A trophic effect of epidermal growth factor (EGF) on rat colonic mucosa in organ culture

The development of an organ-culture system for rat colonic mucosa has enabled a direct assessment of the effect of epidermal growth factor (EGF) on cell division. An augmented mitotic index (AIm) has been employed to identify changes in cell proliferation. Explants of colonic mucosa from four animals were maintained in a medium containing serum for five days. On the fifth day of culture, half of the explants received fresh medium containing EGF (40 ng/ml) and the remainder (controls) fresh medium only. At 6, 12, 24 and 48 hr thereafter groups of both experimental and control explants received the metaphase-arresting drug vincristine (4 micrograms/ml) for 3 hr prior to fixation. The proportions of vincristine-arrested metaphases within the explants were determined. Analysis of the data indicates that when serum is present exogenous EGF exerts a trophic effect which increases with time (P less than 0.001). In a second experiment colonic explants from four animals were maintained for five days in a serum-free medium and were then divided into groups, each of which received one of a range of concentrations of EGF. The AIm was determined for each group after 36 hr. It was found that increasing concentrations of EGF produce a small but significant increase in cell proliferation (P less than 0.01). This effect, however, was less pronounced than that seen when serum was present. These results suggest that EGF has a trophic action on the colon and interacts with additional factors found in serum.     

Protoplast isolation and callus production from leaves of tissue-cultured Vitis spp.

Protoplasts were isolated from mesophyll of axenic cultures of grape, Vitis rotundifolia cv. Summit and V. vinifera cv. Cabernet Sauvignon. Enzymes effective for protoplast isolation were Macerozyme R-10 (0.5% and 0.1%) and Cellulase Onozuka R-10 (1.0% and 0.5%) for V. rotundifolia and V. vinifera, respectively. Polyvinylpyrrolidone and 2-(N-morpholino)ethanesulfonic acid were essential in the isolation media. Protoplasts were purified using flotation/centrifugation. The protoplasts of V. rotundifolia cultured in Gamborg's B5 basal medium with 2.2 M 6-benzyladenine, 4.5 M 2,4-dichlorophenoxyacetic acid and 0.4% agarose gave the best plating efficiency of conditions tried in this study. Cell division occurred within 5 to 6 days and visible microcalli developed within one month. After 6 weeks in culture, microcalli transferred to liquid medium exhibited active callus growth. Protoplasts of V. vinifera cultured under these conditions had similar results.Abbreviations MES 2-(N-morpholino)ethanesulfonic acid monohydrate - PVP polyvinylpyrrolidone - PDS potassium dextran sulfate - BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate     

Rapid changes in the phospholipid composition of gill membranes during thermal acclimation of the rainbow trout,Salmo gairdneri

Summary The phospholipid composition of gill tissue was determined in rainbow trout (Salmo gairdneri) undergoing thermal acclimation between 5°C and 20°C for a period of up to 28 days. Proportions of phosphatidylethanolamine (PE) and cardiolipin (CL) increased during cold acclimation and decreased during warm acclimation; proportions of phosphatidylcholine (PC) changed in the opposite direction (i.e., decreased during cold acclimation). In contrast, levels of phosphatidylserine,-inositol, and sphingomyelin did not vary significantly. Thermal modulation of headgroup composition occurred rapidly as reflected by changes in the ratio of PC-to-PE, which rose significantly from 2.40±0.09 to 2.92±0.09 within 72 h of transfer from 5 to 20°C; adaptation to 5°C was equally rapid. Proportions of PE changed more rapidly than those of PC during cold adaptation, whereas the opposite was true during warm acclimation. Both the time course and the direction of the observed changes in phospholipid composition suggest that such adjustments may contribute to the homeoviscous regulation of membrane properties, particularly during the initial stages of thermal adaptation.     

Monoclonal antibody-based immunoassay for type A Clostridium botulinum toxin is comparable to the mouse bioassay

A monoclonal antibody (BA11) has been produced against Clostridium botulinum type A neurotoxin by the fusion of myeloma cells (P3 NS1/1-Ag4-1) with spleen cells from BALB/c mice immunized with botulinum type A neurotoxoid. The antibody bound specifically to botulinum type A neurotoxin, showing no cross-reactivity with types B and E botulinum toxins or with any of several other bacterial toxins tested. The monoclonal antibody did not bind to botulinum type A neurotoxin which had been denatured with sodium dodecyl sulfate and bound only weakly to each of the separated heavy and light subunits of the neurotoxin, suggesting a conformational requirement for the antigenic determinant of the antibody. A sensitive immunoassay for C. botulinum type A toxin with monoclonal antibody BA11 in conjunction with an enzyme amplication system has been developed which allows detection of 5 to 10 mouse 50% lethal doses ml-1 of purified neurotoxin. The assay was equally sensitive when applied to the detection of crude toxin in food stuffs; the average value for the minimum level of detectable toxin in extracts of tinned salmon or corned beef was 9 +/- 3.1 mouse 50% lethal doses ml-1.     

A Cytokinetic Study of Small-Intestinal and Colonic Mucosa After Resection of 70% of the Small Intestine

Abstract. Pulse-labelling with tritiated thymidine and a fraction of labelled mitoses experiments have been performed in order to investigate the proliferative changes induced at various sites in the hyperplastic small-intestinal mucosa of rats previously subjected to resection of 70% of the small intestine. Proliferative activity in the colon was also studied.
In the distal ileum there is a significant reduction in cell cycle time (T_c) of cells at all levels within the crypt and the growth fraction falls. In the jejunum and proximal ileum the crypts contain an increased number of proliferating cells, but as the size of the maturation zone is also increased, there is no significant alteration in the relative number of proliferating cells per crypt. Nor does the distribution of proliferating cells in these crypts seem to alter. There is no general reduction in T_c at these sites, but there does appear to be a significant reduction in T_c on the part of the cells in the stem-cell zone at the crypt base.
In neither proximal nor distal colon was there any significant proliferative change apparent after small-intestinal resection.     

VARIATION IN THE DURATION OF MITOSIS IN THE CRYPTS OF LIEBERKUHN OF THE RAT; A CYTOKINETIC STUDY USING VINCRISTINE

The variation in the duration of mitosis ( t _m) with cell position in the small intestinal crypts of the adult rat has been measured by a stathmokinetic technique using vincristine. The value for the whole crypt column was 0.43 hr, or 26 min. At the bottom of the crypt t _m was in excess of 1 hr, but rapidly decreased and throughout the greater part of the proliferative compartment was between 0.40 and 0.50 hr. At the top of the proliferative compartment an increase in t _m was demonstrated.
If the value of 0.43 hr for the whole crypt column is correct, then one argument for postulating the formation of metabolic DNA during differentiation in the small bowel epithelium of the rat becomes invalid. Variations in t _m within the crypt have been shown to increase the values of cell velocity obtained from cumulative birth rate diagrams. Finally further evidence has been presented for the existence of a slowly dividing subpopulation of cells at the base of the crypt. These cells may be important in crypt repopulation after damage with phase specific anti-tumour drugs.     

Malignant Neoplasms of the Nasopharynx

A mass in the neck, facial pain and cranial nerve palsies are the most common presenting symptoms of malignant tumours of the nasopharynx. Surgery is limited to biopsy for histological diagnosis. External irradiation is the treatment of choice, both for the primary lesion and the regional and distant metastases. Illustrative case histories are presented in detail. Experience over a 20-year period demonstrates that a 20% five-year survival rate has been obtained.     

Development and germination of pecan somatic embryos derived from liquid culture

Aggregates of globular and pre-globular stage somatic embryos from suspension cultures of pecan (Carya illinoensis Koch) were cultured on solidified media for embryo development. Embryo aggregates and pre-globular stage embryo masses were given various treatments to further ontologic development. A 2- to 4-wk mild dehydration of the embryo aggregates suppressed recurrent embryogenesis, promoted development of globular embryos into cotyledonary stage embryos, and enhanced plant development beyond germination. Fine embryogenic tissue masses filtered from suspension formed cotyledonary-staged embryos when the collection filters were plated on solified medium. The embryogenic capacity of preglobular stage embryo masses was compared between media supplemented with varying concentrations of polyethylene glycol (molecular weight 8 000) vs. filter overlays. The filter paper overlays were not necessary for embryo development. An inverse relationship was found between the number of embryos that developed and the concentration of polyethylene glycol in the medium. However, this relationship was reversed for ability of embryos to germinate and develop into a plant.