RESUMEN
OBJECTIVE: The aim of this study was to evaluate the effect and performance of the new algorithm in cervical cancer screening program in two years' experience of Latina (Italy). MATERIALS AND MTHODS: The female population was divided into two groups, the first group was referred to PAP test and the second one to hr-HPV test according to national guidelines. RESULTS: In two years the participation mean rate increased among women aged 35-64 compared to women aged 25-34. The primary PAP test positive rate and hr-HPV test positive rate were 4.0% and 5.2%, respectively. The PAP test positive rate among hr-HPV+ women decreased from 2012 to 2013. Women with hr-HPV+/PAP+ were referred immediately to colposcopy and this rate was 1.2%. The predictive positive value for CIN2+ to colposcopy was 10.9% in 2012 and 9.1% in 2013, while the detection rate for CIN2+ was 1.6% in 2012 and 1.4% in 2013. CONCLUSION: The stratification of the female population leads to a decreased inappropriate therapeutic path while the combination of hr-HPV test with PAP test in woman aged 35-64 lets obtain high levels specificity and sensitivity results.
Asunto(s)
Detección Precoz del Cáncer , Papillomaviridae/aislamiento & purificación , Neoplasias del Cuello Uterino/virología , Adulto , Femenino , Humanos , Italia , Persona de Mediana Edad , Neoplasias del Cuello Uterino/diagnósticoRESUMEN
Infection with high-risk human papillomavirus (hr-HPV) 16, 18, and 45 causes 94% of cervical carcinoma. In the present screening center the authors perform the hr-HPV test followed by Pap test to women aged 35-64 years if they result hr-HPV+. The authors' aimed to provide data regarding the genotyping test and eventually to propose this test as alternative to triage cytology. They used a genotyping test to identify HPV 16, 18, and 45 in 22 women with histological diagnosis of CIN2+, 22 women with histological diagnosis of CIN1 and 22 women hr-HPV+/Pap-. The group of CIN2+ showed the higher positivity to the test and the higher positivity to HPV 16 than other groups. Analyzing the clinical performance of the genotyping test the authors observed that the specificity was 64%. From these data they concluded that the identification of HPV 16 is predictive for high-grade lesions but this test could not be used alternatively to triage cytology.
Asunto(s)
Papillomavirus Humano 16/aislamiento & purificación , Papillomavirus Humano 18/aislamiento & purificación , Infecciones por Papillomavirus/complicaciones , Displasia del Cuello del Útero/virología , Neoplasias del Cuello Uterino/virología , Adulto , Femenino , Genotipo , Humanos , Persona de Mediana EdadRESUMEN
In this study, purified P-glycoprotein molecules, a membrane drug pump responsible for the multidrug resistance phenomenon, were incorporated in model membranes deposited onto solid supports, according to the method described by Puu and Gustafson (1997). The insertion of proteins into planar supported model membranes is of interest, as the films are fundamental in biosensor applications and for the investigation of how proteins conform and aggregate in a lipid environment. In our investigation, two model membranes were prepared by transferring liposomes containing P-glycoprotein to different hydrophobic supports: (a) thin amorphous carbon films; (b) Langmuir-Blodgett lipid monolayers on mica. After the labelling of P-glycoprotein with two well-characterised monoclonal antibodies, MM4.17 and MRK-16, samples (a) were observed by transmission electron microscopy (TEM) and samples (b) by atomic force microscopy (AFM). The comparative analysis performed by TEM and AFM allowed us to demonstrate the successful insertion of P-glycoprotein in the model membranes and their stability under different environmental conditions (vacuum, air and water). P-glycoprotein appeared to maintain, after purification and insertion in lipid bilayers, a good part of its conformational features as shown by the P-glycoprotein segments bearing the specific monoclonal antibody epitopes.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/análisis , Membrana Dobles de Lípidos/química , Humanos , Inmunohistoquímica , Liposomas/química , Microscopía de Fuerza Atómica , Microscopía Inmunoelectrónica , Células Tumorales CultivadasRESUMEN
A fluorescent derivative of a chimeric toxin between human pro-urokinase and the plant ribosome-inactivating protein saporin (p-uPA-Sap(TRITC)), has been prepared in order to study the endocytosis of this potentially antimetastatic conjugate in the murine model cell line LB6 clone19 (Cl19) transfected with the human urokinase receptor gene. The physiological internalization of urokinase-inhibitor complexes is triggered by the interaction of plasminogen inhibitors (PAIs) with receptors belonging to the low density lipoprotein-related receptor protein (LRP) family, and involves a macro-quaternary structure including uPAR, LRP, and PAIs. However, in contrast to this mechanism, we observed a two-step process: first, the urokinase receptor (uPAR) acts as the anchoring factor on the plasma membrane; subsequently, LRP acts as the endocytic trigger. Once the chimera is bound to the plasma membrane by interaction with uPAR, we suggest that a possible exchange may occur to transfer the toxin to LRP via the saporin moiety and begin the internalization. So an unusual endocytic process is described, where the toxin enters the cell via a receptor different from that used to bind the plasma membrane.
Asunto(s)
Inmunotoxinas , N-Glicosil Hidrolasas , Proteínas de Plantas/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Membrana Celular/metabolismo , Clorpromazina/farmacología , Células Clonales , Endocitosis , Filipina/farmacología , Colorantes Fluorescentes , Humanos , Inmunohistoquímica , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Ratones , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Proteínas de Plantas/toxicidad , Inactivadores Plasminogénicos/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Inmunológicos/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/toxicidad , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidad , Proteínas Inactivadoras de Ribosomas Tipo 1 , Saporinas , Activador de Plasminógeno de Tipo Uroquinasa/toxicidadRESUMEN
The 2.0 A resolution crystal structure of the ribosome inactivating protein saporin (isoform 6) from seeds of Saponaria officinalis is presented. The fold typical of other plant toxins is conserved, despite some differences in the loop regions. The loop between strands beta7 and beta8 in the C-terminal region which spans over the active site cleft appears shorter in saporin, suggesting an easier access to the substrate. Furthermore we investigated the molecular interaction between saporin and the yeast ribosome by differential chemical modifications. A contact surface inside the C-terminal region of saporin has been identified. Structural comparison between saporin and other ribosome inactivating proteins reveals that this region is conserved and represents a peculiar motif involved in ribosome recognition.
Asunto(s)
Inmunotoxinas , N-Glicosil Hidrolasas , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas/química , Ribosomas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Secuencia Conservada , Cristalografía por Rayos X , Lisina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Inactivadoras de Ribosomas Tipo 1 , Ribosomas/química , Saporinas , Semillas/química , Alineación de Secuencia , Anhídridos Succínicos/metabolismo , Tripsina/metabolismo , Levaduras/citologíaRESUMEN
UNLABELLED: Air-filled human serum albumin microspheres are ultrasonic contrast tracers that pass through the right ventricle, traverse the lungs, and effectively opacify the left heart chambers in spontaneously breathing patients. In this clinical study, we assessed whether they also do so in anesthetized patients during and after mechanical ventilation. In 20 anesthetized patients undergoing intermittent positive pressure ventilation (IPPV) for elective peripheral neurosurgical procedures, a sonicated ultrasound contrast drug (0.06 mL/kg) was injected i.v. before inducing anesthesia in spontaneously breathing patients (baseline), during IPPV, and 5 and 30 min after tracheal extubation. Transthoracic echocardiograms were obtained in the four-chamber apical view and were recorded for off-line analysis. Time to contrast appearance in the right ventricle and pulmonary transit time were measured in cardiac cycles. The peak intensity of right and left ventricular chamber opacification were scored on a scale ranging from 1 (no contrast or traces only) to 5 (attenuation). After each injection, the time for contrast appearance in the right ventricle was similar in all patients. Pulmonary transit time increased significantly during IPPV and was normal 5 min and 30 min after extubation. Right ventricular chamber opacification achieved high-grade intensity and remained constant before, during, and after IPPV. Conversely, although the baseline contrast injection resulted in high-grade left ventricular chamber opacification, the intensity decreased significantly during IPPV, remained low 5 min after extubation, and was normalized 30 min after extubation. IMPLICATIONS: During intermittent positive pressure ventilation, i.v. sonicated albumin microbubbles pass through the lungs poorly and inefficiently opacify the left ventricle compared with the effects observed during spontaneous ventilation.
Asunto(s)
Anestesia General , Medios de Contraste/farmacocinética , Ecocardiografía , Ventilación con Presión Positiva Intermitente , Pulmón/irrigación sanguínea , Albúmina Sérica/farmacocinética , Transporte Biológico , Permeabilidad Capilar , Medios de Contraste/administración & dosificación , Ventrículos Cardíacos/diagnóstico por imagen , Humanos , Inyecciones Intravenosas , Periodo Intraoperatorio , Microesferas , Procedimientos Neuroquirúrgicos , Estudios Prospectivos , Albúmina Sérica/administración & dosificaciónRESUMEN
With the electro-driven import of rhodamine 123, we used single cell fluorescence microscopy to single out the contribution of nitric oxide (NO) in controlling mitochondrial membrane potential expressed by (stationary growing) rhabdomyosarcoma and neuroblastoma cells in culture. The experimental design and the computer-aided image analysis detected and quantitated variations of fluorescence signals specific to mitochondria. We observed that 1) the two cell lines display changes of fluorescence dependent on mitochondrial energization states; 2) mitochondrial fluorescence decreases after exposure of the cells to a NO releaser; 4) the different fluorescence intensity measured under stationary growing conditions, or after activation and inhibition of constitutive NO synthase, is consistent with a steady-state production of NO. Direct comparison of single cell fluorescence with bulk cytofluorimetry proved that the results obtained by the latter method may be misleading because of the intrinsic-to-measure lack of information about distribution of fluorescence within different cell compartments. The kinetic parameters describing the reactions between cytochrome oxidase, NO, and O2 may account for the puzzling (20-fold) increase of the KM for O2 reported for cells and tissues as compared to purified cytochrome c oxidase, allowing an estimate of in vivo NO flux.
Asunto(s)
Mitocondrias/fisiología , Óxido Nítrico/metabolismo , Fluorescencia , Colorantes Fluorescentes , Humanos , Microscopía Fluorescente , Rodamina 123 , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Single crystals of the protein saporin isolated from the seeds of S. officinalis have been grown by the vapor-diffusion method using ammonium sulfate as precipitant. The crystals are tetragonal, space group P4122 (P4322), with cell dimensions a = b = 67.53 and c = 119. 67 A, and diffract to 2.0 A resolution on a rotating-anode X-ray source. The asymmetric unit contains one molecule, corresponding to a volume of the asymmetric unit per unit mass (Vm) of 2.38 A3 Da-1.
Asunto(s)
Inmunotoxinas , N-Glicosil Hidrolasas , Proteínas de Plantas/química , Cristalización , Cristalografía por Rayos X , Proteínas de Plantas/aislamiento & purificación , Conformación Proteica , Proteínas Inactivadoras de Ribosomas Tipo 1 , SaporinasRESUMEN
The toxicity of two conjugates containing ribosome-inactivating proteins (RIPs, i.e. saporin and ricin-A chain x-linked to transferrin) has been measured on a prostatic cancer line (PC3) naturally overexpressing the transferrin receptor, in the presence of monensin and chloroquine. This paper investigates whether the increased toxicity of Tf-RIPs induced by monensin and chloroquine may be due to alterations of the normal endocytotic pathway of the complexes mediated by the transferrin receptor. Monensin, besides inducing alkalinization of normally acid intracellular compartments, causes an accumulation of the receptor-bound Tf-RIP in a perinuclear region contiguous to the cisternae of the trans-Golgi network. Chloroquine, though increasing the intracellular pH, seems not to modify the endocytotic pathway of these chimeric molecules. We believe that the enhanced toxicity of the Tf-RIPs may be related to intracellular alkalinization (i.e., endosomal or lysosomal pH) rather than to the effects on the recycling of transferrin receptor-bound toxins. We conclude that the efficacy of chimeric toxins may be modulated not only by the carrier used for their engineering but also by addition of drugs able to influence the stability and activation of the toxins inside the cell.
Asunto(s)
Cloroquina/farmacología , Endocitosis/efectos de los fármacos , Inmunotoxinas/efectos de los fármacos , Inmunotoxinas/toxicidad , Monensina/farmacología , Humanos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Inmunotoxinas/metabolismo , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/metabolismo , Masculino , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Inhibidores de la Síntesis de la Proteína/farmacología , Receptores de Transferrina/biosíntesis , Ricina/toxicidad , Saponinas/genética , Transferrina/genética , Células Tumorales CultivadasRESUMEN
A liposomal carrier system able to interact specifically with HL60 leukaemia cells was produced using small unilamellar liposomes made of pure phospholipids chemically cross-linked to human transferrin. The conjugation of transferrin to liposomes was carried out using N-succinimidyl 3-(2-pyridyldithio)-propionate and 2-iminothiolane as activating agents for the liposomes and the protein. The reaction occurred under conditions set to covalently link on the surface of a single vesicle a limited number (one to ten) of transferrin molecules, as verified by means of electron microscopy and immunoenzymic measurements. Before conjugation, the ultrastructure of the liposomes, and the content and distribution of the amino groups within the bilayer, were determined. The reactivity of the liposomes towards amino-derivatizing or thiolating compounds was also measured. Kinetic spectroscopic measurements confirmed that the distribution of the phosphatidylethanolamine in the vesicle bilayer is asymmetrical: 22% of phosphatidylethanolamine was found exposed to the external surface of the liposomes and accessible to the cross-linker. The modified liposomes were able to interact specifically with the cells and to be internalized by active receptor-mediated endocytosis, as demonstrated by the full inhibition of internalization induced by free transferrin.
Asunto(s)
Endocitosis/fisiología , Leucemia Promielocítica Aguda/patología , Receptores de Transferrina/fisiología , Transferrina/farmacología , Endocitosis/efectos de los fármacos , Humanos , Liposomas , Células Tumorales CultivadasRESUMEN
Saporin, a single-chain, non-cytotoxic, ribosome-inactivating protein from Saponaria officinalis, was chemically linked to the hormone insulin in a 1:1 complex. To follow by dynamic video microscopy the endocytosis and intracellular transport in vivo, a second covalent conjugate with a saporin derivative labelled with fluorescein isothiocyanate was also prepared. Both conjugates were characterized with reference to homogeneity, stoichiometry, optical spectroscopy and toxicity. Both were found to exhibit scarce toxicity toward both CHO and HEP G2 cells; optical video microscopy on living cells indicates that reduced toxicity may be (partly) due to a very limited binding of the saporin-insulin conjugate to membrane receptors. These results suggest a strategy for new possible covalent conjugates of saporin with alternative and specific macromolecular carriers.
Asunto(s)
Antineoplásicos Fitogénicos/aislamiento & purificación , Células CHO/efectos de los fármacos , Insulina/metabolismo , N-Glicosil Hidrolasas , Proteínas de Plantas/aislamiento & purificación , Animales , Antineoplásicos Fitogénicos/metabolismo , Antineoplásicos Fitogénicos/toxicidad , Sitios de Unión , Células CHO/citología , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cricetinae , Electroforesis en Gel de Poliacrilamida , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/metabolismo , Inmunotoxinas/aislamiento & purificación , Inmunotoxinas/metabolismo , Inmunotoxinas/toxicidad , Microscopía Fluorescente , Proteínas de Plantas/metabolismo , Receptor de Insulina/metabolismo , Proteínas Inactivadoras de Ribosomas Tipo 1 , SaporinasRESUMEN
Human transferrin (Tf) and saporin-6 (Sap), a ribosome inactivating protein from Saponaria officinalis, were chemically conjugated: the reaction generated two chimeras (called Tf-Sap) that proved to be cytotoxic to HepG2 cells. Electrophoretic and chromatographic analysis revealed that the two conjugates contained saporin and Tf in a 2:1 or 1:1 molar ratio (140 and 110 KDa, respectively). Free saporin is essentially nontoxic, whereas Tf-Sap efficiently kills HepG2 cells, although its ID50 (= 6 nM) is 1000-fold greater than that of ricin. Intracellular transport of these toxins was followed by in vivo fluorescence video microscopy, preparing the conjugates starting from rhodamine isothiocyanate-labeled saporin. Image analysis of living HepG2 cells exposed to fluorescent Tf-Sap revealed that the endocytotic pathway involving passage through secondary endosomes is dictated by Tf and is different from that of ricin (the dimeric toxin from Ricinus communis), which is delivered to the Golgi apparatus, the probable site of activation. We discuss whether differences in toxicity between ricin and Tf-Sap can be attributed to the different mechanisms of transport and activation.
Asunto(s)
Antineoplásicos Fitogénicos/toxicidad , Inmunotoxinas , N-Glicosil Hidrolasas , Proteínas de Plantas/farmacología , Ricina/toxicidad , Transferrina/farmacología , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/metabolismo , Transporte Biológico , Muerte Celular , Línea Celular Transformada/efectos de los fármacos , Portadores de Fármacos , Humanos , Microscopía por Video , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Biosíntesis de Proteínas , Proteínas Inactivadoras de Ribosomas Tipo 1 , Ricina/química , Ricina/metabolismo , Saporinas , Transferrina/química , Transferrina/metabolismoRESUMEN
Spectroscopic measurements in single living cells are made possible by the development of computer controlled light detectors, which, when applied to optical microscopes, yield spatial, temporal and eventually spectroscopic information about the sample. This minireview describes some experiments in which the distribution and concentration of specific intracellular markers (proteins, protein complexes, RNA) has been followed by quantitative microscopy. The examples chosen have contributed to shed light on a biochemical process as it happens in vivo; because of the non ideal conditions of the intracellular milieu, the comparison of in vivo and in vitro experiments is of great relevance to the understanding of cellular physiology.
Asunto(s)
Células/metabolismo , Microscopía/instrumentación , Espectrofotometría/instrumentación , Animales , Endocitosis/fisiología , Eritrocitos/metabolismo , Humanos , Procesamiento de Imagen Asistido por Computador , Técnicas In Vitro , Oligodendroglía/metabolismo , Oocitos/metabolismo , Huso Acromático/metabolismoRESUMEN
The intracellular dynamics of fluorescent conjugates of the toxic lectin ricin was followed by video fluorescence microscopy on living CHO cells, demonstrating that the ricin heterodimer and its isolated B chain, after binding to the plasma membrane receptors, migrate to and accumulate in the Golgi apparatus following internalization. A ricin derivative labelled with fluorescein on the A chain and rhodamine on the B chain did not display significant splitting of the A-B heterodimer during translocation of the toxin to the Golgi; this novel finding provides support for the hypothesis that further processing of ricin takes place in this cellular compartment.
Asunto(s)
Aparato de Golgi/metabolismo , Microscopía Fluorescente , Ricina/metabolismo , Animales , Células CHO/metabolismo , Membrana Celular/metabolismo , Cricetinae , Fluoresceína , Fluoresceína-5-Isotiocianato , Fluoresceínas , Colorantes Fluorescentes , Sustancias Macromoleculares , Rodaminas , Grabación en VideoRESUMEN
Chlorocruorin is a cooperative respiratory pigment found in the blood of polychaete worms; its prosthetic group is a derivative of the iron protoporphyrin IX, in which the vinyl group at position 2 is substituted by a formyl group. The quaternary structure of chlorocruorins is complex: myoglobin-like subunits are grouped in tetramers and tetramers in dodecamers; 12 dodecamers are assembled in the 3500-kDa particle. Chlorocruorin from Spirographis spallanzanii displays the following overall functional properties: (i) the oxygen affinity is lower than in human hemoglobin, while that of CO is similar if not higher; (ii) the rates of combination with oxygen and carbon monoxide are low; and (iii) the off rate of oxygen is comparable to that of human hemoglobin, while the off rate of CO is 10 times smaller. When CO is partially photolyzed with a long and powerful light flash (70 microseconds), rebinding is biphasic as in mammalian hemoglobins; however, the slowest rate is faster than that observed by stopped flow, suggesting that the unliganded protein decays from the liganded high affinity state (R) to an intermediate state before reaching the low affinity (T) state. Oxygen binding was followed by stopped-flow and flash photolysis. While partial photolysis yields a fast, second-order time course, stopped-flow experiments yield slow, biphasic, and non-second-order time courses. This pattern of reactivity was attributed to a slow conformational transition(s) which is (are) rare limiting with oxygen, but not with CO.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Hemoproteínas/metabolismo , Oxígeno/metabolismo , Conformación Proteica , Animales , Sitios de Unión , Monóxido de Carbono/metabolismo , Hemoproteínas/química , Cinética , Ligandos , Fotólisis , PoliquetosRESUMEN
New data on the properties of red blood cells (RBC) cross-linked with glutaraldehyde are presented (see also Biochem. Biophys. Res. Commun., 1988, 156, 970-977). Equilibrium and kinetic data show that by carrying out the fixation procedure in the absence of oxygen but in the presence of allosteric effectors (e.g., stabilizing the low-affinity (T) quaternary state of hemoglobin), it is possible to maintain, at least in part, the biochemical functions of the crosslinked hemoglobin inside the cell. Moreover, we show that the oxygen affinity of fixed red blood cells (RBC) is still modulated, even though to a smaller degree, by the allosteric effector bezafibrate (BZF), which is able to cross the fixed RBC membrane. Membrane filtration experiments indicate that the higher rigidity of fixed RBC alters significantly their rheodynamic properties and show that in order to exploit "engineered" RBC as "blood substitutes," more flexible cross-linking reagents may offer significant advantages.
Asunto(s)
Eritrocitos/química , Glutaral/química , Hemoglobinas/metabolismo , Bezafibrato/farmacología , Reactivos de Enlaces Cruzados , Eritrocitos/efectos de los fármacos , Eritrocitos/fisiología , Hemoglobinas/química , Cinética , Oxígeno/metabolismo , Oxihemoglobinas/química , Ácido Fítico/farmacologíaRESUMEN
Hemoglobin Dallas, an alpha-chain variant with a substitution of lysine for asparagine at position 97(G4), was found to have increased oxygen affinity (p1/2 = 1 mmHg at pH 7.3 and 20 degrees C), diminished cooperativity (n, the Hill coefficient = 1.7) and reduced Bohr effect (about 50%). Addition of allosteric effectors (such as 2,3-diphosphoglycerate, inositol hexakisphosphate and bezafibrate) led to a decrease in oxygen affinity and increase in cooperative energy. Kinetic studies at pH 7.0 and 20 degrees C revealed that (i), the overall rate of oxygen dissociation is 1.4-fold slower than that for HbA and (ii), the carbon monoxide dissociation rate is unaffected. The abnormal properties of this hemoglobin variant can be attributed to a more 'relaxed' T-state.
Asunto(s)
Hemoglobinas Anormales/genética , Hemoglobinas Anormales/fisiología , Mutación , Oxígeno/metabolismo , Regulación Alostérica , Hemoglobinas Anormales/metabolismo , Humanos , Cinética , Modelos MolecularesRESUMEN
The ligand-binding properties of hemoglobins from two homozygote phenotypes (AA and BB) of water buffalo (Bubalus bubalis) have been characterized by equilibrium and kinetic techniques. In the case of the BB phenotype, the two constituent hemoglobins have been purified and separately analysed. Buffalo hemoglobins display the reduced sensitivity to organic phosphates characteristic of ruminant hemoglobins, their physiological effector probably being the chloride ion. In contrast to the other known hemoglobins from ruminants, all the hemoglobins from the water buffalo display a significant temperature sensitivity, the delta H for oxygen binding in the presence of physiological effectors approaching that of human hemoglobin (delta H = -30.5 kJ/mol O2). This discrepancy with the other ruminant hemoglobins (e.g. ox, delta H = -10.4 kJ/mol O2), whose primary structure is very similar to that of buffalo, hemoglobins might be correlated to the different habitat and phylogenetic history of the two subfamilies (Bos and Bubalus) of Bovidae.
Asunto(s)
Hemoglobinas/genética , Rumiantes/genética , Animales , Hemoglobinas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Cinética , Oxígeno/metabolismo , Fenotipo , TermodinámicaRESUMEN
Many plants express enzymes which specifically remove an adenine residue from the skeleton of the 28 S RNA in the major subunit of the eukaryotic ribosome (ribosome inactivating proteins, RIPs). The site of action of RIPs (A4324 in the rRNA from rat liver) is in a loop structure whose nucleotide sequence all around the target adenine is also conserved in those species which are completely or partially insensitive to RIPs. In this paper we identify a covalent complex between saporin (the RIP extracted from Saponaria officinalis) and ribosomal proteins from yeast (Saccharomyces cerevisiae), by means of chemical crosslinking and immunological or avidin-biotin detection. The main complex (mol. wt. congruent to 60 kDa) is formed only with a protein from the 60 S subunit of yeast ribosomes, and is not detected with ribosomes from E. coli, a resistant species. This observation supports the hypothesis for a molecular recognition mechanism involving one or more ribosomal proteins, which could provide a 'receptor' site for the toxin and favour optimal binding of the target adenine A4324 to the active site of the RIP.
Asunto(s)
Antineoplásicos Fitogénicos/metabolismo , Proteínas Fúngicas/metabolismo , Inmunotoxinas , N-Glicosil Hidrolasas , Proteínas de Plantas/metabolismo , Proteínas Ribosómicas/metabolismo , Western Blotting , Proteínas Inactivadoras de Ribosomas Tipo 1 , Saccharomyces cerevisiae/metabolismo , SaporinasRESUMEN
A detailed investigation with affinity-chromatography-purified fractions of antihemoglobin serum from rabbit shows that the hemoglobin content of human bones dating back 15 to 3,000 years may be very small. Some of the previous results (Ascenzi et al., 1985) indicating a high hemoglobin titer were +vitiated because of an unexpected cross-reactivity of bone extracts with the hemoglobin-unreactive fraction of the antiserum.