Neuronal nets and nerve cell interactions in vitro in insect systems

Summary The development of a synthetic medium that supports growth and differentiation of insect embryonic tissues afforded the possibility of studying the interactions between nerve and other cell types in long term cultures. The mechanical dissociation of embryonic nerve tissues results in survival of nerve cells but not of glial cells. The dissociated glial-free neurons produce a dense fibrillar network in the presence, but not in the absence, of foregut explants or other tissues from same donors. Nerve fiber bundles outgrowing from dissociated neurons enter foregut segments and establish synaptic connections with muscle cells. Foregut explants undergo differentiation and become contractile in long term cultures when innervated by dissociated nerve cells. The progressive deterioration of similar foregut tissues cultured alone contrasts with the excellent condition of innervated explants and suggests that this is due to trophic factors released by nerve fibers. The same in vitro systems provided the opportunity of studying the interaction between nerve fibers produced by the autonomic ingluvial ganglion, which adheres to the surface of the alimentary tract, and muscle cells. Multiple esophagus explants from cockroach embryos become interconnected by fibers emerging from ingluvial ganglia, when the explants are combined in vitro at short distance from each other. Muscle cells migrating from the esophagi line up on axons branching out in the medium, or form contractile ribbons which, in turn, establish connections with nerve fibers. The thigmotropism of muscle cells and strong affinity for nerve fibers reveal a new aspect of muscle cells-to-fibers interaction, amenable to further analysis in vitro. This work was supported in part by United States Public Health Service grant NS-03777 and grant GB-16330 X from the National Science Foundation     

Mechanism of Excretion of a Bacterial Proteinase: Factors Controlling Accumulation of the Extracellular Proteinase of a Sarcina Strain (Coccus P)

It has been known that the extracellular proteinase of Coccus P is found only in cultures grown in the presence of Ca²⁺. It is now shown that this cation is required neither for synthesis, excretion, or activation of a zymogen nor as a prosthetic factor necessary for enzymatic activity. The only function of Ca²⁺ is to stabilize the active structure of the enzyme molecule, presumably by substituting for absence of S-S bridges. In the absence of Ca²⁺, the excreted proteinase undergoes rapid autodigestion and, instead of the active protein, its hydrolytic products are accumulated in the culture fluid. In minimal medium and under conditions of enzyme stability [presence of Ca²⁺ and Ficoll (Pharmacia)], Coccus P accumulates the proteinase at a gradually reduced speed although the rate of cultural growth remains constant. It is shown that this decline in rate of accumulation is caused by the excreted proteinase itself, possibly acting on its own precursor emerging from the cell in a form susceptible to proteolytic attack and not amenable to Ca²⁺ protection. A proteinase precursor is actually demonstrable in a calciumless culture at the onset of the enzyme accumulation which follows Ca²⁺ addition. It is suggested that excreted proteins require an unfolded (or incompletely folded) structure to cross the cell envelope.     

Growth-associated modifications of low-molecular-weight thiols and protein sulfhydryls in human bronchial fibroblasts

The thiol redox status of cultured human bronchial fibroblasts has been characterized at various growth conditions using thiol-reactive monobromobimane, with or without the combination of dithiotreitol, a strong reducing agent. This procedure has enabled measurement of the cellular content of reduced glutathione (GSH), total glutathione equivalents, cysteine, total cysteine equivalents, protein sulfhydryls, protein disulfides, and mixed disulfides. Passage of cells with trypsin perturbs the cellular thiol homeostasis and causes a 50% decrease in the GSH content, whereas the total cysteine content is subsequently increased severalfold during cell attachment. During subsequent culture, transient severalfold increased levels of GSH, protein-bound thiols, and protein disulfides are reached, whereas the total cysteine content gradually declines. These changes in the redox balance of both low-molecular-weight thiols and protein-bound thiols correlate with cell proliferation and mostly precede the major growth phase. When the onset of proliferation is inhibited by maintenance of cells in medium containing decreased amounts of serum, the GSH content remains significantly increased. Subsequent stimulation of growth by addition of serum results in decreased GSH levels at the onset of proliferation. In thiol-depleted medium, proliferation is also inhibited, whereas GSH levels are increased to a lesser extent than in complete medium. Exposure to buthionine sulfoximine inhibits growth, prevents GSH synthesis, and results in accumulation of total cysteine, protein-bound cysteine, and protein disulfides. For extracellular cystine, variable rates of cellular uptake correlate with the initial increase in the total cysteine content observed following subculture and with the GSH peak that precedes active proliferation. The results strongly suggest that specific fluctuations in the cellular redox balance of both free low-molecular-weight thiols and protein sulfhydryls are involved in growth regulation of normal human fibroblasts.     

Aspects of neural plasticity in the central nervous system—III. Methodological studies on the microdialysis technique

Some methodological aspects of the intracerebral microdialysis technique have been investigated: the existence of a pressure gradient at the level of the dialyzing membrane, the substance diffusion from the microdialysis probe and the extent of tissue damage induced by the implantation of the microdialysis probe. At the level of the dialyzing membrane a rough balance between the pressure inside the probe and the one present in the extracellular fluid compartment has been observed. The pattern of substance diffusion in the tissue showed a large variability depending on the substance used and the experimental conditions. Relevant deductions can be made by the use of labeled markers. By means of this approach, the diffusion pattern of tritiated ganglioside GM1 in the tissue around the probe could be shown to follow a biexponential pattern, suggesting a two-step process of diffusion. The degree of tissue damage induced by the microdialysis probe was assessed by analyzing the glial reaction, and was measured by means of semiquantitative immunocytochemistry of glial fibrillary acidic protein immunoreactivity. Only a limited area of neuronal damage was observed in the region surrounding the microdialysis probe. The amount of glial reaction after probe implantation was shown to be comparable with that induced by the implantation of a microinjection cannula.     

Detection of Water in Lipase-Catalyzed Reactions in Reverse Micelles as Studied by Infrared Spectroscopy

The hydrolytic activity of lipolytic enzymes in reverse micelles can be measured continuously with Fourier Transform infrared spectroscopy (FTIR) by following in the region of the OH-stretching band the water consumption during the reaction. This possibility is unique to reverse micellar solutions, because they are optically transparent and because they contain only a limited amount of water.     

Selenium,zinc, and thyroid hormones in healthy subjects

Iodothyronine 5′ deiodinase, which is mainly responsible for peripheral T₃ production, has recently been demonstrated to be a selenium (Se)-containing enzyme. The structure of nuclear thyroid hormone receptors contains Zinc (Zn) ions, crucial for the functional properties of the protein. In the elderly, reduced peripheral conversion of T₄ to T₃ with a lower T₃/T₄ ratio and overt hypothyroidism are frequently observed. We measured serum Se and RBC GSH-Px (as indices of Se status), circulating and RBC Zinc (as indices of Zn status), thyroid hormones and TSH in 109 healthy euthyroid subjects (52 women, 57 men), carefully selected to avoid abnormally low thyroid hormone levels induced by acute or chronic diseases or calorie restriction. The subjects were subdivided into three age groups. To avoid under- or malnutrition conditions, dietary records were obtained for a sample of 24 subjects, randomly selected and representative of the whole population for age and sex. Low T₃/T₄ ratios and reduced Se and RBC GSH-Px activity were observed only in the older group. A highly significant linear correlation between the T₃/T₄ ratio and indices of Se status was observed in the older group of subjects (r=0.54;p<0.002, for Se;r=0.50;p<0.002, for RBC GSH-Px). Indices of Zn status did not correlate with thyroid hormones, but RBC Zn was decreased in older as compared with younger subjects. We concluded that reduced peripheral T₄ conversion is related to impaired Se status in the elderly.     

NADP+-isocitrate dehydrogenase in germinating cucumber cotyledons: Purification and characterization of a cytosolic isoenzyme

The activity of NADP⁺-dependent isocitrate dehydrogenase (ICDH, EC 1.1.1.42) was investigated during the post-germinative growth of cucumber ( Cucumis sativus L. cv. Marketmore) seedlings. Isoelectric focusing showed the presence of several isoenzymes, two of which represented 70–80% of the total NADP⁺-ICDH activity in cotyledons of seedlings grown in the dark. They had pI values between 4.8 and 5.8. The isoenzyme with higher pI was purified to homogeneity by hydrophobic interaction, affinity, hydroxylapatite and anion exchange chromatography. The purified isoenzyme is a dimeric protein, consisting of two apparently identical 43-kDa subunits. It is specific for NADP⁺, inhibited by ATP and by 2-oxoglutarate, whereas it is not inhibited by citrate, succinate, and glyoxylate. The data indicate that NADP⁺-ICDH from cucumber is structurally similar to ICDHs from other plants, but it shows some peculiar biochemical characteristics.     

Distribution patterns of the paraneuronal endocrine cells in the skin, gills and the airways of fishes as determined by immunohistochemical and histological methods

Summary The neuro-endocrine cells of fish skin and respiratory surfaces, and their bioactive secretion as far as is known, are reviewed, and compared with similar elements in tetrapods, particularly amphibians. In the skin of teleost fish, immunohistochemistry has shown that Merkel cells react for serotonin, neuron-specific enolase and enkephalins. The pharmacology is not established in dipnoans or lampreys. In some teleosts, neuromasts react for substance P and leu-enkephalins; substance P is also reported from some ampullary organs (electroreceptors). Taste buds of teleosts may react for enkephalin and substance P. Basal cells of taste buds react for serotonin and neuron-specific enolase. Some unicellular skin glands of teleosts express bioactive compounds, including serotonin and some peptides; this ectopic expression is paralleled in amphibian skin glands. The dipnoan Protopterus has innervated pulmonary neuro-endocrine cells in the pneumatic duct region with dense-cored vesicles. In Polypterus and Amia the lungs have serotonin-positive neuro-endocrine cells that are apparently not innervated. In fish gills, a closed type of neuro-endocrine cell reacts for serotonin, an open type for enkephalins and some calcium-binding proteins (calbindin, calmodulin and S-100 protein). The functions of neuro-endocrine cells in fishes await investigation, but it is assumed they are regulatory.