Changes in the number of chick ciliary ganglion neuron processes with time in cell culture

The purpose of this study was to describe the shape of chick ciliary ganglion neurons dissociated from embryonic day 8 or 9 ganglia and maintained in vitro. Most of the neurons were multipolar during the first three days after plating, with an average of 6.0 processes extending directly from the cell body. The neurons became unipolar with time. The remaining primary process accounted for greater than 90% of the total neuritic arbor. This striking change in morphology was not due to the selective loss of multipolar cells, or to an obvious decline in the health of apparently intact cells. The retraction of processes was neither prevented nor promoted by the presence of embryonic muscle cells. Process pruning occurred to the same extent and over the same time course whether the cells were plated on a monolayer of embryonic myotubes or on a layer of lysed fibroblasts. Process retraction is not an inevitable consequence of our culture conditions. Motoneurons dissociated from embryonic spinal cords remained multipolar over the same period of time. We conclude that ciliary ganglion neurons breed true in dissociated cell culture in that the multipolar-unipolar transition reflects their normal, in vivo, developmental program.     

Defective T cell response to presented antigen in autoimmune mice

The effect of the single autosomal recessive gene lpr on antigen presentation was studied. MRL/Mp-lpr/lpr, C3H/HeJ-lpr/lpr, C57BL/6J-lpr/lpr, and their normal congenic partners were investigated. Mice bearing the lpr gene were unable to respond to TNP-KLH when presented by syngeneic antigen-presenting cells. The congenic normal partners gave a brisk response. Mixing experiments demonstrated that the defect resided with the lpr responding T cell and not with the lpr antigen-presenting cell. Antigen-presenting cells from lpr mice were capable of inducing T cell proliferation in normal congenic partners, whereas antigen-presenting cells from normal mice failed to stimulate lpr T cells. This defect was intrinsic to an Lyt-1+2- cell. Pharmacologic restoration was attempted by in vivo and in vitro administration of interleukin 2. However, cells from lpr mice remained unaffected. The relationship of these findings to autoimmunity is discussed.     

Glutamate receptor desensitization and its role in synaptic transmission

Responses of excitatory amino acid receptors to rapidly applied glutamate were measured in outside-out membrane patches from chick spinal neurons. The peak current varied with glutamate concentration, with a half-maximal response at 510 microM and a Hill coefficient near 2. Currents activated by 1 mM glutamate desensitized and recovered in two phases. The faster time constant was identical to the time constant of decay of synaptic currents, suggesting that glutamatergic synaptic currents are terminated, in part, by receptor desensitization. Steady-state desensitization was evident following application of only 2-3 microM glutamate, concentrations comparable to levels in the extracellular space in the intact brain. Thus, glutamate receptor desensitization can affect synaptic efficacy in two ways: at high concentrations, rapid desensitization of receptors may curtail synaptic currents; at low concentrations, there is a significant reduction in the number of activatable receptors.     

Induction of the human growth hormone gene placed under human hsp70 promoter control in mouse cells: A quantitative indicator of metal toxicity

An in vitro test method for general metal toxicity screening was designed, based on the cellular response to stress. The expression of a transfected human growth hormone gene sequence driven by the human heat-shock protein 70 promoter in NIH/3T3 cells was used as marker of noxious contact with metal compounds. Out of a series of31 metals, 17 were competentfor inducing this stress response system. According to the effective concentration and to the intensity of the response, three different clusters of positive compounds emerged and were ranked as strong, intermediate strength and weak inducers. These results correlated well with data from other in vivo and in vitro metal toxicity studies, including LD₅₀ in mice. Apparently the positivelnegative compounds also fitted well with data from genotoxicity and carcinogenesis studies on metal salts.Abbreviations hGH human growth hormone - hsp70 70 kDa heat-shock protein     

Cell degeneration in the developing optic lobes of the sine oculis and small-optic-lobes mutants of Drosophila melanogaster

In the small-optic-lobes (sol) and sine oculis (so) mutants of Drosophila melanogaster extensive cell death occurs in the optic lobes during the first half of pupal development. Gynandromorph flies show that the sol mutation acts primarily on cells of the medulla cortex. Degeneration of medullar ganglion cells occurs at an early stage of cellular differentiation, when their axons have not yet participated in the formation of the second optic chiasma. The so gene, on the other hand, acts on the eye anlagen. The analysis of chimeric flies demonstrates that degeneration in the optic lobes of so flies is a consequence of eye reduction. At the level of the second optic chiasma extensive axonal degeneration can be observed in the mutant. Neurons seem to die after their failure to establish a sufficient number of functional contacts. In sol;so double mutants, the mutational effects are cumulative causing complete degeneration of columnar cell types in pupae without any eye anlage. The tiny rudiments of the optic lobes in eyeless double mutants still contain tangential neurons of the medulla and of the lobula complex. The central brain is reduced in size due to the missing visual fibers, however, its overall appearance is surprisingly normal.     

Early events in neuromuscular junction formation in vitro: induction of acetylcholine receptor clusters in the postsynaptic membrane and morphology of newly formed synapses

The development of clusters of acetylcholine (ACh) receptors at newly formed synapses between embryonic chick spinal cord and muscle cells grown in vitro has been studied by iontophoretic mapping with ACh. A semi-automated technique using on-line computer analysis of ACh responses and a photographic system to record the position of each ACh application permit the rapid construction of extensive and detailed maps of ACh sensitivity. Clusters of receptors, evident as peaks of ACh sensitivity, are present on many uninnervated myotubes. The distribution of ACh sensitivity closely parallels the distribution of 125I-alpha-bungarotoxin binding sites on the same muscle cell. In all cases where individual myotubes were adequately mapped before and after synapse formation, ingrowing axons induced new clusters of receptors rather than seeking out preexisting clusters. Synapses can form at active growth cones within 3 h of nerve-muscle contact. New receptor clusters can appear beneath neurites within a few hours. Many of the uninnervated clusters on innervated myotubes disappear with time. In contrast, receptor clusters on uninnervated myotubes remain in the same location for many hours. Synaptic clusters and clusters on uninervated myotubes are stable even though individual receptors are metabolized rapidly. The morphology of several identified sites of transmitter release was examined. At the scanning EM level, synapses appeared as small, rough-surfaced varicosities with filopodia that radiated outwards over the muscle surface. One synapse was studied by transmission EM. Acetylcholinesterase and a basement lamina were present within the synaptic cleft.     

Aldosterone biosynthesis induced by ACTH and angiotensin II in newborn rat adrenocortical cells transfected by c-EJ-Ha-ras oncogene.

Adrenocortical cells were obtained by fractionated trypsination of newborn rat adrenal glands and transfected with a plasmid containing the EJ/T24-Ha-ras oncogene. Isolation of adhesive cells led to a proliferative cell line with an overexpression of 21 kDa ras protein. These cells incubated with corticosterone or deoxycorticosterone as the precursor produced a high level of 18-hydroxycorticosterone and aldosterone as identified by gas chromatography- mass spectrometry. ACTH and angiotensin II increased the basal production of aldosterone nineteen-fold and six-fold respectively. Under ACTH stimulation the ratio between aldosterone and 18-hydroxycorticosterone production was 1:3. The transformation of corticosterone under angiotensin II stimulation yielded up to 41% of 18-hydroxycorticosterone (4.7 micrograms/mg of cell protein per 24h) and 4.4% of aldosterone (0.5 microgram/mg of cell protein per 24h) in a low potassium concentration medium (6 mmol/l). To our knowledge this is the first report of continuous proliferative adrenocortical cells producing aldosterone.     

Acidic-phosphoprotein phosphatase activity of rat ventral prostate nuclei: apparent lack of effect of androgens.

A protein phosphatase activity has been demonstrated in nuclei of rat ventral prostate utilizing 32P-labelled phosvitin as a model acidic phosphoprotein substrate. This phosphoprotein phosphatase has a pH optimum of 6.7, is unaffected by the sulphydryl protecting agent 2-mercaptoethanol, and requires a divalent cation for maximal activity. Of the various divalent cations tested, Mg2+ is the most effective in reactivating the EDTA-inhibited enzyme. The phosphatase is inhibited by sodium flouride, sodium oxalate, N-ethylmaleimide, ATP and ADP but is relatively insensitive to ammonium molybdate. Increased ionic strength of the reaction medium also causes a reduction in the enzyme activity, e.g., by 48% at 200 mM sodium chloride. The activity of the acidic phosphoprotein phosphatase did not change significantly at 48 h or 96 h post-orchiectomy when expressed per unit of nuclear protein. However, it is reduced by approx. 30% at these times after castration if based on DNA content. The decline in activity per nucleus reflects the decrease in the realtive nuclear protein content observed at 48 h or 96 h post-orchiectomy. This suggests that the decline in the phosphorylation of prostatic nuclear acidic proteins which occurs upon androgen withdrawal is not due to increased nuclear phosphatase activity.