Cellulase involvement in the abscission of peach and pepper leaves is affected by salicylic acid

Phenolic compounds appear to be involved in a number of regulatoryactivities in plants. During the last decade an increasing amountof evidence has brought to light the role played by salicylicacid in a number of physiological processes. Particularly interestingis the inhibitory effect of salicylic acid on ethylene biosynthesis,which might make this natural plant compound a useful tool forcontrolling some of the responses usually promoted by ethylene.Our data show that salicylic acid is actually able to reduceleaf abscission in both peach and pepper plants. Biochemicalanalyses have revealed that the enzyme usually involved in thisphenomenon (cellulase, EC 3.2.1.4 [EC] .) does not increase followingactivation of leaf abscission in plants treated with salicylicacid. In contrast, control plants show a marked increase inthe levels of both enzyme activity and cellulase protein. Flushingplants with exogenous ethylene in the presence of salicylicacid induces an increase in cellulase expression which, however,does not equal the level induced in plants without salicylicacid. Key words: Cellulase, leaf abscission, peach, pepper, salicylic acid     

Elucidation of the structure of constrained bicyclopeptides in solution by two-dimensional cross-relaxation spectroscopy: Amatoxin analogues

The evaluation of peptide structures in solution is made feasible by the combined use of two-dimensional NMR in the laboratory (NOESY) and rotating frames (ROESY), and by the use of molecular dynamics calculations. The present paper describes how both the NMR method and molecular dynamics calculations were applied to very rigid synthetic bicyclic peptides that are analogues of natural amatoxins. The NMR theory, which allows the estimate of interatomic distances between interacting nuclei, is briefly discussed. The experimental data were compared with those of known solid-state structures. Three amatoxin analogues have been examined. Of these, one is biologically active (S-deoxo γ[R] OH-Ile³-amaninamide) and its structure in the solid state has recently been worked out. The second and third analogues (S-deoxo-Ile³ -Ala⁵-amaninamide and S-deoxo-D -Ile³ -amaninamide, respectively) are inactive and their solid-state structures are unknown. The data presented confirm the authors' previous hypothesis that lack of biological activity of S-deoxo-Ile³-Ala⁵- amaninamide is due to the masking of the tryptophan ring by the methyl group of L -Ala and not to massive conformational changes of the analogue.     

Indomethacin prevents the long-lasting inhibitory effect of aspirin on human platelet cyclo-oxygenase activity

Aspirin and indomethacin do interact with the same site on cyclo-oxygenase. This suggestion is based on in vitro studies on ram seminal vesicles and in vivo drug interaction studies on rat platelets. The purpose of the present study was to ascertain whether the same interaction also occurred after administration of both drugs to human volunteers. Platelet aggregation induced by sodium arachidonate or by collagen, and formation of platelet MDA and TxB2 were measured before, two and 48 hours after ingestion of either indomethacin (50 mg) or aspirin (500 mg) or of both drugs (30 minutes apart). While the inhibitory effect of indomethacin on these parameters was short lasting, that of aspirin persisted for at least 48 hours. However, when both drugs were given concurrently, the long-lasting effect of aspirin was no longer detectable. Since competition at levels other than platelets was unlikely, this study indicates that indomethacin and aspirin inhibit human platelet cyclo-oxygenase by the same basic mechanism. Acetylation of the enzyme appears to be a secondary mechanism which makes the inhibitory effect of aspirin persistent.     

Mycosporine-like amino acids protect the copepod Boeckella titicacae (Harding) against high levels of solar UVR

Studies were conducted in Lake Titicaca, Bolivia (16°S,69°W, 3810 m a.s.l.) to determine the effects of solar ultravioletradiation (UVR; 280–400 nm) and the protective role ofmycosporine-like amino acids (MAAs) in the calanoid copepodBoeckella titicacae (Harding). Zooplankton samples were takenfrom two different radiation environments: Lake Titicaca anda tank with running lake water, which was covered with layersof neutral-density screen for over a year, receiving ~10% ofincident radiation. Samples collected from the lake had significantlyhigher amounts of MAAs compared with those from the tank. Duringshort-term experiments (6–8 h incubations; six radiationtreatments using sharp cut-off Schott filters), significantlyhigher mortality was observed in B. titicacae from the tankthan from the lake. The higher sensitivity of B. titicacae containinglow amounts of MAAs (compared with specimens collected fromthe lake) was assessed through the determination of biologicalweighting functions (BWFs). A 3-day-long experiment performedwith specimens collected from the lake indicated an increasein mortality as a function of UVR exposure, reaching valuesas high as 70% at the end of day 3 for those individuals thatreceived full radiation compared with <10% for those thatreceived only photosynthetically active radiation (PAR; 400–700nm). Concomitantly, a reduction in the concentration of UV-absorbingcompounds as a function of UVR dose and decreasing wavelengthwas determined. The relatively high UVR dose threshold for mortality(~300 kJ m^–2), as well as a comparison with related species,demonstrated the high resistance of B. titicacae to UVR. Ourresults indicate that this resistance is provided by the presenceof protective compounds, i.e. MAAs, which were efficiently bioaccumulatedthrough the diet.     

Growth Factor–dependent Activation of αvβ3 Integrin in Normal Epithelial Cells: Implications for Tumor Invasion

Integrin activation is a multifaceted phenomenon leading to increased affinity and avidity for matrix ligands. To investigate whether cytokines produced during stromal infiltration of carcinoma cells activate nonfunctional epithelial integrins, a cellular system of human thyroid clones derived from normal glands (HTU-5) and papillary carcinomas (HTU-34) was employed. In HTU-5 cells, αvβ3 integrin was diffused all over the membrane, disconnected from the cytoskeleton, and unable to mediate adhesion. Conversely, in HTU-34 cells, αvβ3 was clustered at focal contacts (FCs) and mediated firm attachment and spreading. αvβ3 recruitment at FCs and ligand-binding activity, essentially identical to those of HTU-34, occurred in HTU-5 cells upon treatment with hepatocyte growth factor/scatter factor (HGF/SF). The HTU-34 clone secreted HGF/SF and its receptor was constitutively tyrosine phosphorylated suggesting an autocrine loop responsible for αvβ3 activated state. Antibody-mediated inhibition of HGF/SF function in HTU-34 cells disrupted αvβ3 enrichment at FCs and impaired adhesion. Accordingly, activation of αvβ3 in normal cells was produced by HTU-34 conditioned medium on the basis of its content of HGF/SF. These results provide the first example of a growth factor–driven integrin activation mechanism in normal epithelial cells and uncover the importance of cytokine-based autocrine loops for the physiological control of integrin activation.     

Unrip is a component of SMN complexes active in snRNP assembly

A macromolecular complex containing survival of motor neurons (SMN), the spinal muscular atrophy protein, and Gemin2-7 interacts with Sm proteins and snRNAs to carry out the assembly of these components into spliceosomal small nuclear ribonucleoproteins (snRNPs). Here we report the characterization of unr-interacting protein (unrip), a GH-WD protein of unknown function, as a component of the SMN complex that interacts directly with Gemin6 and Gemin7. Unrip also binds a subset of Sm proteins, and unrip-containing SMN complexes are necessary and sufficient to mediate the assembly of spliceosomal snRNPs. These results demonstrate that unrip functions in the pathway of snRNP biogenesis and is a marker of cellular SMN complexes active in snRNP assembly.     

First-phase insulin secretion: does it exist in real life? Considerations on shape and function

To fulfill its preeminent function of regulating glucose metabolism, insulin secretion must not only be quantitatively appropriate but also have qualitative, dynamic properties that optimize insulin action on target tissues. This review focuses on the importance of the first-phase insulin secretion to glucose metabolism and attempts to illustrate the relationships between the first-phase insulin response to an intravenous glucose challenge and the early insulin response following glucose ingestion. A clear-cut first phase occurs only when the beta-cell is exposed to a rapidly changing glucose stimulus, like the one induced by a brisk intravenous glucose administration. In contrast, peripheral insulin concentration following glucose ingestion does not bear any clear sign of biphasic shape. Coupling data from the literature with the results of a beta-cell model simulation, a close relationship between the first-phase insulin response to intravenous glucose and the early insulin response to glucose ingestion emerges. It appears that the same ability of the beta-cell to produce a pronounced first phase in response to an intravenous glucose challenge can generate a rapidly increasing early phase in response to the blood glucose profile following glucose ingestion. This early insulin response to glucose is enhanced by the concomitant action of incretins and neural responses to nutrient ingestion. Thus, under physiological circumstances, the key feature of the early insulin response seems to be the ability to generate a rapidly increasing insulin profile. This notion is corroborated by recent experimental evidence that the early insulin response, when assessed at the portal level with a frequent sampling, displays a pulsatile nature. Thus, even though the classical first phase does not exist under physiological conditions, the oscillatory behavior identified at the portal level does serve the purpose of rapidly exposing the liver to elevated insulin levels that, also in virtue of their up-and-down pattern, are particularly effective in restraining hepatic glucose production.     

Adhesion Properties and Integrin Expression of Cultured Human Osteoclast-like Cells

Osteoclast interaction with extracellular matrix drives the sequential events that end with bone resorption. However, the role of matrix proteins is not yet fully understood. We studied this problem on human osteoclast-like cells derived from giant cell tumors of bone (GCT cells). On GCT cells we considered cytoskeletal organization, adhesion properties, and integrin expression upon plating in serum-free medium onto fibronectin (FN), collagen (COL), thrombospondin (TSP), bone sialoprotein (BSPII), and osteopontin (OPN). GCT cells promptly adhered and spread on FN, BSPII, and OPN, while only 50% adhered on COL and none on TSP. The integrin β₁ chain was always associated to focal adhesions, while the α_vβ₃ heterodimer was detected in focal contacts only upon plating on BSPII, OPN, and FN. The focal clustering of β₁ was impaired by monensin treatment, indicating that endogenous FN secretion was required to drive β₁ into focal contacts. Conversely, α_vβ₃ clustering was also not affected by monensin when cells were plated onto plasma FN. Immunoprecipitation of metabolically labeled GCT cell lysates showed that three different heterodimers (α_vβ₃, α₃β₁, and α₅β₁) were assembled. Adhesion to FN was completely inhibited by β₁ antibodies at dilutions up to 1:400, while β₃ antibodies, at similar dilutions, impaired spreading but not adhesion. We conclude that α_vβ₃3 is the main integrin used by GCT cells in bone recognition. We also suggest that selected substrata may induce the release and the organization of endogenous FN that eventually drives the recruitment of a β₁ integrin receptor into focal contacts.     

Evaluation of errors of interproton distances and correlation time determined from NMR cross-relaxation rates

Summary We have analyzed a combined use of the two-dimensional nuclear Overhauser effect in the laboratory frame (NOESY) and in the rotating frame (ROESY) to determine interproton distances and correlation time in medium-sized rigid molecules (Davis, 1987). This method can be applied in the intermediate motional regime, 0.2 < _oc, < 5, (_c, correlation time, (_o resonance frequency). Error limits depend on the motional regime and are smallest near _oc=1.14.The method was tested on six geminal proton pairs in the bicyclic octapeptide (S-deoxo--[R]-OH-Ile³ amaninamide, Mw =870) for which at 297 K in DMSO, a correlation time of 1.0 ns, with a standard deviation of 0.12 ns, and an interproton distance of 1.87 Å, with standard deviation of 0.04 Å, are obtained.     

Metabolism of arachidonic acid in isolated glomeruli from pig kidney

Arachidonic acid metabolism in isolated glomeruli from pig kidney was investigated. Arachidonic acid metabolism via cyclooxygenase was studied by three different methodological approaches: radioimmunoassay (RIA), high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). By all these techniques, the major prostaglandins (PG) formed by pig glomeruli appeared to be 6-keto-PGF1 alpha and PGF2 alpha, the former being the most abundant. RIA and GC-MS also detected lower amounts of thromboxane B2 (TxB2) and PGE2. This emphasises the similarity with human glomeruli, in which the main cyclooxygenase product has indeed been reported to be 6-keto-PGF1 alpha. The lipoxygenase activity in isolated pig glomeruli, as studied by HPLC, generated 15-HETE, 12-HETE and 5-HETE. These data demonstrate that isolated glomeruli from pig kidney possess cyclooxygenase as well as lipoxygenase activity. Since a marked functional similarity exists between human and pig kidney, the pig can be regarded as a good model for studying the influence of arachidonic acid metabolites on glomerular pathophysiology.