RESUMEN

Bombesin/gastrin-releasing peptides (BN/GRP) were shown to bind selectively to cell surface receptors, stimulating the growth of various types of malignancies in murine and human models. The novel BN/GRP synthetic receptor antagonist, RC-3095, was able to produce long-lasting tumor regressions in murine and human tumor models in vitro and in vivo. Animal toxicology studies showed no detectable organ toxicity apart from local irritation at the injection site. The purpose of this study was to determine the safety and feasibility of the administration of RC-3095 by daily subcutaneous injections in patients with advanced and refractory solid malignancies. Twenty-five patients received RC-3095 once or twice-daily at doses ranging from 8 to 96 ug/kg. Dose was escalated in groups of 3-5 patients per dose level. The only toxicity observed was local discomfort in the injection site at the highest doses. A single dose administration of RC-3095 at the highest dose level (96 ug/kg) was tested in a clearly hypergastrinemic individual with the Zollingen-Ellison syndrome and produced a decrease in plasma gastrin down to 50% of basal levels in 6 h. There was no objective tumor responses in patients included in the study. A short-lasting minor tumor response was observed in a patient with a GRP-expressing progressive medullary carcinoma of the thyroid. Due to problems with the analytical method, plasma pharmacokinetic data was obtained only from two patients included at the highest dose level. In these patients, RC-3095 reached plasma concentrations >100 ng/mL for about 8 h, which were within therapeutic levels on the basis of prior data obtained in mice and rats. The plasma elimination half-life was between 8.6-10.9 h. Due to the occurrence of local toxicity at the injection site, the dose escalation procedure could not be fully evaluated up to a maximum tolerated dose. Thus, a recommended dose of RC-3095 for Phase II trials could not be clearly established. Considering the novelty of its mechanism of action and impressive preclinical anti-tumor activity, further studies exploiting new formulations of RC-3095 for human use, such as slow-release preparations, and analogues with a more favorable pharmacokinetics are warranted.

Asunto(s)

Antineoplásicos/farmacocinética , Bombesina/análogos & derivados , Bombesina/antagonistas & inhibidores , Péptido Liberador de Gastrina/antagonistas & inhibidores , Neoplasias/metabolismo , Fragmentos de Péptidos/farmacocinética , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/efectos adversos , Antineoplásicos/uso terapéutico , Bombesina/efectos adversos , Bombesina/farmacocinética , Bombesina/uso terapéutico , Femenino , Gastrinas/sangre , Humanos , Inyecciones Subcutáneas , Masculino , Persona de Mediana Edad , Neoplasias/tratamiento farmacológico , Dolor , Fragmentos de Péptidos/efectos adversos , Fragmentos de Péptidos/uso terapéutico , Piel/efectos de los fármacos , Piel/patología

RESUMEN

The chloroplast signal recognition particle (cpSRP) consists of an evolutionarily conserved 54-kDa subunit (cpSRP54) and a dimer of a unique 43-kDa subunit (cpSRP43). cpSRP binds light-harvesting chlorophyll proteins (LHCPs) to form a cpSRP/LHCP transit complex, which targets LHCP to the thylakoid membrane. Previous studies showed that transit complex formation is mediated through the binding of the L18 domain of LHCP to cpSRP43. cpSRP43 is characterized by a four-ankyrin repeat domain at the N terminus and two chromodomains at the C terminus. In the present study we used the yeast two-hybrid system and in vitro binding assays to analyze the function of different domains of cpSRP43 in protein complex formation. We report here that the first ankyrin repeat binds to the 18-amino acid domain on LHCP that binds to cpSRP43, whereas the third and fourth ankyrin repeats are involved in the dimerization of cpSRP43. We show further that the interaction of cpSRP43 with cpSRP54 is mediated via binding of the methionine-rich domain of cpSRP54 to the C-terminally located chromodomains of cpSRP43. Both chromodomains contain essential elements for binding cpSRP54, indicating that the closely spaced chromodomains together create a single binding site for cpSRP54. In addition, our data demonstrate that the interaction of cpSRP54 with the chromodomains of cpSRP43 is enhanced indirectly by the dimerization motif of cpSRP43.

Asunto(s)

Cloroplastos/metabolismo , Proteínas de Saccharomyces cerevisiae , Partícula de Reconocimiento de Señal/metabolismo , Ancirinas , Sitios de Unión , Proteínas de Cloroplastos , Dimerización , Complejos de Proteína Captadores de Luz , Metionina/metabolismo , Modelos Moleculares , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Unión Proteica , Conformación Proteica , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Ácido Nucleico , Partícula de Reconocimiento de Señal/química , Partícula de Reconocimiento de Señal/genética , Relación Estructura-Actividad

RESUMEN

Western blot analysis revealed a cross reaction of an antibody against the spinach triosephosphate translocator with 29 kDa proteins from envelope membranes of plastids from green and red tomato fruits and also of potato tuber amyloplasts. Envelope membranes from potato tubers were isolated from a homogenate of total membranes by isopycnic sucrose density gradient centrifugation. We were able to demonstrate by reverse transcription and sequencing of the PCR product that the mRNA for the triosephosphate translocator in leaves is also present in green and red tomato fruits. The mature protein consists of 330 amino acid residues and is highly homologous to the triosephosphate translocator proteins from potato and tobacco. The PCR product obtained for potato tubers was partly sequenced. It corresponds entirely to the cDNA sequence encoding the potato leaf triosephosphate translocator protein. Evidence for the expression of the triosephosphate translocator gene in various photosynthetic active and inactive tomato tissues (leaf, green fruit, red fruit, root, petal, sepal) and potato tubers was further confirmed by northern blot analysis.

Asunto(s)

Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Solanum tuberosum/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Western Blotting , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Cloroplastos , Cartilla de ADN , Glucosa-6-Fosfato , Glucofosfatos/metabolismo , Hexosafosfatos/metabolismo , Solanum lycopersicum/metabolismo , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Fosfatos/metabolismo , Proteínas de Plantas/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Solanum tuberosum/metabolismo

RESUMEN

Porins are voltage-gated diffusion pores found in all eukaryotic kingdoms. Here we describe, for the first time, the identification and characterization of two cDNAs encoding porins from plants. Peptide sequences obtained from a 30-kDa protein of envelope membranes from pea root plastids allowed the isolation of two cDNA clones from pea and maize. On the protein level, both proteins are homologous by 58%. Sequence comparison against the Swiss-Prot sequence data base revealed a homology of about 25% to mitochondrial porins from fungi and human. Computer-aided predictions of the secondary structure of the plant porins revealed the presence of 16 antiparallel beta-strands that are also found in mitochondrial porins. Porins from non-green plastids and from the outer mitochondrial membrane were reconstituted into planar lipid bilayers. The proteins showed high pore-forming activities and similar single-channel conductances. In vitro translated porin was preferentially imported only into non-green plastids but not into chloroplasts. To our knowledge, this is the first example of selective import of a plastid protein into different types of plastids. This finding is in line with the observation that an immunoreactive 30-kDa band was only found in non-green plastids and mitochondria but not in chloroplasts. We conclude that mitochondria and non-green plastids possess homologous porin proteins, whereas chloroplasts are characterized by a different type of porin.

Asunto(s)

Genes de Plantas/genética , Membranas Intracelulares , Canales Iónicos/genética , Proteínas de Plantas/genética , Plastidios , Porinas/genética , Verduras/genética , Secuencia de Aminoácidos , Transporte Biológico , Biomarcadores , Compartimento Celular , Fraccionamiento Celular , Clonación Molecular , ADN Complementario/genética , Canales Iónicos/clasificación , Canales Iónicos/aislamiento & purificación , Canales Iónicos/metabolismo , Membrana Dobles de Lípidos/metabolismo , Mitocondrias , Datos de Secuencia Molecular , Familia de Multigenes , Pisum sativum , Proteínas de Plantas/clasificación , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Raíces de Plantas , Porinas/aislamiento & purificación , Porinas/metabolismo , Biosíntesis de Proteínas , Estructura Secundaria de Proteína , Análisis de Secuencia , Homología de Secuencia de Aminoácido , Zea mays

RESUMEN

The kinetic properties of the adenosine 5[prime]-diphosphate/adenosine 5[prime]-triphosphate (ADP/ATP) translocator from pea (Pisum sativum L.) root plastids were determined by silicone oil filtering centrifugation and compared with those of spinach (Spinacia oleracea L.) chloroplasts and pea leaf mitochondria. In addition, the ADP/ATP transporting activities from the above organelles were reconstituted into liposomes. The Km(ATP) value of the pea root ADP/ATP translocator was 10 [mu]M and that for ADP was 46 [mu]M. Corresponding values of the spinach ADP/ATP translocator were 25 [mu]M and 28 [mu]M, respectively. Comparable results were obtained for the reconstituted ATP transport activities. The transport was highly specific for ATP and ADP. Adenosine 5[prime]-monophosphate (AMP) caused only a slight inhibition and phosphoenolpyruvate and inorganic pyrophosphate caused no inhibition of ATP uptake. With pea root plastids and spinach chloroplasts, Km values >1 mM were obtained for ADP-glucose. Since the concentrations of ATP and ADP-glucose in the cytosolic compartment of spinach leaves have been determined as 2.5 and 0.6 mM, respectively, a transport of ADP-glucose by the ADP/ATP translocator does not appear to have any physiological significance in vivo. Although both the plastidial and the mitochondrial ADP/ATP translocators were inhibited to some extent by carboxyatractyloside, no immunological cross-reactivity was detected between the plastidial and the mitochondrial proteins. It seems probable that these proteins derive from different ancestors.

RESUMEN

Plastids have been isolated from pea (Pisum sativum L.) roots with a high degree of purity and intactness. In these plastids, the activity of enzymes involved in carbohydrate metabolism have been analyzed and corrected for cytosolic contamination. The results show that fructose-1,6-bisphosphatase, NAD-glyceraldehyde phosphate dehydrogenase, and phosphoglyceromutase are not present in pea root plastids. Transport measurements revealed that inorganic phosphate, dihydroxyacetone phosphate (DHAP), 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, and glucose-6-phosphate (Glc6p) are transported across the envelope in a counterexchange mode. Transport of glucose-1-phosphate was definitely excluded. The oxidation of Glc6P by intact plastids resulted almost exclusively in the formation of DHAP. The parallel measurement of DHAP formation and NO2- consumption during Glc6P-supported nitrite reduction yielded a ratio of NO2-reduced/DHAP formed of 1.6, which is relatively close to the theoretical value of 2.0. These results show that the oxidation of Glc6P, involving the uptake of Glc6P and the release of DHAP, and the reduction of NO2- are very tightly coupled to each other.