Two distinct regulatory steps in cartilage differentiation

The effect of developmental stage on chondrogenic capacity in high-density cell cultures of chick embryonic wing bud mesenchyme is examined. Mesenchyme from stage 19 embryos forms aggregates of closely associated cells which do not form cartilage matrix, nor contain significant levels of type II collagen that are detectable by immunofluorescence, unless they are treated with dibutyryl cyclic AMP. Mesenchyme from stage 24 embryonic wing buds in high-density cell cultures will spontaneously form cartilage, as defined by electron microscopy and immunofluorescence with antibody to type II collagen. Cultures prepared from stage 26 wings form numerous aggregates which fail to accumulate an Alcian blue-staining matrix and which resemble mesenchyme cells morphologically. However, because these cells show considerable intracellular immunofluorescence for type II collagen, they are actually unexpressed cartilage cells. Several treatments, including exposure to dibutyryl cyclic AMP, ascorbic acid and an atmosphere of 5% oxygen, or mixture with small numbers of stage 24 wing mesenchyme cells, stimulate expression, as determined by the accumulation of an Alcian blue-staining matrix and an ultrastructurally recognizable cartilage matrix. Since the addition of similar numbers of differentiated cartilage cells does not stimulate expression of stage 26 cells, it is proposed that initial cartilage expression is dependent on a mesenchyme-specific influence which might be removed by cell dissociation. These studies demonstrate that there are at least two distinct transitions in cartilage differentiation: one involves the conversion of mesenchyme to unexpressed chondrocytes and the second involves mesenchyme-dependent expression of chondrogenic differentiation.     

Organ culture as a model for investigating the effects of antimetabolites and nucleoside transport inhibitors on rodent colonic mucosa

Summary The in-vitro effects of hydroxyurea 5-FU and 5-FUdR have been extensively studied in experimental systems employing cell-line techniques. In this study we investigated the effects of these drugs on the levels of incorporation of labeled nucleosides into DNA in explants of intact rat colonic mucosa maintained in organ culture. The effects of the nucleoside transport inhibitors nitrobenzylthioinosine (NBMPR) and dipyridamole—which are modulators of antimetabolite cytotoxicity—on the incorporation of tritiated thymidine [(³H]TdR) into DNA were also studied. The incorporation of tritiated TdR into DNA was reduced by hydroxyurea but was not altered by either 5-FU or 5-FUdR. The levels of tritiated deoxyuridine were reduced by 5-FU and 5-FUdR in separate experiments; this is in keeping with thymidylate synthase inhibition. NBMPR and dipyridamole also reduced 3H-TdR incorporation into DNA. These results can be explained in terms of the known mechanisms of action of these drugs. This experimental model is therefore useful in assessing the effects of antimetabolites and nucleoside transport inhibitors in intact colonic mucosa.     

Effects of exogenous wild-type p53 on a human lung carcinoma cell line with endogenous wild-type p53

Several studies have shown that expression of exogenous wild-type p53 is detrimental to the growth of cell lines with absent or mutant p53. In this study, wild-type p53 cDNA expression plasmids were transfected into A549 lung carcinoma cells which had previously been shown by sequencing to contain wild-type p53. When a constitutively expressed wild-type p53 plasmid containing the neomycin resistance gene was transfected into these cells, no G418-resistant colonies contained the exogenous p53 cDNA even though the neomycin resistance gene was integrated. When cells were transfected with a dexamethasone-inducible wild-type p53 cDNA expression plasmid, induction of p53 expression resulted in a decreased growth rate and a decreased proportion of S-phase cells. Continuous treatment with dexamethasone resulted in continued p53 expression for 16 days, but beyond that time expression ceased and could not be reinduced. These data indicated that although the A549 cell line could proliferate in the presence of endogenous wild-type p53 there was a strong selection pressure against continued expression of additional exogenous wild-type p53.     

Murine p53 inhibits the function but not the formation of SV40 T antigen hexamers and stimulates T antigen RNA helicase activity

We have characterized the effects of p53 on several biochemical activities of simian virus 40 (SV40) large tumor (T) antigen. While p53 induced a strong inhibition of the T antigen DNA helicase activity, surprisingly, its RNA helicase activity was stimulated. This supports the liklihood that the DNA and RNA helicase activities of T antigen reflect discrete functions. p53 did not significantly affect the ATP-dependent conversion of T antigen monomers to hexamers. However, the ability of these hexamers to assemble on a DNA fragment containing the viral origin was impaired by p53. Thus, these results suggest that p53 inhibits the function but not the formation of T antigen multimers. This conclusion was further supported by the observation that the addition of a purified p53:T antigen complex was as inhihitory as free p53 to the DNA helicase activity of free T antigen. Thus our data indicates that the targets of p53 inhibition are the functional units of T antigen, namely the hexamers.     

Effects of chemical composition on nitrogen mineralization from green manures of seven tropical leguminous trees

Green manures from seven tropical leguminous trees were incubated with soil to determine the rates and controls of net nitrogen release. Fresh green manure (leaves and succulent twigs) was mixed with moist soil and incubated in polyethylene bags. Net N mineralization from green manures was estimated by the accumulation of extractable ammonium and nitrate minus the accumulation in soil alone. Patterns of N mineralization were complex, differed among species, and at 12 weeks ranged from 10 to 65 percent of original green-manure N. Cumulative net N mineralization was negatively correlated with initial soluble polyphenol content in the early phases of decomposition (1 through 8 weeks) and with initial lignin content in later phases (4 through 12 weeks). Neither initial percent N nor lignin: N ratio were strongly correlated with N mineralization. The best chemical index of N release was the initial polyphenol: N ratio. This study confirms previous findings that N mineralization from tropical legumes is controlled more by soluble polyphenols than by lignin or N content.     

Effect of dilauroylphosphatidylcholine liposomes on motility, induction of the acrosome reaction, and subsequent egg penetration of ram epididymal sperm

The effects of dilauroylphosphatidylcholine (PC12) on ram epididymal sperm motility, acrosome reaction (AR) induction, plasma membrane permeability, mitochondrial function, and sperm penetration into zona-free hamster eggs were determined. PC12 (50 microM) induced cell motility in caput and cauda sperm, as measured by subjective estimation and automated motility analysis. Motion parameters of treated caput sperm approached those of control ejaculated sperm. Flow cytometric analysis revealed that membrane permeability to propidium iodide and mitochondrial uptake of rhodamine 123 changed during epididymal transit. PC12 induced the AR in sperm from all epididymal regions relative to control incubated sperm (caput 17% vs. control 8%; corpus 29% vs. control 13%; proximal cauda 48% vs. control 4%; distal cauda 51% vs. control 9%). After PC12 treatment, egg penetration by sperm was increased for sperm from the corpus (corpus 7% vs. control 0%) and cauda (proximal 48% vs. control 0%; distal 51% vs. control 0%), but not for caput sperm (caput 0% vs. control 0%). These studies establish that some sperm in each region of the epididymis possess the capacity for movement and the AR. Caput sperm, however, were unique in that they could not penetrate eggs. Additional maturational changes must occur in the caput and/or corpus epididymidis before penetration capacity can be expressed.     

Dwarf mutants ofBrassica: Responses to applied gibberellins and gibberellin content

Eight rapid-cyclingBrassica genotypes differing in height were treated with gibberellins (GAs) by syringe application to the shoot tip. The height of two genotypes ofBrassica napus, Bn5-2 and Bn5-8, andB. rapa mutants,dwarf 1 (dwf1) anddwarf 2 (dwf2), was unaffected by exogenous GA₃ at dosages up to 0.1 g/plant, a level which increased shoot elongation of normal genotypes. Thus, these dwarf mutants are GA-insensitive. In contrast to theB. napus dwarfs, twoB. rapa mutants,rosette (ros), anddormant (dor), elongated following GA₃ application. The dwarfros was most sensitive, responding to applications as low as 1 ng GA₃/plant. Furthermore,ros also responded to GA₁ and some of its precursors with decreasing efficacy: GA₃>ent-kaurenoic acid GA₁>GA₂₀GA₁₉=GA₄₄GA₅₃. Endogenous GAs were measured by gas chromatography-selected ion monitoring using [²H₂]GA internal standards for calibration, from shoots of the GA-insensitive genotypes Bn5-2, Bn5-8 which contained theB. napus mutantdwarf 1, and from a normal genotype Bn5-1. Concentrations of GA₁ and GA₂₀ averaged 3.2- and 4.6-fold higher, respectively, and GA₁₉ levels also tended to be higher in the dwarfs than in the normal genotype.