin was found indicating that mitochondria and tubulin are colocalised in zebrafish ovarian follicles. The exposure of ovarian follicles to inhibitors induced the loss of mitochondrial structural integrity showing that mitochondria distribution in granulosa cells of stage III zebrafish ovarian follicles is determined by the microtubules network.     

Characterization of human fungiform papillae cells in culture

The ability to maintain human fungiform papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Here we describe a method for enzymatically isolating human cells from fungiform papillae obtained by biopsy and maintaining them in culture for more than 7 passages (7 months) without loss of viability and while retaining many of the functional properties of acutely isolated taste cells. Cells in these cultures exhibited increases in intracellular calcium when stimulated with perceptually appropriate concentrations of several taste stimuli, indicating that at least some of the native signaling pathways were present. This system can provide a useful model for molecular studies of the proliferation, differentiation, and physiological function of human fungiform papillae cells.     

An Investigation of the Structural Diversities of Lithiated HMPA Complexes of o-Mercaptopyridine and Trithiocyanuric Acid: Syntheses, Crystal structures and Model Molecular Orbital Calculations

Reaction of o-mercaptopyridine (o-MPH) and trithiocyanuric acid (TTCyH₃) with one equivalent of BuⁿLi in the presence of HMPA yields the mono-lithiated salts MPLi.HMPA (1) and TTCyH₂Li.2HMPA (2) respectively, which have been characterised by NMR spectroscopy and X-ray crystallography. Reaction of three equivalents of BuⁿLi with anhydrous TTCyH₃ in THF yields the tri-lithiated species TTCyLi₃.4THF (3). In all three compounds the lithium centres have N,S-bridged coordination modes. Whereas 1 is dimeric in the solid state, 2 has an unusual monomeric structure and 3, which is a very rare example of a structurally characterised tri-lithiated compound, has an unprecedented polymeric structure incorporating (NCSLi) _n (n = 1, 2) rings. The structural diversities displayed by 1 and 2 have been probed, and thereby in part rationalised, by ab initio (6-31G*/RHF, 6-31G**/RHF and 6-31G*/MP2 levels) MO calculations on both their thio-keto and thiol isomers and on their uncomplexed and complexed lithiated derivatives. In particular, the optimised structures predict and reproduce the N,S-bridging coordination modes found for lithium and explain why structure 1 is dimeric whereas 2 is monomeric.Electronic Supplementary Material available.     

ras and neu oncogenes reverse serum inhibition and epidermal growth factor dependence of serum-free mouse embryo cells

Serum-free mouse embryo cells, cultured in basal nutrient medium supplemented with insulin, transferrin, epidermal growth factor, fibronectin, and high-density lipoprotein, do not exhibit growth crisis, lack detectable chromosomal aberrations, are nontumorigenic in vivo, are dependent on epidermal growth factor for survival, and are growth inhibited by serum or platelet-free plasma. These cells after transfection with the human Ha-ras or rat neu oncogenes no longer required epidermal growth factor for survival, were tumorigenic in vivo, and also proliferated in serum-containing medium. Autocrine activity capable of replacing epidermal growth factor was detected in conditioned medium from ras-transformed cultures, but little such activity was detected in medium from neu-transformed cultures. In addition, the capability of ras or neu-transformed cells to grow in serum-containing medium could not be mimicked in untransformed cells by the addition of growth factors or conditioned medium from transformed cells. These results suggest that the known structural similarity of the neu gene product to the EGF receptor is also reflected in a functional similarity by which the mutationally activated neu protein can replace the ligand-activated EGF receptor. These results also suggest that the ability of ras- and neu-transformed cells to escape the effect of the inhibitory serum activity is a nonautocrine property distinct from the acquisition of EGF autonomy.     

Death of serum-free mouse embryo cells caused by epidermal growth factor deprivation is prevented by cycloheximide, 12-O-tetradecanoylphorbol-13-acetate, or vanadate

Serum-free mouse embryo cells cultured in medium supplemented with insulin, transferrin, high-density lipoprotein, and fibronectin are dependent on epidermal growth factor for survival. Cycloheximide or actinomycin D prevented cell death caused by growth factor deprivation, suggesting that cell death required the synthesis of RNA and protein, a phenomenon similar to that reported for neuronal cell death in the absence of nerve growth factor. Orthovanadate, an inhibitor of phosphotyrosine phosphatases, and 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, also prevented serum-free mouse embryo cell death in the absence of epidermal growth factor.     

Immunological determination of phosphoenolpyruvate carboxylase and the large and small subunits of ribulose 1,5-bisphosphate carboxylase in leaves of the c(4) plant pearl millet

The light-dependent development of the photosynthetic apparatus in the first leaf of the C₄ plant pearl millet (Pennisetum americanum) was monitored by immunologically determining the concentration of phospho-enolpyruvate carboxylase and ribulose 1,5-bisphosphate carboxylase. A competitive enzyme-linked immunosorbent assay procedure using antibodies to the monomeric subunit of phosphoenolpyruvate carboxylase and the large and small subunit of ribulose 1,5-bisphosphate carboxylase was used to quantitate the amounts of these polypeptides in the first leaf of etiolated seedlings and etiolated seedlings exposed to light for varying periods of time. Phosphoenolpyruvate carboxylase was present in etiolated tissue; however, light stimulated its synthesis nearly 23-fold. Maximum accumulation of phosphoenolpyruvate carboxylase occurred approximately 4 days after etiolated plants were placed in the light. Both the large subunit and the small subunit of ribulose 1,5-bisphosphate carboxylase were present in leaves of etiolated seedlings. Light also stimulated the synthesis of both of these polypeptides, but at different rates. In etiolated leaves there was approximately a 3-fold molar excess of the small subunit to large subunit. Exposure of the etiolated leaves to light resulted in the molar ratio of the large subunit to the small subunit increasing to approximately 0.72. These data indicate that the net synthesis of these two polypeptides is not coordinately regulated at all times.     

A note on the effects of associated micro-organisms on the growth and nitrogenase activity of the cyanobacterium Anabaena cylindrica

Pseudomonas spp., Micrococcus spp. and Fusarium oxysporum promote growth and nitrogenase activity in Anabaena cylindrica. Respiratory CO₂ production by the microbial associate appears to be the main benefit to the cyanobacterium. Some stimulation of nitrogenase, however, appears to be due to other causes in associations with Pseudomonas and Fusarium.