Biomedical subjects
Publications and source records attributed to J M Nieto.
We describe the lesions of natural distemper in red foxes (Vulpes vulpes) in Spain. The avidin-biotin-peroxidase complex (ABC) technique and a monoclonal antibody against the nucleocapsid protein of canine distemper virus were successfully used to confirm canine distemper diagnosis.
A mutation in the hha allele results in a large increase in the production of intracellular as well as extracellular haemolysin in Escherichia coli cells harbouring the haemolytic recombinant plasmid pANN202-312. This single gene mutation was located between 490 and 491.6 kb on the physical map of the E. coli chromosome. From the DNA sequence of hha a small polypeptide of 8629 Da was predicted and was expressed in minicells. The deduced polypeptide sequence did not show significant similarities to other characterized proteins related to the regulation of gene expression in E. coli, although it was shown that the hha mutation increases cytoplasmic synthesis of haemolysin.
A morphological and morphometrical study has been carried out on glomerular lesions in mink with spontaneous Aleutian disease, using the WHO classification for Systemic Lupus Erytematous Nefritis. 154 renal samples from sick animals and 10 samples from uninfected mink were processed by routine histopathological techniques and metacrylate inclusions. The samples were studied quantitatively with an automatic image analyzer. 5 forms of glomerulonephritis (GN) were identified: mesangial glomerulonephritis (n = 13), focal and segmental GN (n = 10), diffuse GN (n = 99), membranous GN (n = 12) and advanced sclerosing GN (n = 10) and were associated with the degree of interstitial plasmocytosis. Glomerule morphometry was shown to be an excellent method for identifying the type of lesion, while it quantified the participation of various glomerular elements in the lesion.
Antibodies to synthetic peptides from the alpha and beta-tubulin sequences were employed to study zones of this protein active in microtubule assembly. In purified calf brain tubulin, six short sequences, selected according to their hydrophilicity and conservation, were found to be accessible to their affinity-purified immunoglobulin G (IgG) antibodies, in a competition radioimmunoassay performed under non-assembly native conditions. This indicated that the six sequences are exposed on the surface of the tubulin alpha beta heterodimer. IgG antibodies to the alpha(430-443) and beta(412-431) sequences perturbed substoichiometrically the assembly of purified tubulin, inducing microtubule bundling and the formation of opened up structures. These positions, which are close to the C termini, were accessible to the anti-peptide antibodies in taxol-induced microtubules, Zn2(+)-induced tubulin sheets, Mg2(+)-induced tubulin rings and in PtK2 cell microtubules. This, together with the comparison of the sizes and gross shapes of the antibody probes and microtubules, suggested that these sequences might be located at the protruding parts of the protofilaments. Antibodies to positions alpha(155-168) did not react with microtubules, while the equivalent zone beta(153-165) was accessible. The alpha(214-226) and beta(241-256) sequences were antigenically occluded in the taxol microtubules, Zn2(+)-induced sheets and Mg2(+)-induced ring arrays, as well as in native microtubules from PtK2 cells, though they became reactive by fixation. This result strongly suggested that these two zones are close to tubulin-tubulin contact sites. A working model is proposed in which the positions alpha(214-226) and beta(241-256) are close to the axial contacts between heterodimers, which lead to protofilament formation, while the positions alpha(241-256) and beta(214-226) are suggested to be related to the alpha-beta binding interface within the heterodimer.
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We have characterized the binding of trans-1-(2,5-dimethoxyphenyl)-3-[4-(dimethylamino)phenyl]-2-methyl-2- propen- 1-one (MDL 27048) to purified procine brain tubulin, and the inhibition of microtubule assembly by this compound in vitro and using cultured cells. Binding measurements were performed by difference absorption and fluorescence spectroscopy. MDL 27048 binds to one site/tubulin heterodimer with an apparent equilibrium constant Kb = (2.8 +/- 0.8) X 10(6) M-1 (50 mM 2-(N-morpholino)ethanesulfonic acid, 1 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, 0.5 mM MgCl2, 0.1 mM GTP buffer, pH 6.7, at 25 degrees C). Podophyllotoxin displaced the binding of MDL 27048, suggesting an overlap with the colchicine-binding site. Assembly of purified tubulin into microtubules was inhibited by substoichiometric concentrations of MDL 27048, which also induced a slow depolymerization of preassembled microtubules. The cytoplasmic microtubules of PtK2 cells were disrupted in a concentration and time-dependent manner by MDL 27048, as observed by indirect immunofluorescence microscopy. Maximal depolymerization took place with 2 X 10(-6) M MDL 27048 in 3 h. When the inhibitor was washed off from the cells, fast microtubule assembly (approximately 8 min) and complete reorganization of the cytoplasmic microtubule network (15-30 min) were observed. MDL 27048 also induced mitotic arrest in SV40-3T3 cell cultures. Due to all these properties, this anti-tumor drug constitutes a new and potent microtubule inhibitor, characterized by its specificity and reversibility.
A comparative study of canine testicular tumors in descended and cryptorchid testes was performed in order to associate pathologic features with clinical signs. Observation on 80 canine testicular tumors revealed the following distribution: in undescended testes, 4 tumors were classified as Seminomas (mean age: 8.8 +/- 3 years) and 14 as Sertoli cell tumors (8.7 +/- 1.7 years); in descended testes, 21 tumors were classified as seminomas (8.8 +/- 3 years), 13 as Sertoli cell tumors (9.8 +/- 1.8 years), 22 as Leydig cell tumors (11.5 +/- 2 years), 5 multiple primary tumors and 1 as an immunoblastic lymphoma. Histological features were studied and correlated with other clinical parameters: feminization manifestations, prostatic disease, perineal hernia and perianal gland tumor.
The colchicine analogue 2-methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-on e (MTC) was found to inhibit concanavalin A- and formyl-methionyl-leucyl-phenylalanine-stimulated human neutrophil degranulation and to depolymerize the microtubule network at low concentrations. The inhibitory capacity of MTC for neutrophil degranulation was similar to that of nocodazole and taxol. The mechanistically distinct actions of these three drugs on microtubules support the notion that microtubules are required for neutrophil enzyme release in response to different stimuli. MTC affected both degranulation and microtubule integrity rapidly and reversibly, after only a 5-min preincubation. At these short periods of incubation, colchicine irreversibly affected neutrophil degranulation only at concentrations in the millimolar range and behaved similarly to its microtubule-inactive analogue lumicolchicine. At longer times of incubation (30-60 min), low concentrations of both MTC and colchicine induced a drastic shortening and depolymerization of microtubules, preserving the microtubule-organizing center, but only MTC was able to completely inhibit the secretory response of neutrophils. These results suggest that the colchicine effect on neutrophil degranulation is not specifically mediated by its action on the microtubule network of these cells. In contrast, the specific and reversible effects of the colchicine analogue MTC suggest that it may be a useful agent with which to study the role of microtubules in this cellular function.
We synthesized five peptides homologous to the potentially antigenic positions alpha(214-226), alpha(430-443), alpha(415-443), beta(241-256), and beta(412-431) of the porcine brain tubulin sequences. These peptides were successfully employed to raise tubulin-cross-reactive antibodies. The antibodies are specific of the regions of tubulin spanned by the peptides. They react specifically with the tubulin bands in immunoblots and with microtubules in immunofluorescence assays of cytoskeletons. The peptides of the C-terminal regions have also been employed to localize determinants recognized by two available monoclonal antibodies to tubulin in the positions alpha(415-430) and beta(412-431), respectively. In a first application of the anti-peptide antibodies, we have mapped the fragments of limited proteolysis of purified calf brain tubulin by trypsin, chymotrypsin, papain, thermolysin, subtilisin, and protease V8 from Staphylococcus aureus. Thirty-seven peptides have been identified, of which 32 have been unequivocally aligned into the tubulin sequences on the basis of their antigenic reactivity. There are three major, well-defined zones of preferential cleavage by the proteases: the C-termini and two internal zones in each chain. C-Terminal cleavages of both chains by subtilisin do not remove the antigenic reactivity of the zones alpha(415-430) and beta(412-431). C-Terminal cleavages by protease V8 are preferential of beta-tubulin. All six proteases tested cleave alpha- and/or beta-tubulin at one or both of the internal zones. These zones are located roughly at one-third and two-thirds of the chain length in both subunits. Therefore, a model of the tubulin monomers is proposed which consists of three major, proteolytically defined, compact regions (N-terminal, middle, and C-terminal thirds) and the cleavable zones. This model is discussed with the tubulin structural information presently available.
2-methoxy-5-(2,3,4-trimethoxyphenyl) 2,4,6-cycloheptatrien-1-one (MTC) is a synthetic colchicine analogue, lacking the B ring of the alkaloid (Fitzgerald: Biochem. Pharmacol. 25:1381-1387, 1976). MTC has been shown to bind reversibly to the colchicine binding site of tubulin and to inhibit microtubule assembly in vitro (Andreu et al: Biochemistry 23:1742-1752, 1984; Bane et al: J. Biol. Chem. 259:7391-7398, 1984). Its action on different cultured cell lines (PtK2, Pk15, and SV-3T3) has now been studied. 0.2 X 10(-6) M MTC stopped Pk15 and SV-3T3 cell growth, inducing an accumulation of mitoses in a few hours. Removal of MTC from the culture medium rapidly restored normal mitotic index and growth rates. Partial depolymerization of the cytoplasmic microtubules of PtK2 cells was observed at concentrations ranging from 2 to 5 X 10(-7) M. Maximal microtubule network depolymerization was obtained after 4 h of treatment with 2 to 5 X 10(-6) M MTC or at a higher MTC concentration (2 X 10(-5) M) for less than 2 h. Removal of 2 X 10(-5) M MTC (the highest MTC concentration used) from the culture medium resulted in almost complete microtubule polymerization after 10 min of drug recovery and a normal microtubule network in 20-30 min. MTC constitutes an antimitotic drug directed to the colchicine site. It is water-soluble, shows a fast and reversible action, and may therefore be employed as a convenient tool to study cellular microtubule-dependent functions.
Rupture of the uterus is an uncommon obstetric emergency that usually occurs after 36 weeks gestation in a woman with a previous cesarean section. Complete rupture of the uterus with extrusion of the fetus into the peritoneal cavity is associated with high fetal mortality and with hypovolemic shock in the mother. Incomplete ruptures are less catastrophic and are often found incidentally at routine elective cesarean section. Management of uterine rupture consists of prompt recognition, rapid replacement of maternal blood volume, and early laparotomy and hysterectomy or, in selected cases, uterine repair. Disseminated intravascular coagulation has been reported in association with such obstetrical emergencies as abruptio placentae, intrauterine fetal demise, septic abortion, and amniotic fluid embolism. We report a case in which there was clinical and laboratory evidence of DIC in a patient with uterine rupture. The patient was successfully managed with prompt hysterectomy and replacement of coagulation factors.
Because patients given 50% nitrous oxide and 50% oxygen failed to show any significant clinical response, a mixture of 70% nitrous oxide and 30% oxygen was administered to 124 patients at an altitude of 5,280 feet in the emergency department at Denver General Hospital. Seventy-five percent of the patients were 25 years of age or younger. Twenty-two (17.7%) had minimal or no response, while 71 (57.3%) had good to excellent response. More than 20% of the patients received concomitant analgesic medications. Six patients experienced side effects during administration. Of these patients, four had received narcotic medication prior to administration of nitrous oxide. The mixture of 70% nitrous oxide and 30% oxygen was safe and effective in treating juvenile and adult patients presenting with pain in the emergency department at a higher elevation.