Differentiation of myoblasts and CNS cells grown either separately or as co-cultures on microcarriers

Dispersed neuronal and muscular elements from fetal or neonatal origin, can organize and mature in culture when grown on positively charged cylindrical microcarriers (MCS), to a stage which simulatein vivo maturation. Cells arrange themselves on the MCS to form aggregates which remain floating in the nutrient medium. In such a tridimensional organization, the neuronal tissue is capable of regenerating a network of nerve fibers which establish synapse interconnections and undergo myelination. Oligodendrocytes organize on MCS in a tridimensional pattern and produce extensive myelin-like membranes. Myoblasts in MC-cultures fuse into polynucleated myotubes which become striated and contract spontaneously. Creatine kinase and acetylcholine receptor (AChR) are formed during myogenesis in similar quantities in MC-cultures and in monolayers. When both neuronal and muscle tissues are prepared from the same fetus (autologous nerve-muscle co-cultures) and are cultured on MCS, they interconnect to form neuro-muscular junctions. Cells from both tissues, exhibit better differentiation, for longer periods in MC-cultures than they do in monolayers. The floating functional entities are easy to sample and can be harvested for ultrastructural, immunocytochemical and biochemical analysis. In addition, MC-cultures can be used as a good tool for the study of acute and chronic exposures to toxicological agents, as well as for implantation into demyelinated, injured or dystrophic tissues. In this case the MCS in the implanted entities will serve as identifiable markers.     

Characteristics of carrot plant regenerated from two cell line selected as either ionic-Al tolerant cell or Al-phosphate utilizing cells

Plantlets of carrot (Daucus carota L.) were regenerated from two types of cell lines. One type was selected as ionic-Al tolerant (IAT) cells, while the second type featured Al-phosphate utilizing cells (IPG). Their tolerance characteristics were investigated. The plantlets from IAT were directly regenerated, whereas those from IPG were regenerated after somatic hybridization with wild-type cells previously inactivated with iodacetamide, because the IPG cells had completely lost the ability to regenerate naturally.The sexual progeny of IAT showed Al-tolerant properties, established by testing their root elongation in the presence of 500 µM Al ions. Most of the calli obtained from the somatic hybrids grew more rapidly than the wild-type cells when Al-phosphate was used as a sole source of phosphorus. Thus, we obtained two types of carrot plantlets, regenerated from IAT and IPG. Both possessed the tolerant characteristics as observed with the stress-selected cells.     

Lateral connections between heart muscle cells as revealed by conventional and high voltage transmission electron microscopy

Summary The lateral surfaces of heart muscle cells are interconnected by a varied and extensive network of structures that exist in addition to intercalated discs. Ultrastructural images of this network are vastly improved over those from epoxy-embedded material, particularly for low density components, through the application of a method for removing the embedding matrix from thin or thick sections that are then stereoscopically analyzed with standard or high voltage transmission electron microscopy. The connections include cables, 3–20 nm in diameter, multi-strand cables, 10–40 nm-granules, meshlike mats, and sheets, all extensively interwoven. It is suggested that intercellular connections of varying strength and distribution aid in the integration of mechanical performance of the large population of myocytes during the contractile cycle of the heart.This study was supported by a grant from NIH Biotechnology Resources through the University of Colorado High Voltage E.M. Laboratory, NIH Research Grant HL 24336, a N.Y. Heart Association Grant-in-Aid, and NIH Research Career Development Award HL 00568I thank Dr. E.H. Sonnenblick for continual aid and encouragement and Dr. R. Terry, Ms. Y. Kress, and Ms. J. Fant for use of facilities. I also thank Dr. K.R. Porter for guidance in the use of the HVEM technique, Dr. J.J. Wolosewick and Dr. M. Fotino for valuable suggestions, and Ms. J. Fleming, Mr. G. Wray, and Mr. G. Charlie of HVEM staff at Boulder. I acknowledge Dr. F. Pepe for use of facilities, Dr. R. Bloodgood for comments, and Mrs. L. Cohen-Gould, Ms. T. Downey, Mr. F. Reingold, Mrs. T. Maio, and Mrs. R. Shamoon for excellent assistance     

p16INK4a/Ki‐67 dual labelling as a marker for the presence of high‐grade cancer cells or disease progression in urinary cytopathology

E. Piaton, A. S. Advenier, C. Carré, M. Decaussin‐Petrucci, F. Mege‐Lechevallier and A. Ruffion
p16 ^INK4a /Ki‐67 dual labelling as a marker for the presence of high‐grade cancer cells or disease progression in urinary cytopathology Objective: Overexpression of p16^INK4a independent of the presence of E6–E7 oncoproteins of high‐risk papillomaviruses has been identified in bladder carcinoma in situ lesions with or without concurrent papillary or invasive high‐grade (HG) urothelial carcinoma. As p16^INK4a and Ki‐67 co‐expression clearly indicates deregulation of the cell cycle, the aim of this study was to investigate the frequency of p16^INK4a/Ki‐67 dual labelling in urinary cytology samples. Methods: Immunolabelling was performed in demounted, destained Papanicolaou slides after ThinPrep^® processing. A total of 84 urinary cytology samples (18 negative, 10 low grade, 19 atypical urothelial cells and 37 high grade) were analysed for p16^INK4a/Ki‐67 co‐expression. We assessed underlying urothelial malignancy with cystoscopy, histopathology and follow‐up data in every case. Results: Compared with raw histopathological results, p16 ^INK4a/Ki‐67 dual labelling was observed in 48 out of 55 (87.3%) HG lesions and in 11 out of 29 (37.9%) negative, papillary urothelial neoplasia of low malignant potential or low‐grade carcinomas (P = 0.05). All cases with high‐grade/malignant cytology were dual labelled. Sixteen out of 17 (94.1%) carcinoma in situ cases and eight out of 14 (57.1%) cases with atypical urothelial cells matching with HG lesions were dual labelled. Extended follow‐up allowed three cases of progression to be diagnosed in dual‐labelled cases with negative/low‐grade cytology results after a 9‐ to 11‐months delay. Conclusions: The data show that p16^INK4a/Ki‐67 co‐expression allows most HG cancer cells to be detected initially and in the follow‐up period. Additional studies are needed in order to determine whether dual labelling can be used as a triage tool for atypical urothelial cells in the urine.