Membrane recycling and calcium dynamics during settlement and adhesion of zoospores of the green alga Ulva linza.

Recruitment of individuals of the marine alga Ulva linza on to a suitable habitat involves the settlement of motile zoospores on to a substratum during which a preformed adhesive is secreted by vesicular exocytosis. The fluorescent styryl dye FM 1-43 and fluorescent Ca(2+) indicators were used to follow membrane cycling and changes in cytosolic Ca(2+) ([Ca(2+)](cyt)) associated with settlement. When swimming zoospores were exposed continuously to FM 1-43, the plasma membrane was preferentially labelled. During settlement, FM 1-43-labelled plasma membrane was rapidly internalized reflecting high membrane turnover. The internalized membrane was focused into a discrete region indicating targeting of membrane to an endosome-like compartment. Acetoxymethyl (AM)-ester derivatives were found to be unsuitable for monitoring [Ca(2+)](cyt) because the dyes were rapidly sequestered from the cytoplasm into sub-cellular compartments. [Ca(2+)](cyt) was, however, reliably measured using dextran-conjugated calcium indicators delivered into cells using a biolistic technique. Cells loaded with Oregon Green BAPTA-1 dextran (Invitrogen, Paisley, UK) showed diffuse cytosolic loading and reliably responded to imposed changes in [Ca(2+)](cyt). During settlement, zoospores exhibited both localized and diffuse increases in [Ca(2+)](cyt) implying a role for [Ca(2+)](cyt) in exocytosis of the adhesive.

Ascorbic acid in plants: biosynthesis and function.

Ascorbic acid (vitamin C) is an abundant component of plants. It reaches a concentration of over 20 mM in chloroplasts and occurs in all cell compartments, including the cell wall. It has proposed functions in photosynthesis as an enzyme cofactor (including synthesis of ethylene, gibberellins and anthocyanins) and in control of cell growth. A biosynthetic pathway via GDP-mannose, GDP-L-galactose, L-galactose, and L-galactono-1,4-lactone has been proposed only recently and is supported by molecular genetic evidence from the ascorbate-deficient vtc 1 mutant of Arabidopsis thaliana. Other pathways via uronic acids could provide minor sources of ascorbate. Ascorbate, at least in some species, is a precursor of tartrate and oxalate. It has a major role in photosynthesis, acting in the Mehler peroxidase reaction with ascorbate peroxidase to regulate the redox state of photosynthetic electron carriers and as a cofactor for violaxanthin de-epoxidase, an enzyme involved in xanthophyll cycle-mediated photoprotection. The hypersensitivity of some of the vtc mutants to ozone and UV-B radiation, the rapid response of ascorbate peroxidase expression to (photo)-oxidative stress, and the properties of transgenic plants with altered ascorbate peroxidase activity all support an important antioxidative role for ascorbate. In relation to cell growth, ascorbate is a cofactor for prolyl hydroxylase that posttranslationally hydroxylates proline residues in cell wall hydroxyproline-rich glycoproteins required for cell division and expansion. Additionally, high ascorbate oxidase activity in the cell wall is correlated with areas of rapid cell expansion. It remains to be determined if this is a causal relationship and, if so, what is the mechanism. Identification of the biosynthetic pathway now opens the way to manipulating ascorbate biosynthesis in plants, and, along with the vtc mutants, this should contribute to a deeper understanding of the proposed functions of this multifaceted molecule.

A coumarin-based prodrug strategy to improve the oral absorption of RGD peptidomimetics.

In recent years, major progress has been made in the design and synthesis of fibrinogen antagonists, which are peptidomimetic Arg-Gly-Asp (RGD) analogs. These RGD analogs are very promising antiplatelet agents. However, the clinical development of orally active RGD analogs has been hindered by the low oral bioavailability of many such RGD analogs. Aimed at enhancing their oral bioavailability, we have synthesized several coumarin-based cyclic prodrugs of RGD analogs, which have the two most polar functional groups, a carboxyl and an amino group, masked as an ester and an amide, respectively. As expected, these cyclic prodrugs have higher membrane interaction potentials as estimated by determining their partitioning between aqueous buffer and an immobilized artificial membrane than the corresponding RGD analogs. Consequently, these cyclic prodrugs are 5-6-fold more able to permeate monolayers of Caco-2 cells, an in vitro cell culture model of the intestinal mucosa barrier. Preliminary studies using dog also indicate the promising potential of using this coumarin-based prodrug strategy to improve the oral bioavailability of such RGD analogs.

Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis.

Vitamin C (L-ascorbic acid; AsA) acts as a potent antioxidant and cellular reductant in plants and animals. AsA has long been known to have many critical physiological roles in plants, yet its biosynthesis is only currently being defined. A pathway for AsA biosynthesis that features GDP-mannose and L-galactose has recently been proposed for plants. We have isolated a collection of AsA-deficient mutants of Arabidopsis thaliana that are valuable tools for testing of an AsA biosynthetic pathway. The best-characterized of these mutants (vtc1) contains approximately 25% of wild-type AsA and is defective in AsA biosynthesis. By using a combination of biochemical, molecular, and genetic techniques, we have demonstrated that the VTC1 locus encodes a GDP-mannose pyrophosphorylase (mannose-1-P guanyltransferase). This enzyme provides GDP-mannose, which is used for cell wall carbohydrate biosynthesis and protein glycosylation as well as for AsA biosynthesis. In addition to genetically defining the first locus involved in AsA biosynthesis, this work highlights the power of using traditional mutagenesis techniques coupled with the Arabidopsis Genome Initiative to rapidly clone physiologically important genes.

The biosynthetic pathway of vitamin C in higher plants.

Vitamin C (L-ascorbic acid) has important antioxidant and metabolic functions in both plants and animals, but humans, and a few other animal species, have lost the capacity to synthesize it. Plant-derived ascorbate is thus the major source of vitamin C in the human diet. Although the biosynthetic pathway of L-ascorbic acid in animals is well understood, the plant pathway has remained unknown-one of the few primary plant metabolic pathways for which this is the case. L-ascorbate is abundant in plants (found at concentrations of 1-5 mM in leaves and 25 mM in chloroplasts) and may have roles in photosynthesis and transmembrane electron transport. We found that D-mannose and L-galactose are efficient precursors for ascorbate synthesis and are interconverted by GDP-D-mannose-3,5-epimerase. We have identified an enzyme in pea and Arabidopsis thaliana, L-galactose dehydrogenase, that catalyses oxidation of L-galactose to L-galactono-1,4-lactone. We propose an ascorbate biosynthesis pathway involving GDP-D-mannose, GDP-L-galactose, L-galactose and L-galactono-1,4-lactone, and have synthesized ascorbate from GDP-D-mannose by way of these intermediates in vitro. The definition of this biosynthetic pathway should allow engineering of plants for increased ascorbate production, thus increasing their nutritional value and stress tolerance.

Photoaffinity identification of colchicine-solubilized regulatory subunit from rat brain adenylate cyclase.

Five GTP binding proteins in rat cerebral cortex synaptic membranes were identified by photoaffinity labelling with [3H] or [32P](P3-azido-anilido)-P1-5' GTP (AAGTP). When AAGTP-treated membranes were incubated with colchicine or vinblastine and subsequently washed, a single AAGTP-labelled protein of 42 kD was released into the supernatant. About 30% of the total labelled 42-kD protein was released into supernatants from membranes pretreated with colchicine or vinblastine compared with 15% released from control membranes. The amount of adenylate cyclase regulatory subunit (G unit) remaining in these membranes was assessed with reconstitution studies after inactivating the adenylate cyclase catalytic moiety with N-ethylmaleimide (NEM). Forty to fifty percent of functional G units were lost from membranes treated with colchicine prior to washing. This 40-50% loss of functional G unit after colchicine treatment corresponds to the previously observed 42% loss of NaF and guanylyl-5'-imidodiphosphate [Gpp(NH)p]-activated adenylate cyclase. Release of the AAGTP-labelled 42-kD protein from colchicine-treated synaptic membranes is double that from lumicolchicine-treated membranes. This colchicine-mediated release of 42-kD protein correlates with a doubling of functional G unit released from synaptic membranes after colchicine treatment. These findings suggest multiple populations of the G unit within the synaptic plasma membrane, some of which may interact with cytoskeletal components.

Limited trypsin proteolysis of photoreceptor GTP-binding protein. Light- and GTP-induced conformational changes.

The amphibian photoreceptor rod outer segment contains a guanine nucleotide-binding complex which consists of a 39,000-dalton polypeptide that binds guanine nucleotides (G protein), a 36,000-dalton polypeptide (H protein), and an approximately 6,500-dalton polypeptide. Sensitivity to trypsin proteolysis was utilized as a probe of structure-function relationships for these polypeptides. Digestion of the H protein generated fragments of 26,000 and 15,000 daltons whose proteolytic susceptibility was not altered by guanosine triphosphates, light, or membranes. The approximately 6,500-dalton polypeptide was not trypsin sensitive. When the G protein was eluted from illuminated membranes by GTP, trypsin proteolysis cleaved a terminal 1,000-dalton fragment (G1) to yield a 38,000-dalton fragment (G38). With increased digestion time, a 6,000-dalton fragment (G6) was removed from G38 to yield a 32,000-dalton fragment (G32). G32 was subsequently digested to fragments of 23,000 and 12,000 daltons. However, when the G protein was eluted from illuminated membranes by hydrolysis-resistant analogues of GTP, G32 was protected from further digestion. This is consistent with a GTP-induced conformational change in the G protein which is altered by GTP hydrolysis. Proteolysis of the G protein after covalent labeling with a photoaffinity analogue of GTP demonstrated that the analogue is bound to first G38 and then G32, indicating the GTP-binding site is associated with G32. Fragment G6 was cleaved when the G protein was soluble or bound to unilluminated membranes. However, when bound to illuminated membranes, fragments were generated reflecting the loss of 7,500, 9,000, or 11,000 daltons from the G protein. This light-induced alteration in proteolytic susceptibility indicates there is a light-induced conformational change in the G protein. Fragment G1 was not removed from the G protein when it was membrane bound, suggesting G1 is involved in binding to a membrane structure. These data suggest that the light-induced binding of the G protein to illuminated membranes and the reversal of this binding by GTP are mediated through conformational changes in the G protein and that three conformations exist: 1) a basal, inactive conformation; 2) a primed conformation induced by binding to photolyzed rhodopsin, with a high affinity for GTP; and 3) an active conformation, induced by binding of GTP, which activates the catalytic complex of light-activated phosphodiesterase.

Functional exchange of components between light-activated photoreceptor phosphodiesterase and hormone-activated adenylate cyclase systems.

Previous studies have noted profound similarities between the regulation of light-activated 3',5'-cyclic nucleotide phosphodiesterase (3',5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) in retinal rods and hormone-activated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in a variety of tissues. We report here the functional exchange of components isolated from the photoreceptor system, which displayed predicted functional characteristics when incubated with recipient adenylate cyclase systems from rat cerebral cortical and hypothalamic synaptic membranes and frog erythrocyte ghosts. We demonstrate functional exchange of photoreceptor components at each of three loci: the hormone receptor, the GTP-binding protein (GBP), and the catalytic moiety of adenylate cyclase. Illuminated (but not unilluminated) rhodopsin was fund to mimic the hormone-receptor complex, causing GTP-dependent activation of adenylate cyclase. The photoreceptor GBP complexed with guanosine 5'-[beta, gamma)imidotriphosphate (p[NH]ppG) produced a marked activation of recipient adenylate cyclase systems. Much smaller activation was observed when GBP was not complexed with p[NH]ppG. A heat-stable photoreceptor phosphodiesterase inhibitor reduced both basal and Mn2+-activated adenylate cyclase activities and this inhibition was reversed by photoreceptor GBP.p[NH]ppG. These data demonstrate a remarkable functional compatibility between subunits of both systems and furthermore imply that specialized peptide domains responsible for protein-protein interactions are highly conserved.

A GTP-protein activator of phosphodiesterase which forms in response to bleached rhodopsin.

A specific protein associated with rod-outer-segment disc membranes binds GTP only in the presence of bleached rhodopsin. Once formed the protein-GTP complex becomes a soluble activator of cGMP phosphodiesterase. It is shown that this activator complex can be completely separated from rhodopsin and retain its ability to activate phosphodiesterase when added to a pool of totally dark (unilluminated) disc membranes. The photoreactive GTP analogue p3-(4-azidoanilido)-5' GTP (AAGTP) is shown to be a more effective substrate than GTP, Gpp(NH)p or 8-azido GTP. [8, 5' 3H] AAGTP was used to specifically covalently label the GTP-binding protein. The protein labeled exhibits a mass of 40,000 daltons when analyzed by SDS-PAGE.

Complex partial status epilepticus (psychomotor status).

A 20-year-old mentally retarded woman, who had a history of intractable epileptic seizures since early childhood, had prolonged episodes of confusion, decreased responsiveness and automatic behavior lasting as long as 2 days. These are believed to represent epileptic status of the complex partial (psychomotor) type because the electroencephalogram (EEG) recorded during two such periods showed continuous high amplitude, semirhythmic, 4 to 6 cycles per second (cps), spike activity over both frontotemporal regions; several interictal tracings revealed a consistent spike focus in the right anterior temporal area. Diazepam given intravenously during one such episode terminated both the abnormal behavior and the ictal discharges in the EEG.

A light-activated GTPase in vertebrate photoreceptors: regulation of light-activated cyclic GMP phosphodiesterase.

We have been studying the mechanism by which light and nucleoside triphosphates activate the discmembrane phosphodiesterase (oligonucleate 5'-nucleotidohydrolase; EC 3.1.4.1) in frog rod outer segments. GTP is orders of magnitude more effective than ATP as a cofactor in the light-dependent activation step. GTP and the analogue guanylyl-imidodiphosphate function equally as allosteric activators of photoreceptor phosphodiesterase rather than participating in the formation of a phosphorylated activator. Moreover, we have found a light-activated (5-fold) GTPase which participates in the modulation of photoreceptor phosphodiesterase. This GTPase activity appears necessary for the reversal of phosphodiesterase activation in vitro and may play a critical role in the in vivo regulation of light-sensitive phosphodiesterase. The K(m) for GTP in the light-activated GTPase reaction is <1 muM. The light sensitivity of this GTPase (number of photons required for half-maximal activation) is identical to that of light-activated phosphodiesterase. The GTPase action spectrum corresponds to the absorption spectrum of rhodopsin. There is, in addition, a light-insensitive GTPase activity with a K(m) for GTP of 90 muM. At GTP concentrations above 5 muM, there is no appreciable activation of GTPase activity by light. The substrate K(m) values for guanylate cyclase, light-activated GTPase, and light-activated phosphodiesterase order an enzyme array that might permit light to simultaneously cause the hydrolysis of both the substrate and product of guanylate cyclase. These findings reveal yet another facet of light regulation of photoreceptor/cyclic GMP levels and also provide a striking analogy to the GTP regulation of nonphotoreceptor, hormone-sensitive adenylate cyclase.