Fluorescence resonance energy transfer detection of synaptophysin I and vesicle-associated membrane protein 2 interactions during exocytosis from single live synapses

To investigate the molecular interactions of synaptophysin I and vesicle-associated membrane protein 2 (VAMP2)/synaptobrevin II during exocytosis, we have used time-lapse videomicroscopy to measure fluorescence resonance energy transfer in live neurons. For this purpose, fluorescent protein variants fused to synaptophysin I or VAMP2 were expressed in rat hippocampal neurons. We show that synaptophysin I and VAMP2 form both homo- and hetero-oligomers on the synaptic vesicle membrane. When exocytosis is stimulated with alpha-latrotoxin, VAMP2 dissociates from synaptophysin I even in the absence of appreciable exocytosis, whereas synaptophysin I oligomers disassemble only upon incorporation of the vesicle with the plasma membrane. We propose that synaptophysin I has multiple roles in neurotransmitter release, regulating VAMP2 availability for the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex and possibly participating in the late steps of exocytosis.     

Effect of 2,4-D and 4-CPPU on somatic embryogenesis from stigma and style transverse thin cell layers of Citrus

Callus induction, somatic embryogenesis and plant regeneration were obtained in lemon [Citrus limon (L.) Burm. cv `Femminello'] and sweet orange [C. sinensis (L.) Osb. cv `Washington Navel GS'] from cultures of stigma and style transverse thin cell layer explants [(t)TCLs]. Explants were cultured on 16 different media, based on the nutrients and vitamins of Murashige and Tucker medium (MT) supplemented with different combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and N-(2-chloro-4-pyridyl)-N-phenylurea (4-CPPU). Sucrose (146 mM) was used as the sole carbon source. Somatic embryos arose from callus at the surface of stigma and style (t)TCLs 3–5 months after culture initiation of both sweet orange and lemon. The percentages of embryo formation from style (t)TCLs ranged from 0% (the media containing 2,4-D) to 16.0% (the medium supplemented with 4 M 4-CPPU) for C. limon. Better results were obtained when stigma (t)TCLs from C. limon were used; in fact, percentages ranged from 0% on the media containing 2,4-D, with the only exception for the medium supplemented with 0.4 M 2,4-D, to 24.8% on medium with 4 M 4-CPPU. The embryogenic response of lemon (t)TCLs was usually higher than for sweet orange (t)TCLs. After about 3 months, somatic embryos developed into plantlets at high frequencies ranging from 53% to 75% for sweet orange and lemon style derived embryos, respectively.     

Structure,function and floristic relationships of plant communities in stressful habitats marginal to the Brazilian Atlantic rainforest

The Brazilian Atlantic rainforest consists of a typical tropical rainforest on mountain slopes, and stands out as a biodiversity hotspot for its high species richness and high level of species endemism. This forest is bordered by plant communities with lower species diversity, due mostly to more extreme environmental conditions than those found in the mesic rainforest. Between the mountain slopes and the sea, the coastal plains have swamp forests, dry semi-deciduous forests and open thicket vegetation on marine sand deposits. At the other extreme, on top of the mountains (>2000 m a.s.l.), the rainforest is substituted by high altitude fields and open thicket vegetation on rocky outcrops. Thus, the plant communities that are marginal to the rainforest are subjected either to flooding, drought, oceanicity or cold winter temperatures. It was found that positive interactions among plants play an important role in the structuring and functioning of a swamp forest, a coastal sandy vegetation and a cold, high altitude vegetation in the state of Rio de Janeiro. Moreover, only a few species seem to adopt this positive role and, therefore, the functioning of these entire systems may rely on them. Curiously, these nurse plants are often epiphytes in the rainforest, and at the study sites are typically terrestrial. Many exhibit crassulacean acid metabolism. Conservation initiatives must treat the Atlantic coastal vegetation as a complex rather than a rainforest alone.     

Human alpha-thrombin stimulates proliferation of interferon-gamma differentiated,growth-arrested U937 cells,overcoming differentiation-related changes in expression of p21CIP1/WAF1 and cyclin D1

In addition to its central role in blood coagulation and hemostasis, human alpha-thrombin is a growth factor for a variety of cell types. We recently demonstrated that interferon-gamma (IFNgamma)-differentiated U937 cells show increased expression of the proteolytically activated receptor for thrombin (PAR-1) relative to undifferentiated U937. In the present study we show that cell proliferation is inhibited in IFNgamma-differentiated cells relative to undifferentiated U937. Addition of thrombin to the differentiated cells, however, overcomes the inhibition and restores the cells to a highly proliferative state. Ribonuclease protection assays indicate that the IFNgamma-induced growth arrest is associated with an increased expression of the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) and downregulation of cyclin D(1). Treatment of cells with thrombin downregulates p21(CIP1/WAF1) expression in these cells and upregulates cyclin D(1) mRNA expression, thus overcoming the differentiation-related effects in a coordinated manner. Treating differentiated cells with the PAR-1 activation peptide, SFLLRN, stimulates proliferation and has effects similar to those of thrombin on expression of p21(CIP1/WAF1). Thus, it appears that these thrombin stimulated proliferative effects are mediated through activation of PAR-1. These results may help explain how thrombin can overcome growth arrest in normal tissue to initiate tissue repair and why thrombin and thrombin-like enzymes may contribute to unrestricted proliferation observed in certain malignancies.     

Molecular recognition between Azotobacter vinelandii rhodanese and a sulfur acceptor protein

The occurrence of rhodanese-like proteins in the major evolutionary phyla, together with the observed abundance of these proteins also within the same genome, suggests that their function cannot be limited to cyanide scavenging. The aim of the present study was to investigate whether Azotobacter vinelandii RhdA, an enzyme possessing unique biochemical and structural features with respect to other members of rhodanese homology superfamily, could recognize a suitable protein as a potential acceptor of the sulfane sulfur held on its catalytic Cys residue. Both the potential sulfur-delivery RhdA-S and the sulfur-deprived RhdA were found to interact with either holo- or apo-adrenodoxin, the 'substrate' protein used in this work. Interaction of RhdA-S with apo-adrenodoxin led to mobilization of RhdA-S sulfane sulfur. Under appropriate conditions, the sulfur released from RhdA-S was productively used for 2Fe-2S cluster reconstitution to yield holo-adrenodoxin from apo-adrenodoxin in the absence of any other sulfur source. A comparison of the reactivity of RhdA-S with protein and non-protein thiols allowed also some insights into the accessibility of the sulfane sulfur carried by RhdA.     

Functional expression of murine V2R pheromone receptors involves selective association with the M10 and M1 families of MHC class Ib molecules

The vomeronasal organ (VNO) of the mouse has two neuronal compartments expressing distinct families of pheromone receptors, the V1Rs and the V2Rs. We report here that two families of major histocompatibility complex (MHC) class Ib molecules, the M10 and the M1 families, show restricted expression in V2R-expressing neurons. Our data suggest that neurons expressing a given V2R specifically co-express one or a few members of the M10 family. Biochemical and immunocytochemical analysis demonstrates that in VNO sensory dendrites M10s belong to large multi-molecular complexes that include pheromone receptors and beta2-microglobulin (beta2m). In cultured cells, M10s appear to function as escort molecules in transport of V2Rs to the cell surface. Accordingly, beta2m-deficient mice exhibit mislocalization of V2Rs in the VNO and a specific defect in male-male aggressive behavior. The functional characterization of M10 highlights an unexpected role for MHC molecules in pheromone detection by mammalian VNO neurons.     

Proteins from bovine tissues and biological fluids: defining a reference electrophoresis map for liver,kidney, muscle,plasma and red blood cells

A number of high resolution two-dimensional electrophoresis (2-DE) reference maps for bovine tissues and biological fluids have been determined for animals in basal state. Among the 1863 distinct protein features detected in samples of liver, kidney, muscle, plasma and red blood cells, 509 species were identified and associated to 209 different genes. Difficulties in the identification were related to the poorly characterized Bos taurus genome and were solved by a combined matrix-assisted laser desorption/ionisation-mass spectrometry and liquid chromatography-electrospray ionization tandem mass spectrometry approach. The experimental output allowed us to establish a 2-DE database accessible through the World Wide Web network at the URL address (http://www.iabbam.na.cnr.it/Biochem). These reference maps may serve as a tool in future veterinary medical studies aimed at the evaluation of changes in protein repertoire for altered animal physiological conditions and infectious diseases, to the definition of molecular markers for novel diagnostic kits and vaccines, as well as the characterization of protein modifications in bovine materials following technological processes used in the food industry.     

Synthesis of radiometal-labeled and fluorescent cell-permeating peptide-PNA conjugates for targeting the bcl-2 proto-oncogene

The B-cell lymphoma/leukemia-2 (bcl-2) proto-oncogene has been associated with the transformation of benign lesions to malignancy, disease progression, poor prognosis, reduced survival, and development of resistance to radiation and chemotherapy in many types of cancer. The objective of this work was to synthesize an antisense peptide nucleic acid (PNA) complementary to the first six codons of the bcl-2 open reading frame, conjugated to a membrane-permeating peptide for intracellular delivery, and modified with a bifunctional chelating agent for targeting imaging and therapeutic radiometals to tumors overexpressing bcl-2. Four peptide-PNA constructs were synthesized by a combination of manual and automated stepwise elongation techniques, including bcl-2 antisense conjugates and nonsense conjugates with no complementarity to any known mammalian gene or DNA sequence. The PNA sequences were synthesized manually by solid-phase 9-fluorenylmethoxycarbonyl (Fmoc) techniques. Then a fully protected lysine monomer, modified with 1,4,7,10-tetraazacyclododecane-N,N',N',N'"-tetraacetic acid (DOTA) for radiometal chelation, was coupled manually to each PNA sequence. Synthesis of the DOTA-PNA conjugates was followed by automated elongation with a peptide sequence (PTD-4-glycine, PTD-4-G), known to mediate cellular internalization of impermeable effector molecules, or its retro-inverso analogue (ri-PTD-4-G). Preparation of the four conjugates required an innovative synthetic strategy, using mild acid conditions to generate hydrophobic, partially deprotected intermediates. These intermediates were purified by semipreparative reversed-phase HPLC and completely deprotected to yield pure peptide-PNA conjugates in 6% to 9% overall yield. Using modifications of this synthetic strategy, the ri-PTD-4-G conjugate of bcl-2 antisense PNA was prepared using a lysine derivative of tetramethylrhodamine (TMR) for fluorescence microscopy. Plasma stability studies showed that (111)In-DOTA-labeled ri-PTD-4-G-anti-bcl-2 PNA was stable for 168 h at 37 degrees C, unlike the conjugate containing the parent peptide sequence. Scanning confocal fluorescence microscopy of TMR-labeled ri-PTD-4-G-anti-bcl-2 PNA in Raji lymphoma cells demonstrated that the retro-inverso peptide was active in membrane permeation and mediated cellular internalization of the antisense PNA into the cytoplasm, where high concentrations of bcl-2 mRNA are expected to be present.     

Helicobacter pylori Infection and Gastric Autoimmune Diseases: Is There a Link?

Background. Helicobacter pylori is thought to be involved in atrophic body gastritis. We explored the prevalence of H. pylori infection in asymptomatic subjects with gastric parietal cell antibodies, as well as in patients with pernicious anemia, to evaluate a possible role of H. pylori gastric infection in gastric autoimmunity. Patients and Methods. We studied 79 consecutive asymptomatic subjects with parietal cell antibodies, 24 patients with pernicious anemia, and 66 parietal cell antibody‐negative controls. All patients underwent gastric biopsies for histology and detection of H. pylori. Red blood cell count and volume, serum levels of gastrin, pepsinogen I, iron, folic acid, vitamin B₁₂, and circulating antibodies to H. pylori and to intrinsic factor were also determined. Results. We found an atrophic body gastritis in 14 of the 79 asymptomatic subjects with parietal cell antibodies (18%) and in 2 of the 66 controls (3%) (p = .01). Mean levels of gastrin were increased (p < .0001), while those of pepsinogen were reduced (p < .001) compared with controls. H. pylori was identified at the gastric level and/or circulating anti‐H. pylori antibodies were detected in 46 parietal cell antibody‐positive subjects (58%) compared with 26 controls (39%) (p = .03). In patients with pernicious anemia we found an atrophic body gastritis in 18 of 24 cases (75%) (p < .001 vs. controls). Mean levels of gastrin were markedly increased (p < .0001) and those of pepsinogen I decreased (p < .0001) relative to controls. Only five of these patients (21%) had evidence of H. pylori infection compared with 46 of the parietal cell antibody‐positive subjects (58%) (p = .003) and 26 of the controls (39%). Considering all patients with gastric autoimmunity (i.e. with parietal cell antibodies and/or with pernicious anemia), H. pylori was found in 44 of 72 of those without atrophy (61%) but in 6 of 31 with gastric body atrophy (19%) (p < .001), indicating that H. pylori infection is greatly reduced when gastric acid secretion decreases. Conclusions. The frequent detection of H. pylori infection in subjects with early gastric autoimmunity, indicated by the presence of parietal cell antibodies, suggests that H. pylori could have a crucial role in the induction and/or the maintenance of autoimmunity at the gastric level.