The building up of the cell wall is tightly dependent on the functionality of the secretory pathway. Syntaxins as well as other SNARE proteins play important roles during vesicle secretion and fusion. We have compared the secretion of newly synthesised cell-wall polysaccharides to that of secretory marker proteins such as secreted green-fluorescent protein (sec-GFP) and secreted rat preputial beta-glucuronidase (secRGUS) in leaf protoplasts and roots of wild-type and transgenic Nicotiana tabacum plants, overexpressing a syntaxin homologue NtSyr1 (Sp1) and its soluble variant Sp2 that interferes specifically with Sp1 function, affecting post-Golgi transport. In protoplasts transiently transformed with secGFP and Sp1, no variation was observed in the pattern of fluorescence with respect to control; on the contrary, GFP fluorescence accumulate within the cells in protoplasts co-transformed with secGFP and Sp2. Sp2 reduced the percentage of marker protein secretion to 53% as quantified with secRGUS. In protoplasts obtained from leaves of wild-type and transformed tobacco plants expressing Sp1, Sp2 and Sp1 plus Sp2, no remarkable differences in the percentage of newly synthesised polysaccharides incorporated into the regenerating cell walls were observed. The same results were confirmed in roots of whole transformed seedlings. Tests with cytochalasin D (CD) showed a marked decrease in the amount of newly synthesised polysaccharides into the wall and a simultaneous sharp increase in membrane-associated polysaccharides. SecRGUS secretion was also inhibited by CD. The data indicate that marker proteins and matrix polysaccharides, as well as cellulose synthase complexes, are secreted through the involvement of different secretory machineries.

Exocytosis molecular mechanisms in plant cells are not fully understood. The full characterization of molecular determinants, such as SNAREs, for the specificity in vesicles delivery to the plasma membrane should shed some light on these mechanisms. Nicotiana tabacum Syntaxin 1 (NtSyr1 or SYP121) is a SNARE protein required for ABA control of ion channels and appears involved in the exocytosis of exogenous markers.NtSyr1 is mainly localized on the plasma membrane, but when over expressed the protein also appears on endomembranes. Since NtSyr1 is a tail-anchored protein inserted into the target membrane post-translationally, it is not clear whether its initial anchoring site is the ER or the plasma membrane.In this study, we investigated the sorting events of NtSyr1 in vivo using its full-length cDNA or its C-terminal domain, fused to a GFP tag and transiently expressed in protoplasts or in the leaves of Nicotiana tabacum cv. SR1. Five chimeras were produced of which two were useful to investigate the protein sorting within the endomembrane system. One (GFP-H3M) had a dominant negative effect on exocytosis; the other one (SP1-GFP) resulted in a slow targeting to the same localization of the full-length chimera (GFP-SP1). The insertion of signal peptides on SP1-GFP further characterized the insertion site for this protein. Our data indicates that NtSyr1 is firstly anchored on ER membrane and then sorted to plasma membrane.

Two vacuolar green fluorescent proteins (GFP) were stably inserted in Nicotiana tabacum and Nicotiana benthamiana genome, with unexpected difficulties, and compared with A. thaliana cv. Wassilewskaja transgenic plants expressing the same constructs. GFP fluorescence was strong in all tissues of A. thaliana but it was barely visible in Nicotiana. Confocal microscopy analysis revealed a variable distribution of the marker in those cells where GFP fluorescence was visible. The role of light dependent proteases was the variable pointing out more inter-species diversity. GFPs degradation was much higher in Nicotiana spp. than in A. thaliana. The version of GFP used appeared not to be a good vacuolar marker for Nicotiana differentiated tissues, although it can efficiently label vacuoles in protoplasts or calli. Nevertheless the sensitivity of the reporter protein can be used as an indicator of hidden characteristics of the plant vacuoles, revealing differences otherwise invisible. One of the markers in our system, GFP-Chi, evidenced a clear morphological difference in the vacuolar system of guard cells of the three species.

The uptake of (8-14c)hypoxanthine by human erythrocytes suspended in a phosphate-free medium has been studied. The erythrocytes freshly drawn from human healty donors were washed twice and resuspended in isotonic NaCl medium containing 1-30 uM labelled hypoxanthine to give a 50% hematocrit. At regular intervals, the cells were separated from the bathing medium by rapid centrifugation through a layer of dibutyl phthalate in order to determine hypoxanthine uptake. Under these conditions, more than 90% o f labelled hypoxanthine taken up by the cells was present in the cytosol a s such. The time course o f hypoxanthine uptake behaved a s a single exponential process, the relaxation time being reversely related to the temperature o f the cell suspension ( 5 = 130 sec at o'c).Hypoxanthine was not appreciably taken up by erythrocytes when the external site of the cell membranes was partially degested by proteinase K. PRIMARY GOUT IN AGING PATIENTS26 A. Carcassi, S. Boschi, F. D8Ubaldo and S. Campagna.The onset of primary gout in aging patients has been rarer in the past than it is today. We observed late onset-over 65 years of age-of gout in 15 patients or in 14% out of 104 patients. Comparative studies of clinic and physiopatological manifestations of these 15 patients revealed the following:-prevalence of females was higher (26.5%), familial incidenceo'i the disease was present only in 6%, overweight was uncommon (13%). In most cases hyperuricemia was present (mean value 8.3 mg%), with significant reduction of uric acid clearance (mean value 6.7 ml/min) while uricosuria was normal (mean value 672 mg/24h.) The site of initial attack was the first metatarsal falangeal joint (podagra) in 60%. Tophi were never found. Renal calculi were observed only in 2 cases (13%). Cholesterol and tryglicerides mean levels were normal meanwhile significantly lower levels of HDL-Cholesterol and of Apo-A Lipoproteins were observed in patients with coronary heart disease (20%). Hypertension was present in 33% of the patients. In these patients levels of ionized calcium were significantly lower (mean value 2.04 mEq/l) than in patients with normal blood pressure (mean value 2.18 mEq/l) . HYPOXANTHINE UPTAKE BY ISOLATED BRAIN MICRO-Giacomello, R.Strom, C.Salerno Department of Human Biopathology, Institutes of Biological Chemistry and Rheumatology, University of Rome, and C.N.R. Centre for Molecular Biology, Rome, Italy.Isolated bovine brain microvessels were used as in -vitro model of blood/brain barrier. In a phosphatefree Krebs Ringer medium at 37'~, (8-14~)hypoxanthine is taken up by the microvessels and converted for about 45% in the corresponding nucleotide. The initial rate of the uptake is a hyperbolic function of hypoxanthine concentration in the suspending medium. Inorganic phosphate (up to 1 0 mM) decreases the apparent Km without affecting the apparent Vmax of hypoxanthine uptake. The presence of inorganic phosphate, which (in the absence of hypoxanthine) causes a raise in the ATP concentration in the endothelial cells, induces al...

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