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1. The morphology of porcine submandibular gland tissue is demonstrated by light and electron microscopy.2. Subcellular particles from porcine submandibular glands were prepared by a procedure adapted to the presence of viscous mucus, and were fractionated on continuous and discontinuous sucrose gradients.3. Six particulate fractions were obtained by discontinuous gradient centrifugation. These were identified by electron microscopy, marker enzymes and nucleic acid content and were designated as follows: (1) plasma membranes, (2) smooth membranes and light Golgi membranes, (3) heavy Golgi membranes, (4) membrane mixture and mitochondria, ( 5 ) mitochondria and (6) membrane mixture and mucin residues. The bulk of rough membranes and cell nuclei was found in the 3000 x g sediment before fractionation.4. Sialyltransferase and N-acetylneuraminate monooxygenase showed highest activities in the Golgi membrane fractions representing 45 % and 36 x, respectively, of total tissue activity. The specific activities of these membrane-bound enzymes were respectively 13 and 10 times higher when compared with the homogenate. 5. The acylneuraminic acid/protein ratio was lowest in the Golgi membranes when compared with the other particulate fractions and the cytosol glycoprotein. The molar N-acetylneuraminic acid/N-glycolylneuraminic acid ratio was highest in the two Golgi membrane fractions (average 45: 55) and in the pool of free acylneuraminic acids (41 : 59), and lowest in the cytosol glycoprotein (10 : 90).6. Approximately 2.6 o/;l of the total acylneuraminic acids occurring in porcine submandibular glands are freely diffusible, 80 % of them representing free N-acetylneuraminic acid and N-glycolylneuraminic acid in a molar ratio of about 4: 6 and 20 % representing cytidylyl-glycosides of N-acetylneuraminic acid and N-glycolylneuraminic acid in a ratio of 6 : 4.7. In radioactive labelling experiments with surviving slices of porcine submandibular glands the highest specific radioactivities were found in the pool of free acylneuraminic acids, followed by Nacetylneuraminic acid and N-glycolylneuraminic acid bound to the Golgi membranes. N-Acetylneuraminic acid and N-glycolylneuraminic acid of the cytosol glycoproteins have lowest turnover rates.8. Based on these experiments a model is presented describing two pathways of N-acetylneuramink acid modification in the course of glycoprotein biosynthesis. At least 40 x of N-acetylneuraminic acid is hydroxylated before its transfer onto Golgi membranes and linkage to growing glycoprotein molecules and about 50 thereafter. This results in a molar N-acetylneuraminic acid/N-glycolylneuraminic acid relationship of 1 : 9 in the secreted glycoprotein. It is assumed that free N-acetylneuramink acid is modified by the soluble cytosol monooxygenase whereas the glycoprotein-bound N-acetylneuraminic acid is hydroxylated by the corresponding membrane-bound enzyme.

Tumour necrosis factor (TNF)-alpha-induced apoptosis is associated with several nuclear and cell surface alterations, in particular with the condensation of chromatin and the fragmentation of the cell nucleus, formation of blebs on the cell surface and breakdown of the plasma membrane. However, there is little information about the relationship between the cell surface alterations and the nuclear changes during apoptosis. To study this, cultured WEHI cells were exposed to TNF-alpha over different time periods. The cytological changes were studied using a correlative approach, which allowed observation of the same cell consecutively under light, scanning and transmission electron microscopy. The earliest sign of cell alteration was a reduction of the number of microvilli after 15 min of TNF-alpha exposure. This reaction was reversible (reappearance of microvilli) and took place during the first hour, in which neither nuclear alterations nor plasma membrane breakdown were observed. The changes in the nucleus began with condensation of chromatin after approximately 1 h of TNF-alpha-exposure. After 4-5 h the microvilli disappeared again, particularly in areas where the formation of blebs (blebbing) was observed. Strikingly, cell surface alterations (bleb formation) were detected only in those cells that presented with condensed chromatin, and not in cells with a normal chromatin pattern, proving at least a close correlation between nuclear and cell surface changes during the process of apoptosis.

The epithelium of amphibian urinary bladder is readily dissociated into single cells by exposure to solutions of low Ca²⁺ activity. Using a novel device for mounting bladders on the microscope stage, we studied the net movement of membrane material in freshly dissociated viable cells. Fluorescent labelling of apical and basolateral membrane glycoconjugates and proteins with Lucifer yellow CH, tetramethyl rhodamine B isothiocyanate (TRITC), and lectin-TRITC showed pronounced redistribution. In response to opening of the tight junctions and subsequent cell dissociation, apical label expanded over most of the cell surface. In contrast, the larger part of the basolateral membrane was invaginated and became the limiting membrane of internalized vesicles. The vesicles fused and expanded into large vacuoles within 2 h. Cell rounding and vacuolation were inhibited by cytochalasin B, i.e. dependent on functions of microfilaments. We suggest that the membrane redistribution occurs by disruption of recycling pathways, initiated by withdrawal of the tight junction: the trafficking of apical material appears interrupted at the internalization step, that of basolateral material at the reinsertion step.

Sensors and multi-sensor arrays are the basis of new technologies for the non-label monitoring of cell activity. In this paper we show that choroid plexus cells can be cultured on silicon chips and that sensors register in real time changes in their activity, constituting an interesting experimental paradigm for cell biology and medical research. To validate the signals recorded (metabolism = peri-cellular acidification, oxygen consumption = respiration; impedance = adhesion, cell shape and motility) we performed experiments with compounds that act in a well-known way on cells, influencing these parameters. Our in vitro model demonstrates the advantages of multi-sensor arrays in assessment and experimental characterization of dynamic cellular events—in this case in choroid plexus functions, however with applicability to other cell types as well.