Cell wall regeneration of individual Candida albicans yeast and mycelial protoplasts was studied with confocal and electron microscopy using polyclonal antibodies and lectins. Quantitative measurements of the fluorescence emitted by individual protoplasts during the process of regeneration indicate that chitin is the first polymer to be laid down, whereas beta (1,3)- and beta (1,6)glucan are incorporated at a later stage. Mannoproteins were found on the surface of fresh protoplasts and those newly synthesized were then deposited with time. During the first steps of wall regeneration, the proteins that interacted covalently with chitin or glucan were different, but the same species were found linked to each polymer in yeast and mycelial regenerating forms. The aggregates formed by regenerating protoplasts were shown to be due to the chitin and mannoprotein network initially laid.

Analyses of freeze-fracture replicas of mouse olfactory bulb reveal the presence of gap junctions in the plasma membranes of the cell bodies of mitral cells. Due to their localization and morphology we presume that they interconnect mitral and granule cells. Since the quality of electrical transmission between neurons is considered to be determined by the biochemical nature of the gap junction channel forming proteins (connexins) we performed immunohistochemistry and in situ hybridization using probes for connexin43 (Cx43), the most abundant connexin in brain tissue. Attribution of Cx43 immunolabel to specific neurons could not definitely be assessed by means of immunohistochemistry. In situ hybridization, however, using a specific cRNA probe for Cx43 revealed a label confined to cell bodies of mitral and tufted cells of the olfactory bulb. These data indicate that Cx43 is expressed by bulbar neurons and suggest that Cx43 is a molecular constituent of gap junction channels in neurons.

The localization of the neural cell adhesion molecule L1 in the male urogenital tract (including seminal vesicles and prostate) of the mouse and bull was investigated using immunocytochemical and immunochemical methods in order to better understand the function of this glycoprotein in non-neural tissues. L1 antibodies labeled non-myelinated nerves in all portions of the urogenital tract investigated. However, L1 immunoreactivity was also found between epithelial cells of several regions of the urogenital system including epididymal tail, deferent duct, ejaculatory duct and seminal vesicles. Some L1 immunoreactivity was also demonstrated between epithelial cells of murine urinary bladder and urethra. The specificity of the immunoreaction was verified by western blots. There was no correlation between L1 expression and proliferating activity as revealed by double immunocytochemistry using various markers of cell proliferation. This unexpected expression of L1 in nonneural tissue is mainly restricted to non-proliferating epithelia of those portions of the urogenital tract that are derived from the Wolffian duct. It is suggested that L1 in these epithelia could enhance the mechanical resistance and reduce transepithelial permeability.

The olfactory system is a unique part of the central nervous system since it retains neuronal turnover and regenerative capacities in adulthood. Thus it provides an ideal model to study plasticity of membrane moities involved in cell-cell interactions. One structure particularly involved in cell-cell interaction is the tight junction, which establishes polarization of epithelial cells and creates diffusion barriers to paracellular passages. ZO-1 is a phosphoprotein peripherally associated with tight junctions. We have studied expression of ZO-1 protein in the developing and adult olfactory system of the mouse in order to get information about the localization and developmental expression of this tight junction component. ZO-1 expression has also been determined in cell cultures of olfactory bulbs.ZO-1 was present in the olfactory placode prior to formation of tight junctions. ZO-1 was localized in the developing and mature olfactory epithelium at heterotypic contacts between supporting cells and olfactory neurons as well as at homotypic contacts between both these cell types. Confocal microscopy showed quantitative differences in the ZO-1 expression among different olfactory dendrites. In the olfactory nerves ZO-1 immunolabeling was detectable between olfactory ensheathing cells. From the seventh postnatal day ZO-1 immunolabeling was detected at the mitral cell layer of the bulb on cells tentatively identified as oligodendrocytes. Myelinated tracts of the bulb were ZO-1 negative. Cell cultures of olfactory bulbs showed ZO-1 immunoreaction, mostly localized on glial fibrillary acidic protein (GFAP)-positive cells. Our results provide further evidence that ZO-1 serves functions unrelated to the tight junction complex and indicate molecular heterogeneity of these cell-cell contacts. (C) 1994 Wiley-Liss, Inc.

The localization of Ca++-independent cell adhesion molecules (CAMs) in the developing and mature olfactory epithelium and bulb is reviewed. The CAMs included in this article are the neural cell adhesion molecule (N-CAM), the 180 kD component of N-CAM (N-CAM 180), the embryonic form of N-CAM (E-N-CAM), L1 glycoproteins, J1 glycoproteins, and the adhesion molecule on glia (AMOG). In addition, the expression of the L2-HNK-1 carbohydrate epitope, shared by N-CAM, L1, J1 and myelin-associated glycoprotein (MAG) in the adult olfactory epithelium and bulb has also been documented. For the localization of these molecules at the light and electron microscopic levels, immunocytochemical techniques were used and are described in detail.During development and organogenesis, the olfactory system exhibits a pattern of CAM expression similar to the general pattern described for the developing nervous system. In the adult olfactory system, however, a significant retention of CAMs characteristic for developmental and morphogenetic processes, such as E-N-CAM, AMOG, as well as the high molecular weight components of J1 glycoproteins, can be observed. The retention of these embryonic features are most likely associated with the cell turnover and high plasticity of this system. Moreover, the predominance of N-CAM 180 with respect to other components of N-CAM, as well as the absence of the L2/HNK-1 carbohydrate epitope, are also particular traits of the primary olfactory system which could be associated with its exceptional properties.

To gain insight into the function of gap junctions' connexin43, connexin32 and connexin26 in a neural structure that retains neuronal turnover capacities throughout adulthood, the expression of these molecules has been investigated in the developing and adult olfactory system by immunocytochemical and biochemical methods.Connexin43 was detectable from the olfactory placode stage. During early embryonic development, the levels of connexin43 expression remained low. An increase in the expression of this connexin occurred perinatally. Expression of connexin43 became very high during the postnatal stages and adulthood. Electron microscopy (EM) immunocytochemistry of the olfactory system showed connexin43 expression in non-neuronal cells. Strong regional differences in the expression of connexin43 in the olfactory epithelium were observed. No apparent relationship between connexin43 expression and turnover activity of olfactory neurons was detected. Western blots of olfactory tissues revealed the presence of three different isoforms of connexin43.Connexin32 was detected in the olfactory bulb at late postnatal stages including adulthood. Connexin32 was observed on some cells tentatively identified as oligodendrocytes.Connexin26 was localized onto leptomeninges. Some immunofluorescence was also obtained in the periglomerular region and in the subependymal layer of the bulb. Northern blot analysis revealed the presence of mRNA of connexin32 and connexin26 in the adult olfactory system. Our results substantiate the cell specific expression of these three types of connexins and they document the primacy of connexin43 in olfactory tissues. Moreover, our findings indicate that although expression of connexin43 in the olfactory system is developmentally regulated, it is not directly associated with the neuronal cell turnover of the olfactory epithelium.

Depending upon growth temperature, Candida albicans can exhibit two different morphologies, a budding yeast or a mycelium. By studying the distribution of concanavalin A-ferritin particles on the cell wall surface during bud and germ tube formation, we have elucidated the way cell wall extension occurs. Both processes initially require the localized lysis of the wall in order to allow the incorporation of the newly synthesized material. Later on, the cell wall behaves as an elastic structure, allowing extension by an intosusception process and, as a consequence, cell growth.

Vesicles containing invertase activity were obtained from protoplast homogenates of Saccharomyces cerevisiae by differential centrifugation followed by gel chromatography. These vesicles were similar in size and shape to yeast coated vesicles, and appear to have a complex polypeptide composition. Most of these polypeptides were seemingly bound to the surface of the vesicular structures, being released by treatment with alkali. A protein with an electrophoretic mobility similar to that of yeast clathrin (molecular mass of 185 kDa) co-purified with vesicles containing invertase activity, and exhibited cross-reactivity with antimmalian (pig) clathrin antibodies.

To gain insight into the cellular and molecular mechanisms underlying neurogenesis in adult mouse olfactory bulb, several adhesion molecules expressed by glial cells and neurons were investigated. In the germinal zone of the olfactory bulb, the subependymal layer of the rostral region of the lateral ventricles, two adhesion molecules are detectable that are characteristic of early morphogenetic events: J1/tenascin and the polysialylated form, the so-called embryonic form, of N-CAM. The polysialylated form of N-CAM is expressed by most cells in the subependymal layer, and by some astrocytes and neurons in the granular layer adjacent to the subependymal layer. This suggests that bipotential precursor cells retain expression of the embryonic form during their migration from the subependymal layer and during the first stages of differentiation into neurons and glia. Expression of the polysialylated form of N-CAM is also retained in monolayer cultures of six-day-old olfactory bulbs, 55 days after seeding in vitro. J1/tenascin was detectable in the subependymal layer using two monoclonal antibodies. The immunostaining pattern was different between the two antibodies and more restricted to the subependymal layer than when staining with polyclonal J1 antibodies was performed, indicating that J1/tenascin exists in distinct isoforms. Finally, our observations suggest that, in the adult olfactory bulb, L1 is not only a neuron-neuron adhesion molecule, but it may also be involved in neuron-glia interactions, since it is found at contact sites between these two cell types. L1, therefore, may be a neuron-glia adhesion molecule in some parts of the CNS, while it is not in others.

The expression of the neural adhesion molecules L1 and N-CAM has been studied in the embryonic and early postnatal olfactory system of the mouse in order to gain insight into the function of these molecules during development of a neural structure which retains neuronal turnover capacities throughout adulthood. N-CAM was slightly expressed and L1 was not significantly expressed in the olfactory placode on Embryonic Day 9, the earliest stage tested. Rather, N-CAM was strongly expressed in the mesenchyme underlying the olfactory placode. In the developing nasal pit, L1 and N-CAM were detectable in the developing olfactory epithelium, but not in regions developing into the respiratory epithelium. At early developmental stages, expression of the so-called embryonic form of N-CAM (E-N-CAM) coincides with the expression of N-CAM, whereas at later developmental stages and in the adult it is restricted to a smaller number of sensory cell bodies and axons, suggesting that the less adhesive embryonic form is characteristic of morphogenetically dynamic neuronal structures. Moreover, E-N-CAM is highly expressed at contact sites between olfactory axons and their target cells in the glomeruli of the olfactory bulb. L1 and N-CAM 180, the component of N-CAM that accumulates at cell contacts by interaction with the cytoskeleton are detectable as early as the first axons extend toward the primordial olfactory bulb. L1 remains prominent throughout development on axonal processes, both at contacts with other axons and with ensheathing cells. Contrary to N-CAM 180 which remains detectable on differentiating sensory neuronal cell bodies, L1 is only transiently expressed on these and is no longer detectable on primary olfactory neuronal cell bodies in the adult. Furthermore, whereas throughout development L1 has a molecular form similar to that seen in other parts of the developing and adult central nervous systems, N-CAM and, in particular, N-CAM 180 retain their highly sialylated form at least partially throughout all ages studied. These observations suggest that E-N-CAM and N-CAM 180 are characteristic of developmentally active structures and L1 may not only be involved in neurite outgrowth, but also in stabilization of contacts among fasciculating axons and between axons and ensheathing cells, as it has previously been found in the developing peripheral nervous system.

The effects of ethanol on development: the fetal alcohol syndrome (FAS) 346 The animal model 346 Alcoholic liver disease 348 Effect of prenatal exposure to ethanol on liver development 350 Qualitative and quantitative ultrastructural alterations 350 Hepatocyte ph osphatases 350 Goigi appa ratus 352 Protein synthesis and glycosylation 354 Plasma membrane and cell junctions 357 Hepatocyte subpopulations 358 References 359

The expression of the neural cell adhesion molecules N-CAM and L1 was investigated in the olfactory system of the mouse using immunocytochemical and immunochemical techniques. In the olfactory epithelium, globose basal cells and olfactory neurons were stained by the polyclonal N-CAM antibody reacting with all three components of N-CAM (N-CAM total) in their adult and embryonic states. Dark basal cells and supporting cells were not found positive for N-CAM total. The embryonic form of N-CAM (E-N-CAM) was only observed on the majority of globose basal cells, the precursor cells of olfactory neurons, and some neuronal elements, probably immature neurons, since they were localized adjacent to the basal cell layer. Differentiated neurons in the olfactory epithelium did not express E-N-CAM. In contrast to N-CAM total, the 180-kDa component of N-CAM (N-CAM180) and E-N-CAM, L1 was not detectable on cell bodies in the olfactory epithelium. L1 and N-CAM180 were strongly expressed on axons leaving the olfactory epithelium. Olfactory axons were also labeled by antibodies to N-CAM180 and L1 in the lamina propria and the nerve fiber and glomerular layers of the olfactory bulb, but only some axons showed a positive immunoreaction for E-N-CAM. Ensheathing cells in the olfactory nerve were observed to bear some labeling for N-CAM total, L1, and N-CAM180, but not E-N-CAM. In the olfactory bulb, L1 was not present on glial cells. In contrast, N-CAM180 was detectable on some glia and N-CAM total on virtually all glia. Glia in the nerve fiber layer were labeled by E-N-CAM antibody only at the external glial limiting membrane. In the glomerular layer, E-N-CAM expression was particularly pronounced at contacts between olfactory axons and target cells. The presence of E-N-CAM in the adult olfactory epithelium and bulb was confirmed by Western blot analysis. The continued presence of E-N-CAM in adulthood on neuronal precursor cells, a subpopulation of olfactory axons, glial cells at the glia limitans, and contacts between olfactory axons and their target cells indicates the retention of embryonic features in the mammalian olfactory system, which may underlie its remarkable regenerative capacity.

Mannoproteins from cell walls of Saccharomyces cerevisiae synthesized at successive stages of the population growth cycle have been solubilized with Zymolyase and subsequently analyzed. The major change along the population cycle concerned a large size mannoprotein material; the size of the newly-synthesized molecules varied from 120,000–500,000 (mean of about 200,000) at early exponential phase to 250,000–350,000 (mean of about 300,000) at late exponential phase. These differences are due to modifications in the amount of N-glycosidically linked mannose residues, since the size of the peptide moiety was 90,000–100,000 at all growth stages and the level of O-glycosylation changed only slightly. After, incubation of the purified walls with concanavalin A-ferritin and subsequent analysis by electron microscopy, labelling was localized at the external and internal faces of the walls. The middle space of these was labelled after digestion of the glucan network with Zymolyase, which demonstrate the presence of mannoproteins in close contact with the structural glucan molecules throughout the wall.

The effects of prenatal exposure to ethanol on the lateral (contiguous) plasma membrane of newborn rat hepatocytes was investigated using qualitative and quantitative freeze-fracture. The number of free intramembranous particles decreased by 11% and 17% in P- and E-faces, respectively. Alcohol also induced the appearance of particle-free areas within the gap junctions, in addition to a three-fold increase in the mean area and an increment in the percent of plasma membrane occupied by these elements. However, no differences in either the size of gap junction particles or in the mean interparticle distance were found between control and alcohol-treated animals. On the other hand, prenatal exposure to ethanol did not alter the structure of tight junctions.

The effect of prenatal exposure to ethanol on the Golgi apparatus of newborn rat hepatocytes has been studied cytochemically using several trans-Golgi markers (thiamine pyrophosphatase, uridine diphosphatase, inosine diphosphatase, acid phosphatase, and 5'-nucleotidase) as well as a cis-side marker (osmium impregnation). The amount of cerium phosphate formed in the cytochemical reactions was roughly quantitated by stereologic methods. The Golgi apparatus of about 40% of the hepatocytes appeared disorganized after alcohol treatment, and in the other 60%, the electron density of reaction product deposits for all phosphatases investigated was decreased. 5'-Nucleotidase was completely absent in cisternae of Golgi apparatus of treated cells. In control cells impregnated with osmium tetroxide, reduced osmium compounds were observed in most Golgi cisternae and in nearby vesicles. In contrast, only small vesicles appeared positive in treated hepatocytes. These results suggest that prenatal alcohol exposure alters some Golgi functions. Thus, the decrease in nucleoside diphosphatase and 5'-nucleotidase cytochemical activities after ethanol exposure strongly suggests that this treatment could affect glycosylation in the Golgi apparatus of newborn rat hepatocytes.

To assess the effect of ethanol on the planar distribution of cholesterol as well as on the surface architecture of presynaptic terminals of rats, synaptosomes isolated from cerebral cortex of rats chronically exposed to alcohol were incubated with filipin, a cytochemical marker for beta-hydroxycholesterol, and analyzed using both conventional (qualitative and quantitative) and freeze-fracture electron microscopy. Synaptosomes incubated in the absence of filipin were used as cytochemical controls. Biochemical determination indicates a 12% increase of cholesterol in synaptosomal membranes from alcohol treated rats. This increase was confirmed by a significant increment in the number of filipin-cholesterol complexes. Synaptosomes of treated rats showed a reduction in the total number of synaptic vesicles (SV) as well as a decrease in the density and total number of intramembranous particles (IMP) per synaptosome. In control rats, most synaptosomal IMP were distributed in clusters whereas in those of rats exposed to alcohol they were distributed at random. These changes in distribution of IMP were also observed in presynaptic terminals analyzed "in situ." These findings indicate that ethanol acts on the presynaptic terminals. The variations in cholesterol content as well as in the density and distribution of IMP appear to be related to alcohol-induced changes in the physicochemical properties of components of the synaptosomal membrane.

Incorporation of polysaccharides into the walls of regenerating protoplasts of Candida albicans was followed in the presence of papulacandin B, tunicamycin and nikkomycin. With the first drug, chitin was incorporated normally whereas incorporation of glucans and mannoproteins was significantly decreased. Tunicamycin decreased incorporation of all wall polymers when added at the beginning of the regeneration process but blocked only mannan and alkali-insoluble glucan incorporation when added after 5 h. Nikkomycin inhibited chitin synthesis, and the walls formed by the protoplasts were enriched in alkali-soluble glucan. Pulse-chase experiments suggested that a precursor-product relationship between the alkali-soluble and alkali-insoluble glucans existed in the wall. The results obtained with the antibiotics were confirmed and extended by cytological studies using wheat-germ agglutinin labelled with colloidal gold and concanavalin A-ferritin as specific markers of chitin and mannoproteins respectively. The results support the idea that regeneration of walls by protoplasts occurs in two steps: firstly, a chitin microfibrillar skeleton is formed, and in a later step glucan-mannoprotein complexes are added to the growing structure. The chitin skeleton probably allows the orderly spatial arrangement of the other polymers giving rise to the regenerated cell wall.

The presence of orthogonal arrays of particles (OAP) in pneumocytic membranes was studied using freeze-fracture replicas of lung tissue from turtles, frogs and various mammalian species, including man. OAP are typically present in the basal plasma membrane of pneumocytes in turtles and frogs, while they have only been detected in the plasma membrane of type I cells in human lungs among the mammalian species studied. The median of particles per OAP was 10 (range 4-40) in lower vertebrates and 8 (range 4-24) in man. The mean density of OAP per unit cell surface varied between 40 and 60/micron2. The frequent occurrence of OAP in pneumocytic membranes in the lungs of man, turtles, and frogs suggests that they are related to a specific, although undetermined pulmonary function which is restricted to the ciliated airway epithelium in those mammalian species lacking OAP in the alveolar epithelium.

Modifications occurring in the plasma membrane and their relationship to newly synthesized microfibrils were examined in regenerating protoplasts of Candida albicans by freeze-fracture electron microscopy. Freshly prepared protoplasts showed no residual wall material, and long invaginations covered the surface of the plasma membrane. Analysis of the external face (E-face) of the plasma membrane showed a significant decrease in the number of intramembranous particles (IMP) in comparison with the original cells. After 40 min incubation in regeneration medium, newly synthesized microfibrils which seemed to originate from protrusions in the plasma membrane were observed. The plasma membrane showed important modifications with respect to IMP. After 3 h 45 min, the cells were covered by an abnormal wall which showed isolated fibrils partially embedded in the matrix material. The plasma membrane of these partially regenerated protoplasts was similar to that of original cells. After 8 h, regeneration of the protoplasts seemed to be complete as no differences from the original cells were detected in the plasma membrane or the wall. Calcofluor white altered the deposition of wall polymers during regeneration, but did not modify the plasma membrane of the protoplasts.