A chloride-permeable channel from Phaseolus vulgaris roots incorporated into planar lipid bilayers

Ion channels are key participants in physiological processes of plant cells. Here, we report the first characterization of a high conductance, Cl(-)-permeable channel, present in enriched fractions of plasma membranes of bean root cells. The Cl(-) channel was incorporated into planar lipid bilayers and its activity was recorded under voltage clamp conditions. The channel is voltage-dependent, excludes the passage of cations (K(+), Na(+), and Ca(2+)), and is inhibited by micromolar concentrations of Zn(2+). The Cl(-) conductance here characterized represents a previously undescribed channel of plant cells.     

NMR solution structure of viscotoxin C1 from Viscum album species Coloratum ohwi: toward a structure-function analysis of viscotoxins

The high resolution three-dimensional structure of the newly discovered plant viscotoxin C1, from the Asiatic Viscum album ssp. Coloratum ohwi, has been determined in solution by (1)H NMR spectroscopy at pH 3.6 and 285 K. The viscotoxin C1-fold, consisting of a helix-turn-helix motif and a short stretch of an antiparralel beta-sheet is very similar to that found for the highly similar viscotoxins A2 and A3 and for other related thionins. Different functional properties of members of the thionin family are discussed here in light of the structural and electrostatic properties. Among the very homologous family of alpha- and beta-thionins, known for their antimicrobial activity, the viscotoxin subfamily differs from the other members because of its high toxicity against tumoral cells. Key residues for the modulation of viscotoxin cytotoxicity have been identified on the basis of sequence and structural alignment.     

Investigations of Sso7d catalytic residues by NMR titration shifts and electrostatic calculations

Sso7d is a small basic protein consisting of 62 amino acids isolated from the thermoacidophilic archeobacterium Sulfolobus solfataricus. The protein is endowed with DNA binding properties, RNase activity, and the capability of rescuing aggregated proteins in the presence of ATP. In this study, the electrostatic properties of Sso7d are investigated by using the Poisson-Boltzmann calculation of the surface potential distribution and following by NMR spectroscopy the proton chemical shift pH titration of acidic residues. Although the details of the catalytic mechanism still have to be defined, the results from NMR experiments confirm the possible involvement of Glu35 as the proton acceptor in the catalytic reaction, as seen by its abnormally high pK(a) value. Poisson-Boltzmann calculations and NMR titration shifts suggest the presence of a possible hydrogen bond between Glu35 and Tyr33, with a consequent rather rigid arrangement at these positions. Comparison with RNase T1 suggests that Tyr7 may be a good candidate for acting as a proton donor in the active site of Sso7d as shown by its low phenolic pK(a) of approximately 9.3. Titration experiments performed with the UpA, a RNA dinucleotide model, showed that the protein residues affected by the interaction are mainly located in a different region with respect to the surface affected by DNA recognition, in good agreement with the surface potential distribution found with electrostatic calculations.     

Heterologous expression and characterization of mouse spermine oxidase

Polyamine oxidases are key enzymes responsible of the polyamine interconversion metabolism in animal cells. Recently, a novel enzyme belonging to this class of enzymes has been characterized for its capability to oxidize preferentially spermine and designated as spermine oxidase. This is a flavin adenine dinucleotide-containing enzyme, and it has been expressed both in vitro and in vivo systems. The primary structure of mouse spermine oxidase (mSMO) was deduced from a cDNA clone (Image Clone 264769) recovered by a data base search utilizing the human counterpart of polyamine oxidases, PAOh1. The open reading frame predicts a 555-amino acid protein with a calculated M(r) of 61,852.30, which shows a 95.1% identity with PAOh1. To understand the biochemical properties of mSMO and its structure/function relationship, the mSMO cDNA has been subcloned and expressed in secreted and secreted-tagged forms into Escherichia coli BL21 DE3 cells. The recombinant enzyme shows an optimal pH value of 8.0 and is able to oxidize rapidly spermine to spermidine and 3-aminopropanal and fails to act upon spermidine and N(1)-acetylpolyamines. The purified recombinant-tagged form enzyme (M(r) approximately 68,000) has K(m) and k(cat) values of 90 microm and 4.5 s(-1), respectively, using spermine as substrate at pH 8.0. Molecular modeling of mSMO protein based on maize polyamine oxidase three-dimensional structure suggests that the general features of maize polyamine oxidase active site are conserved in mSMO.     

MAPK-dependent degradation of G protein-coupled receptor kinase 2

G protein-coupled receptor kinase 2 (GRK2) is a key modulator of G protein-coupled receptors (GPCR). Altered expression of GRK2 has been described to occur during pathological conditions characterized by impaired GPCR signaling. We have reported recently that GRK2 is rapidly degraded by the proteasome pathway and that beta-arrestin function and Src-mediated phosphorylation are involved in targeting GRK2 for proteolysis. In this report, we show that phosphorylation of GRK2 by MAPK also triggers GRK2 turnover by the proteasome pathway. Modulation of MAPK activation alters the degradation of transfected or endogenous GRK2, and a GRK2 mutant that mimics phosphorylation by MAPK shows an enhanced degradation rate, thus indicating a direct effect of MAPK on GRK2 turnover. Interestingly, MAPK-mediated modulation of wild-type GRK2 stability requires beta-arrestin function and is facilitated by previous phosphorylation of GRK2 on tyrosine residues by c-Src. Consistent with an important physiological role, interfering with this GRK2 degradation process results in altered GPCR responsiveness. Our data suggest that both c-Src and MAPK-mediated phosphorylation would contribute to modulate GRK2 degradation, and put forward the existence of new feedback mechanisms connecting MAPK cascades and GPCR signaling.     

Inhibition of pig liver and Zea mays L. polyamine oxidase: a comparative study

Polyamine oxidase (PAO) is involved in polyamine metabolism and production of hydrogen peroxide in animal and plants, thus representing a key system in development and programmed cell death. In the present study, the inhibitory effect of amiloride, p-aminobenzamidine, clonidine, 4',6-diamidino-2-phenyl-indole (DAPI), gabexate mesylate, guazatine, and N,N'-bis(2,3-butadienyl)-1,4-butane-diamine (MDL72527) on the catalytic activity of pig liver and Zea mays L. PAO, Lens culinaris L. and Pisum sativum L. and swine kidney copper amine oxidase, bovine trypsin, as well as neuronal constitutive nitric oxide synthase (NOS-I) was investigated. Moreover, agmatine and N(3) -prenylagmatine (G3) were observed to inhibit pig liver and Zea mays L. PAO, bovine trypsin, and NOS-I action, but were substrates for Lens culinaris L., Pisum sativum L. and swine kidney copper amine oxidase. Guazatine and G3 inhibited selectively Zea mays L. PAO with K(i) values of 7.5 x 10(-9) M and 1.5 x 10(-8) M, respectively (at pH 6.5 and 25.0 degrees C). As a whole, the data reported here represent examples of enzyme cross-inhibition, and appear to be relevant in view of the use of cationic L-arginine-and imidazole-based compounds as drugs.     

Prevention by delay: nonspecific immunity elicited by IL-12 hinders Her-2/neu mammary carcinogenesis in transgenic mice

As a natural consequence of the expression of the activated transforming rat Her-2/neu oncogene all mammary glands of female transgenic BALB/c (BALB-neuT) mice develop atypical epithelial hyperplasia which progresses to invasive carcinoma. A lobular carcinoma is palpable in all mammary glands of 33-week-old BALB-neuT mice. This progression is markedly delayed by systemic administration of IL-12. In a series of studies the best administration schedule, the lowest dose and the most effective administration time have been defined. The cellular and molecular mechanisms resulting in the delay of carcinogenesis have been established. By means of a series of downstream mediators IL-12 inhibits the angiogenic burst that goes along with the passage from preneoplastic to neoplastic and invasive lesions; it also recruits lymphoid cells in the mammary pad and activates their cytotoxicity towards neoplastic cells and newly formed vessels; and furthermore, it induces lymphoid cells to trigger antiangiogenic activities in neoplastic epithelial cells. Effective, low-dose and non-toxic IL-12 treatments may thus be envisaged as a possible option in the management of preneoplastic mammary lesions and in mammary cancer prevention.     

Molecular mechanics of mouse cardiac myosin isoforms

Two myosin isoforms are expressed in myocardium, alphaalpha-homodimers (V(1)) and betabeta-homodimers (V(3)). V(1) exhibits higher velocities and myofibrillar ATPase activities compared with V(3). We also observed this for cardiac myosin from normal (V(1)) and propylthiouracil-treated (V(3)) mice. Actin velocity in a motility assay (V(actin)) over V(1) myosin was twice that of V(3) as was the myofibrillar ATPase. Myosin's average force (F(avg)) was similar for V(1) and V(3). Comparing V(actin) and F(avg) across species for both V(1) and V(3), our laboratory showed previously (VanBuren P, Harris DE, Alpert NR, and Warshaw DM. Circ Res 77: 439-444, 1995) that mouse V(1) has greater V(actin) and F(avg) compared with rabbit V(1). Mouse V(3) V(actin) was twice that of rabbit V(actin). To understand myosin's molecular structure and function, we compared alpha- and beta-cardiac myosin sequences from rodents and rabbits. The rabbit alpha- and beta-cardiac myosin differed by eight and four amino acids, respectively, compared with rodents. These residues are localized to both the motor domain and the rod. These differences in sequence and mechanical performance may be an evolutionary attempt to match a myosin's mechanical behavior to the heart's power requirements.     

Niemann-Pick type C disease: mutations of NPC1 gene and evidence of abnormal expression of some mutant alleles in fibroblasts

We analyzed Niemann-Pick type C disease 1 (NPC1) gene in 12 patients with Niemann-Pick type C disease by sequencing both cDNA obtained from fibroblasts and genomic DNA. All the patients were compound heterozygotes. We found 15 mutations, eight of which previously unreported. The comparison of cDNA and genomic DNA revealed discrepancies in some subjects. In two unrelated patients carrying the same mutations (P474L and nt 2972del2) only one mutant allele (P474L), was expressed in fibroblasts. The mRNA corresponding to the other allele was not detected even in cells incubated with cycloheximide. The promoter variants (-1026T/G and -1186T/C or -238 C/G), found to be in linkage with 2972del2 allele do not explain the lack of expression of this allele, as they were also found in control subjects. In another patient, (N1156S/Q922X) the N1156S allele was expressed in fibroblasts while the expression of the other allele was hardly detectable. In a fourth patient cDNA analysis revealed a point mutation in exon 20 (P1007A) and a 56 nt deletion in exon 22 leading to a frameshift and a premature stop codon. The first mutation was confirmed in genomic DNA; the second turned out to be a T-->G transversion in exon 22, predicted to cause a missense mutation (V1141G). In fact, this transversion generates a donor splice site in exon 22, which causes an abnormal pre-mRNA splicing leading to a partial deletion of this exon. In some NPC patients, therefore, the comparison between cDNA and genomic DNA may reveal an unexpected expression of some mutant alleles of NPC1 gene.