Non-ependymal cilia in the habenulae and the interpeduncular nucleus of the frog tadpole

Summary Cilia of the 9+2 pattern are found electron microscopically in nonependymal cells of the habenulae and the interpeduncular nucleus of the tadpole of Rana esculenta at an early stage of development (8 mm length, head to tip of tail). A comparison is made between these and the ependymal and sensory cilia in the same specimens. The cilia project into the neuropil emerging from a perikaryon rich in free ribosomes and displaying a prominent Golgi apparatus. These perikarya contain dense core vesicles. Synapses with vesicles of the clear spherical type have been observed along the ciliary shaft. On a purely morphologic basis the authors hypothesize that these cilia, at least in this early ontogenetic stage, may extend considerably the conducting surface of the cell and represent a sensory structure which could be stimulated by terminal processes belonging to distantly located cells. In addition, they could also be involved in the trophic exchange of material with the adjacent structures.     

Genetic Evidence for Interaction between Nonhomologous Proteins in Yeast and a Case of Suppression at the HIS1 Locus

The HIS1 and THR4 loci are the structural genes for phosphoribosyl-ATP pyrophosphorylase and threonine synthetase, respectively. The allele his1–1S has no enzyme activity at 30°, but does have activity at 15° provided the cell contains the wild-type THR4 allele or a suppressing allele at another locus, designated SUP(his1–1S). Under these conditions, cells with the his1–1S mutation are capable of growth on minimal medium at 15°. Three kinds of reversions of a his1–1S thr4 sup(his1–1S) strain to histidine prototrophy have been obtained: (1) his1–1S locus reversions to HIS1 that restore growth without added histidine at 30°, (2) thr4 reversions to THR4 that simultaneously eliminate the requirement for threonine and restore the low-temperature effect on the his1-1S allele, and (3) mutations from sup to SUP. The SUP allele is not an ochre suppressor, and it is not linked to either HIS1, THR4 or a centromere. It may represent a missense suppressor. It is proposed that the effect of THR4 is caused by aggregation of the wild-type threonine synthetase with defective his1–1S monomers, causing a favorable conformational change in the histidine protein that restores limited enzymatic activity. This can be regarded as a case of complementation between nonhomologous proteins.     

The development of competence in resting B cells. The induction of cyclic AMP and ornithine decarboxylase activity after direct contact between B and T helper cells.

In the present communication, an experimental approach is utilized that facilitates the study of biochemical processes induced in B cells after their interaction with Th cells. In this approach, Th cell clones are stimulated for 18 h upon anti-CD3-coated plates, fixed with paraformaldehyde, and added at a 2 to 3:1 ratio to small, resting B cells (isolated from Percoll gradients). Th cells not stimulated on anti-CD3-coated plates, but fixed with paraformaldehyde, serve as controls for these experiments. The activated, fixed Th cells induce a transient, sixfold increase in B cell levels of cAMP, as well as an increase in B cell expression of ornithine decarboxylase (ODC) activity. This enzyme initiates the synthesis of polyamines and has been shown to be increased as cells enter the growth phase. In addition, previous studies have shown that the cellular levels of ODC activity are controlled by a multi-tiered regulatory cascade. To examine this aspect, polyclonally stimulated B cells were studied. Such cells demonstrated a gradual increase in ODC mRNA levels that peaked between 6 and 15 h and can be partially explained by a three- to fourfold increase in mRNA stability but not by changes in the enzyme affinity for substrate. The increase in ODC mRNA occurs in the absence of protein synthesis, suggesting that the ODC gene is a member of the immediate/early gene family. Finally, the early increase in ODC mRNA was enhanced in cells in which cAMP levels were artificially elevated, suggesting the possibility that the cAMP-dependent signaling pathway participates during the regulation of this gene expression. The significance of these experimental results concerning the process of B cell activation is discussed.     

The phase behavior of cholesteryl esters in intracellular inclusions.

Differential scanning calorimetry and polarizing light microscopy have been used to investigate kinetic and thermodynamic properties of the phase behavior of cholesteryl ester contained in Fu5AH rat hepatoma cells and J774 murine macrophages. These cultured cells store cholesteryl esters as cytoplasmic inclusions of approximately 1-micron diameter and thus are models of the foam cells characteristic of atherosclerotic plaque. Simple binary mixtures of cholesteryl palmitate and cholesteryl oleate, the predominant cholesteryl esters in cellular inclusions in both cell types serve as models to explain important aspects of the phase behavior of these inclusions. Although inclusions should exist as stable crystals at 37 degrees C under conditions of thermodynamic equilibrium, microscopic examination of cells indicates that inclusions exist as metastable liquid crystals at 37 degrees C for extended periods of time. Using an analytical model based on nucleation theory, we predict that the cholesteryl ester inclusions should be liquid-crystalline in the cytoplasm of living cells. This may not be true either for lysosomal cholesteryl ester or for extracellular cholesteryl ester present in advanced atherosclerotic plaque where fusion of droplets can enhance the possibility of crystallization. The enhanced metastability of the relatively fluid liquid-crystalline state in cellular inclusions should result in increased activity of the neutral cholesteryl ester hydrolase in living cells.     

Neurite outgrowth on a step gradient of chondroitin sulfate proteoglycan (CS-PG).

Sulfated proteoglycans (PGs) may play a significant role in the regulation of neurite outgrowth. They are present in axon-free regions of the developing nervous system and repel elongating neurites in a concentration-dependent manner in vitro. The addition of growth-promoting molecules, such as laminin, can modify the inhibitory effect of PGs on neurite outgrowth (Snow, Steindler, and Silver, 1990b). Substrata containing a high-PG/low-laminin ratio completely inhibit neurite outgrowth, while normal, unimpeded outgrowth is observed on low-PG/high-laminin substrata. Therefore, different patterns of neurite outgrowth may result from regulation of the ratio of growth-promoting molecules to growth-inhibiting molecules. Using video microscopy, embryonic chicken dorsal root ganglia neurons (DRG), chicken retinal ganglia neurons (RGC), and rat forebrain neurons (FB) were analyzed as they extended processes from a substratum consisting of laminin alone onto a step gradient of increasing concentrations of chondroitin sulfate proteoglycan (CS-PG) bound to laminin. In contrast to neurite outgrowth inhibition that occurs at the border of a single stripe of high concentration of CS-PG (Snow et al., 1990b and this study), growth cones grew onto and up CS-PG presented in a step-wise graded distribution. Although the behavior of the different cell types was unique, a common behavior of each cell type was a decrease in the rate of neurite outgrowth with increasing CS-PG concentration. These data suggest that appropriate concentrations of growth-promoting molecules combined with growth-inhibiting molecules may regulate the direction and possibly the timing of neurite outgrowth in vivo. The different responses of different neuronal types suggest that the presence of sulfated PG may have varying effects on different aspects of neuronal development.     

Increased Expression of β-Amyloid Protein Precursor and Microtubule-Associated Protein τ During the Differentiation of Murine Embryonal Carcinoma Cells

Expression of the genes encoding the beta/A4 amyloid protein precursor (APP) and microtubule-associated protein tau was studied in an embryonal carcinoma cell line (P19) that differentiates in vitro into cholinergic neurons after treatment with retinoic acid. Expression of APP increased 34- (mRNA) and 50-fold (protein) during neuronal differentiation; APP-695 accounted for most of this increase. These remarkable increases in APP expression coincided with a proliferation of neuronal processes and with an increase in content of tau mRNA. Moreover, subsequent decreases in the levels of APP and tau mRNA coincided with the onset of the degeneration of the neuronal processes. Immunocytochemical staining suggested that greater than 85% of the P19-derived neurons are cholinergic and that APP is present in the neuronal processes and cell bodies. These results suggest that APP may play an important role in construction of neuronal networks and neuronal differentiation and also indicate that this embryonal carcinoma cell line provides an ideal model system to investigate biological functions of APP and the roles of APP and tau protein in development of Alzheimer's disease in cholinergic neurons.     

Peripheral distribution of dermatan sulfate proteoglycans (decorin) in amyloid-containing plaques and their presence in neurofibrillary tangles of Alzheimer's disease.

We used a polyclonal antibody and a mixture of three monoclonal antibodies (MAb), all recognizing the protein core of the small dermatan sulfate proteoglycan (DSPG) (known as PG-II or decorin) derived from human skin fibroblasts, to immunolocalize this molecule in the characteristic lesions in Alzheimer's brain. All antibodies demonstrated positive decorin immunostaining in both the amyloid deposits of neuritic plaques (NPs) and the filamentous structures within neurofibrillary tangles (NFTs). Unlike heparan sulfate proteoglycans (HSPGs), which tend to be evenly distributed throughout NPs containing amyloid fibrils, decorin was primarily localized to the periphery of the spherically shaped amyloid plaques and to the edges of amyloid fibril bundles within the plaque periphery. Decorin was also immunolocalized to the paired helical and straight filaments within NFTs and to collagen fibrils surrounding blood vessels. The unusual distribution of decorin confined to the periphery of amyloid plaques in AD brain suggests that this particular PG may play an important role in the development of the amyloid plaque.     

An Escherichia coli plasmid-based, mutational system in which supF mutants are selectable: Insertion elements dominate the spontaneous spectra

A new system is described to determine the mutational spectra of mutagens and carcinogens in Escherichia coli; data on a limited number (142) of spontaneous mutants is presented. The mutational assay employs a method to select (rather than screen) for mutations in a supF target gene carried on a plasmid. The E. coli host cells (ES87) are lacI⁻ (am26), and carry the lacZΔM15 marker for α-complementation in β-galactosidase. When these cells also carry a plasmid, such as pUB3, which contains a wild-type copy of supF and lacZ-α, the lactose operon is repressed (off). Furthermore, supF suppression of lacl^um26 results in a lactose repressor that has an uninducible, lacl^S genotype, which makes the cells unable to grow on lactose minimal plates. In contrast, spontaneous or mutagen-induced supF⁻ mutations in pUB3 prevent suppresion of lacl^am26 and result in constitutive expression of the lactose operon, which permits growth on lactose minimal plates. The spontaneous mutation frequency in the supF gene is 0.7 and 1.0 × 10⁻⁶ without and with SOS induction, respectively. Spontaneous mutations are dominated by large insertions (67% in SOS-uninduced and 56% in SOS-induced cells), and their frequency of appearance is largely unaffected by SOS induction. These are identified by DNA sequencing to be Insertion Element: IS1 dominates, but IS4, IS5, gamma-delta and IS10 are also obtained. Large deletions also contribute significantly (19% and 15% for - SOS and +SOS, respectively), where a specific deletion between a 10 base pair direct repeat dominates; the frequency of appearance of these mutations also appears to be unaffected by SOS induction. In contrast, SOS induction increases base pairing mutations (13% and 27% for -SOS and +SOS, respectively), The ES87/pUB3 system has many advantages for determining mutational spectra, including the fact that mutant isolation is fast and simple, and the determination of mutational changes is rapid because of the small size of supF.     

Separation of dissociated thyroid follicular and parfollicular cells: association of serotonin binding protein with parafollicular cells

Parafollicular cells (PC) of the sheep thyroid gland are neural crest derivatives that synthesize and release the biogenic amine serotonin (5-HT) as well as the hormone calcitonin. The thyroid also contains a highly specific serotonin-binding protein (SBP). Separation of dissociated thyroid cells was done to study the cellular localization of SBP and to develop a means of isolating PC for study. Various methods were used to obtain an enriched and purified population of PC. Minced thyroid glands were enzymatically dissociated and the cells were layered on a Ficoll linear density gradient. Fractions obtained from the gradient were examined for cell number, viability, 5-HT concentration, SBP activity, and morphology by electron microscopy. One of the fractions was found to be enriched in PC. High levels of 5-HT and SBP were also found in this fraction, whereas these levels were low where the majority of cells were found. This PC-rich fraction, however, contained numerous follicular cells (FC); therefore, additional approaches to cell separation were used. FC can be stimulated in vitro with thyroid stimulating hormone (TSH) to become intensely phagocytic. When stimulated cells were incubated in the presence of silica microspheres, the FC engulfed the microspheres, which were toxic to them. PC did not become phagocytic and were unharmed by the microspheres. Suspended cells, after incubation with microspheres, were centrifuged on a discontinuous gradient, and a PC-rich fraction was obtained. Silica, however, interfered with analysis of SBP. Another method to take advantage of the phagocytic potential of FC was therefore used. TSH-stimulated cell suspensions were passed through a column of sepharose to which thyroglobulin had been coupled. Stimulated FC apparently adhered to the beads and were retained by the columns. Fractions eluting from the columns were greatly enriched with PC. These fractions contained high levels of 5-HT and SBP, and considerably reduced FC contamination was found by quantitative electron microscopy. It is concluded that SBP is localized to PC in the sheep thyroid. The idea that these cells resemble serotonergic neurons in their mechanisms of 5-HT storage is supported.