Vascular endothelial growth factor receptor-3 activity is modulated by its association with caveolin-1 on endothelial membrane

Vascular endothelial growth factor receptor-3 (VEGFR-3) is constitutively expressed in lymphatic vessels and transiently in endothelial cells of blood vessels during angiogenesis. Here we report that VEGFR-3 localizes in the caveolae membrane of endothelial cells and co-immunoprecipitates with caveolin-1. Caveolin-1 silencing or its depletion from the cell membrane by cholesterol increases VEGFR-3 autophosphorylation, suggesting that caveolin acts as a negative regulator of VEGFR-3 activity. Receptor activation induces caveolin-1 phosphorylation on tyrosine residues including tyrosine 14. Cell treatment with Src or Abl inhibitors PP2 or STI571, prior to receptor stimulation, affects caveolin-1 phosphorylation without affecting receptor autophosphorylation, suggesting that both Src and Abl are involved in VEGFR-3-dependent caveolin-1 phosphorylation. Caveolin-1 phosphorylation in Src/Fyn/Yes knockout cells demonstrated that Abl phosphorylates caveolin-1 independently from Src family members. These results suggest a functional interaction between VEGFR-3 and caveolin-1 to modulate endothelial cell activation during angiogenesis.     

Site-directed mutagenesis of two aromatic residues lining the active site pocket of the yeast Ltp1

We mutated Trp(134) and Tyr(135) of the yeast LMW-PTP to explore their catalytic roles, demonstrating that the mutations of Trp(134) to Tyr or Ala, and Tyr(135) to Ala, all interfere with the formation of the phosphorylenzyme intermediate, a phenomenon that can be seen by the decrease in the kinetic constant of the chemical step (k(3)). Furthermore, we noted that the Trp(134) to Ala mutation causes a dramatic drop in k(cat)/K(m) and a slight enhancement of the dissociation constant K(s). The conservative mutant W134Y shows a k(cat)/K(m) very close to that of wild type, probably compensating the two-fold decrease of k(3) with an increase in substrate affinity. The Y135A mutation enhances the substrate affinity, but reduces the enzyme phosphorylation rate. The replacement of Trp(134) with alanine interferes with the partition between phosphorylenzyme hydrolysis and phosphotransfer from the phosphorylenzyme to glycerol and abolish the enzyme activation by adenine. Finally, we found that mutation of Trp(134) to Ala causes a dramatic change in the pH-rate profile that becomes similar to that of the D132A mutant, suggesting that an aromatic residue in position 134 is necessary to assist the proper positioning of the proton donor in the transition state of the chemical step.     

Antibody-based targeting of the tumor vasculature

Conventional cytotoxic therapies of cancer often suffer from a lack of specificity, leading to a poor therapeutic index and considerable toxicities to normal organs. An elegant way to overcome the disadvantages of conventional tumor therapy is the selective delivery of therapeutics to the tumor site by their conjugation to a carrier molecule specific for a tumor-associated molecular marker. Markers expressed on the tumor's vasculature represent particularly attractive targets for a site-specific pharmacodelivery due to their inherent accessibility for blood-borne agents and the various therapeutic options that they allow, ranging from intraluminal blood coagulation to the recruitment of immune cells. In this review, we will outline advances in the preclinical and clinical evaluation of antibody-based vascular targeting agents, describe technologies for the discovery of novel vascular targets and discuss future prospects for vascular targeting applications.     

Heterogeneity of photosynthetic membranes from Rhodobacter capsulatus: size dispersion and ATP synthase distribution

The density distribution of photosynthetic membrane vesicles (chromatophores) from Rhodobacter capsulatus has been studied by isopicnic centrifugation. The average vesicle diameters, examined by electron microscopy, varied between 61 and 72 nm in different density fractions (70 nm in unfractionated chromatophores). The ATP synthase catalytic activities showed maxima displaced toward the higher density fractions relative to bacteriochlorophyll, resulting in higher specific activities in those fractions (about threefold). The amount of ATP synthase, measured by quantitative Western blotting, paralleled the catalytic activities. The average number of ATP synthases per chromatophore, evaluated on the basis of the Western blotting data and of vesicle density analysis, ranged between 8 and 13 (10 in unfractionated chromatophores). Poisson distribution analysis indicated that the probability of chromatophores devoid of ATP synthase was negligible. The effects of ATP synthase inhibition by efrapeptin on the time course of the transmembrane electric potential (evaluated as carotenoid electrochromic response) and on ATP synthesis were studied comparatively. The ATP produced after a flash and the total charge associated with the proton flow coupled to ATP synthesis were more resistant to efrapeptin than the initial value of the phosphorylating currents, indicating that several ATP synthases are fed by protons from the same vesicle.     

The glutamine/amino acid transporter (ASCT2) reconstituted in liposomes: Transport mechanism, regulation by ATP and characterization of the glutamine/glutamate antiport

The glutamine/amino acid transporter solubilized from rat renal apical plasma membrane (brush-border membrane) with C₁₂E₈ and reconstituted into liposomes has been previously identified as the ASCT2 transporter. The reconstituted transporter catalyses an antiport reaction in which external glutamine and Na⁺ are cotransported in exchange with internal glutamine (or other amino acids). The glutamine-Na⁺ cotransport occurred with a 1:1 stoichiometry. The concentration of Na⁺ did not influence the Km for glutamine and vice versa. Experimental data obtained by a bi-substrate analysis of the glutamine-Na⁺ cotransport, together with previous report on the glutamine_ex/glutamine_in pseudo bi-reactant analysis, indicated that the transporter catalyses a three-substrate transport reaction with a random simultaneous mechanism. The presence of ATP in the internal compartment of the proteoliposomes led to an increase of the Vmax of the transport and to a decrease of the Km of the transporter for external Na⁺. The reconstituted glutamine/amino acid transporter was inhibited by glutamate; the inhibition was more pronounced at acidic pH. A kinetic analysis revealed that the inhibition was competitive with respect to glutamine. Glutamate was also transported in exchange with glutamine. The external Km of the transporter for glutamate (13.3 mM) was slightly higher than the internal one (8.3 mM). At acidic pH the external but not the internal Km decreased. According with the Km values, glutamate should be transported preferentially from inside to outside in exchange for external glutamine and Na⁺.     

Sphingosine 1-phosphate receptors modulate intracellular Ca2+ homeostasis

Ligation of sphingosine 1-phosphate (S1P) to a set of specific receptors named S1P receptors (S1PRs) regulates important biological processes. Although the ability of S1P to increase cytosolic Ca2+ in various cell types is well known, the role of the individual S1PRs has not been fully characterized. Here, we provide a complete analysis of S1P-dependent intracellular Ca2+ homeostasis in HeLa cells. Overexpression of S1P2, or S1P3, but not S1P1, leads to a significant increase in cytosolic and mitochondrial [Ca2+] in response to S1P challenge. Moreover, cells ectopically expressing S1P2, or S1P3 exhibited an appreciable decrease of the free Ca2+ concentration in the endoplasmic reticulum, dependent on stimulation of receptors by S1P endogenously present in the culture medium which was accompanied by a reduced susceptibility to C2-ceramide-induced cell death. These results demonstrate a differential contribution of individual S1PRs to Ca2+ homeostasis and its possible implication in the regulation of cell survival.     

Hypertrophy and atrophy inversely regulate Caveolin-3 expression in myoblasts

Caveolin-3 (Cav-3) is a muscle-specific membrane protein crucial for myoblast differentiation, as loss of the protein due to mutations within the gene causes an autosomal dominant form of limb girdle muscular dystrophy 1-c. Here we show that along with p38 activity the PI3-kinase/AKT/mTOR pathway is required for proper Cav-3 up-regulation during muscle differentiation and hypertrophy, as confirmed by the marked increase of Cav-3 expression in hypertrophied C2C12 cells transfected with an activated form of AKT. Accordingly, Cav-3 expression was further increased during hypertrophy of L6C5 myoblasts treated with Arg(8)-vasopressin and in hypertrophic muscles of MLC/mIGF-1 transgenic mice. In contrast, Cav-3 expression was down-regulated in C2C12 myotubes exposed to atrophic stimuli such as starvation or treatment with dexamethasone. This study clearly suggests that Cav-3 expression is causally linked to the maturation of muscle phenotype and it is tightly regulated by hypertrophic and atrophic stimuli.     

Interleukin 1 in oviductal tissues of viviparous, oviparous, and ovuliparous species of amphibians

In previous reports, we have shown that interleukin 1 (IL1), a cytokine associated with implantation in mice, is also expressed in reproductive tissues of viviparous squamate reptiles and cartilaginous fishes. In the present study, we investigated the expression of IL1B and its functional membrane receptor type I (IL1R1) in amphibians, a class of vertebrates that is characterized by different reproductive modes, including internal and external fertilization. In particular, we investigated the oviductal tissues of the aplacental viviparous Salamandra lanzai, the oviparous Triturus carnifex, and the ovuliparous Bufo bufo. In immunohistochemistry with anti-human IL1B and IL1R1 polyclonal antibodies we found that in S. lanzai, most cells in the uterine mucosa were immunoreactive for IL1B and IL1R1. In T. carnifex, IL1B and IL1R1 were present in ciliated luminal cells, and there was evidence of IL1B in glandular cells. In B. bufo, the expression of IL1B and IL1R1 was limited to the apical cytoplasm of the ciliated oviductal cells. Western blot analysis showed that a putative mature form of IL1B, similar to that seen in mammals, was present in the oviductal tissues of S. lanzai, whereas different forms, which probably correspond to an inactive pro-IL1B protein, were found in T. carnifex and B. bufo. A band that corresponded to the predicted 80-kDa human IL1R1 was found in S. lanzai and T. carnifex. Although the present study shows that IL1B and IL1R1 expression occurs in all reproductive modes, the differential expression patterns noted between ovuliparity and oviparity and viviparity may reflect the different roles of IL1 in the various reproductive modes.     

Introduction of the exopolysaccharide gene cluster from Streptococcus thermophilus Sfi6 into Lactococcus lactis MG1363: production and characterization of an altered polysaccharide

Streptococcus thermophilus Sfi6 produces an exopolysaccharide (EPS) composed of glucose, galactose and N-acetylgalactosamine in the molar ratio of 1:2:1. The genes responsible for the EPS biosynthesis have been isolated previously and found to be clustered in a 14.5 kb region encoding 13 genes. Transfer of this gene cluster into a non-EPS-producing heterologous host, Lactococcus lactis MG1363, yielded an EPS with a similar high molecular weight, but a different structure from the EPS from the native host. The structure of the recombinant EPS was determined by means of 1H homonuclear and 1H-13C heteronuclear two-dimensional nuclear magnetic resonance (NMR) spectra and was found to be --> 3)-beta-D-Glcp-(1 --> 3)-alpha-D-Galp-(1 --> 3)-beta-D-Galp-(1 --> as opposed to --> 3)[alpha-D-Galp-(1 --> 6)]-beta-D-Glcp-(1 --> 3)-alpha-D-GalpNAc-(1 --> 3)-beta-D-Galp-(1 --> for the wild-type S. thermophilus Sfi6. Furthermore, L. lactis MG1363 (pFS101) was also lacking a UDP-N-acetylglucosamine C4-epimerase activity, which would provide UDP-GalNAc for a GalNAc incorporation into the EPS and probably caused the substitution of GalNAc by Gal in the recombinant EPS. This modification implies that (i) bacterial glycosyltransferases could potentially have multiple specificities for the donor and the acceptor sugar molecule; and (ii) the repeating unit polymerase can recognize and polymerize a repeating unit that differs in the backbone as well as in the side-chain from its native substrate.     

Separation and purification of periplasmic cytochrome c553 using reversed micelles

Surfactant concentration, ionic strength, and pH were optimised for the selective separation and purification of periplasmic cytochrome c553 from recombinant E. coli TG2 cells using response surface methodology. Back-extraction was accomplished using counter-ionic surfactant addition. Optimum forward extraction conditions were: 65 mM bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT), 0.07 M NaCl, and pH 8.4, while the optimum back-extraction conditions were 80 mM trioctylmethylammonium chloride, 0.85 M KCl, and pH 9.62. In comparison to a conventionally purified sample using column chromatography (10 mg cytochrome c553 l–1 with a purity of 0.66), reverse micelles achieved the same concentration and similar purity (0.50) in only two simple steps.