Analysis of chromosomes with restriction endonucleases and DNase hypersensitivity.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
J de la Torre
Publications and source records attributed to J de la Torre.
Intracranial tuberculosis in AIDS: CT and MRI findings.
CT and MRI findings in 35 patients with the acquired immune deficiency syndrome (AIDS) and proven intracranial tuberculosis (TB) are presented. Over 90% of the patients were intravenous drug abusers and in two-thirds TB was the first manifestation of AIDS. CT was normal in one quarter, the most frequent findings being hydrocephalus (51%) and meningeal enhancement (41%), commonly seen together (31.5%). Meningeal enhancement was seen in 48% of the CT studies with intravenous contrast medium and in 3 cases studied with MRI and i.v. gadolinium DTPA, in 2 of which CT was negative. Parenchymal involvement was found in 37% of cases; MRI was more sensitive than CT for its detection. One quarter of the patients had ischaemic lesions, mainly in the basal ganglia. We confirm the usefulness of CT and the superiority of MRI in the diagnosis of intracranial TB and in differential diagnosis from other conditions likely to be found in these patients.
Acquired Immunodeficiency Syndrome↗
The distribution of genes on human chromosomes as studied by in situ nick translation.
We have studied the distribution of potentially active genes on human chromosomes, using two methods: DNAse I hypersensitivity and restriction enzyme--nick translation with enzymes sensitive to methylation of CpG doublets. DNAse hypersensitivity is known to be associated with potentially active genes, and, when the reaction is detected by "in situ" nick translation, produces an R-banding pattern. Digestion of chromosomes with HpaII or CfoI, both of which should preferentially cut unmethylated sequences in the CpG islands associated with the majority of genes, also produces R-banding patterns. Deviations are attributable to overdigestion of the chromosomes, leading to extraction of DNA and loss of the specific sites that were to be detected. Contrary to the results of a number of previous workers, we have failed to demonstrate any differences between the DNAse I hypersensitivity or the degree of methylation of the active and inactive X chromosomes in metaphases from females.
Chromosome Mapping↗
Heterochromatin characterization of sex chromosomes in Triturus marmoratus (Urodela, Salamandridae).
The sex chromosomes of the Iberian marbled newt, Triturus marmoratus, were studied using various banding techniques, including restriction enzyme/nick translation (RE/NT) procedures. Four types of heterochromatin on the sex chromosomes could be distinguished: (1) distamycin A/DAPI and chromomycin A3/distamycin A positive, EcoRI/NT negative, and HaeIII/NT and HinfI/NT positive; (2) distamycin A/DAPI and chromomycin A3/distamycin A positive, but RE/NT negative; (3) AT rich, but RE/NT negative; and (4) distamycin A/DAPI and chromomycin A3/distamycin A positive, EcoRI/NT and HinfI/NT negative, but HaeIII/NT positive. These data suggest a common origin for the terminal heterochromatic domains of both the X and Y chromosomes in this species.
Animals↗
Restriction endonuclease/nick translation of fixed mouse chromosomes: a study of factors affecting digestion of chromosomal DNA in situ.
We used a restriction endonuclease/nick translation procedure to study the ability of certain enzymes, known to cleave mouse satellite DNA in solution, to attack satellite DNA in fixed mouse chromosomes. Although AvaII and Sau96I readily attack the mouse major satellite in fixed chromosomes, BstNI and EcoRII do not normally do so, although if the heterochromatin is uncondensed as a result of culture in the presence of 5-azacytidine, BstNI can attack it. No clear evidence was obtained for digestion in situ of the minor satellite of mouse chromosomes by MspI, the only enzyme reported to cleave this satellite. Our results show that the DNA of mouse heterochromatin is not merely not extracted by certain restriction enzymes, but is actually not cleaved by them. Chromatin conformation is therefore shown to be an important factor in determining patterns of digestion of chromosomes by restriction endonucleases.
Animals↗
Meiotic chromosome structure. Kinetochores and chromatid cores in standard and B chromosomes of Arcyptera fusca (Orthoptera) revealed by silver staining.
The behaviour of two chromosome structures in silver-stained chromosomes was analyzed through the first meiotic division in spermatocytes of the acridoid species Arcyptera fusca. Results showed that at diakinesis kinetochores and chromatid cores are individualized while they associate in bivalents of metaphase I; only kinetochores and distal core spots associate in the sex chromosome. Metaphase I is characterized by morphological and localization changes of both kinetochores and cores which define the onset of anaphase I. These changes analyzed in both autosomes and in the sex chromosome allow us to distinguish among three different substages in metaphase I spermatocytes. B chromosomes may be present as univalents, bivalents, or trivalents. Metaphase I B univalents are characterized by separated cores except at their distal ends and individualized and flat sister kinetochores. At anaphase I sister kinetochores of lagging B chromatids remain connected through a silver-stained strand. The behaviour of cores and kinetochores of B bivalents is identical with that found in the autosomal bivalents. The differences in the morphology of kinetochores of every chromosome shown by B trivalents at metaphase I may be related to the balanced forces acting on the multivalent. The results show dramatic changes in chromosome organization of bivalents during metaphase I. These changes suggest that chromatid cores are not involved in the maintenance of bivalents. Moreover, the changes in morphology of kinetochores are independent of the stage of meiosis but correlate with the kind of division (amphitelic-syntelic) that chromosomes undergo.
Animals↗
Discontinuous undercondensation of centromeric heterochromatin in mouse chromosomes: evidence in Hoechst 33258-treated cells.
Mouse chromosomes from the L929 cell line have been treated with Hoechst 33258 to induce undercondensation of centromeric heterochromatin. The morphological changes induced by this fluorochrome were analyzed in electron micrographs of whole-mounted chromosomes. Results show that the condensation inhibition of centromeric heterochromatin caused by Hoechst 33258 is not produced homogeneously and suggest compositional differences within an individual centromere.
Animals↗
Determination of acetoacetate in urine by solid-phase spectrophotometry.
A method for the determination of acetoacetate has been developed based on solid-phase spectrophotometry (SPS). The acetoacetate reacts with nitroprusside and glycine and the reaction product is sorbed on Dowex 1-X8 resin. The absorbance of the resin phase at 590 and 720 nm is measured directly. The calibration graph is linear up to 3.3 mg l-1 and the RSD is 1.9%. The detection limit is 7.6 micrograms l-1. The method has been applied to the determination of acetoacetate in normal and diabetic subjects' urine without pretreatment of the samples, and the results compared with those of 1H-NMR and homogeneous nitroprusside methods.
Acetoacetates↗
Effect of specific proteolytic cleavages on tubulin polymer formation.
The capacity for self-polymerization and shape of the tubulin polymers assembled after digestion with trypsin, Pronase, chymotrypsin, subtilisin, Staphylococcus aureus proteinase V8 and proteinase K were investigated. Digestion with trypsin, Pronase or chymotrypsin resulted in a decrease in the ability of tubulin for self-assembly, whereas limited proteolysis with subtilisin, S. aureus proteinase V8 or proteinase K resulted in an increase in such ability. The shape of the assembled polymers varied from typical microtubules (after the treatment with trypsin or Pronase) to sheets (after the treatment with chymotrypsin) and from hooked microtubules with a constant polarity (after the treatment with subtilisin) to the disappearance of a defined polarity of such polymers (after the treatment with S. aureus V8 proteinase or proteinase K). These results indicate that the tubulin C-terminal regions are involved in the regulation of microtubule polymerization, shape, directional growth and lateral interactions between tubulin protofilaments.
Chymotrypsin↗
The TILT formula for brain lesions.
A formula to evaluate and grade brain lesions is reported. The formula is based on 4 descriptive factors which characterize the lesion severity and potential outcome on the subject. The factors are, type of lesion, intensity of lesion, location and time of lesion duration before therapy is applied (TILT). A relative value is assigned to each TILT factor and the sum of these values determines the severity of the injury or traumindex. The relative benefit of specific therapy can then be assessed quickly and objectively by applying its effect on TILT. Three examples picked at random from the literature, illustrate application of TILT comparing "claimed" and "apparent" benefit of specific treatments. The formula is useful for both model-induced basic and sustained clinical injuries of the brain.
Animals↗
A model for quantifying genetic recombination in chromosome polymorphisms due to supernumerary heterochromatic segments.
Heterochromatic supernumerary segments can modify chiasma distribution in the affected bivalents. A model is developed for quantifying the genetic recombination in populations polymorphic for such segments. This paper describes this model for a monochiasmate bivalent, and its application in different populations of the grasshopper Chorthippus parallelus. The observations suggest that these polymorphic systems have evolutionary implications.
Animals↗
Interaction of tubulin with octyl glucoside and deoxycholate. 2. Protein conformation, binding of colchicine ligands, and microtubule assembly.
The structural change induced by binding of mild detergents to cytoplasmic calf brain tubulin and the effects on the functional properties of this protein have been characterized. Massive binding of octyl glucoside or deoxycholate monomers induces circular dichroism changes indicating a partial alpha-helix to disordered structure transition of tubulin. The protein also becomes more accessible to controlled proteolysis by trypsin, thermolysin, or V8 protease. This is consistent with the looser protein structure proposed in previous binding and hydrodynamic studies [Andreu, J. M., & Muñoz, J. A. (1986) Biochemistry (preceding paper in this issue)]. Micelles of octyl glucoside and deoxycholate bind colchicine and its analogue 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC). This impedes the determination of colchicine binding in the presence of detergents. Both detergents cause a reduction in the number of tubulin equilibrium binding sites for the colchicine site probe MTC. Deoxycholate monomers bind poorly to the tubulin-colchicine complex, but deoxycholate above the critical micelle concentration effectively dissociates the complex. Microtubule assembly in glycerol-containing buffer is inhibited by octyl glucoside, which raises the critical protein concentration. Low concentrations of deoxycholate enhance tubulin polymerization, allowing it to proceed without glycerol. The polymers formed are microtubules, pairwise associated open microtubular sheets, and macrotubules possibly generated by helical folding of the sheets, as indicated by the optical diffraction patterns. Saturation of tubulin with octyl glucoside, followed by full dissociation of the detergent, allowed the recovery of binding to the colchicine site and microtubule assembly, indicating the reversibility of the protein structural change.
Animals↗
The removal of the carboxy-terminal region of tubulin favors its vinblastine-induced aggregation into spiral-like structures.
Vinblastine induces brain tubulin to assemble into spirals. This process is stimulated by microtubule-associated proteins (MAPs) which copolymerize with brain microtubules assembled in vitro. When the carboxy terminal of tubulin is removed by subtilisin digestion, vinblastine readily induces the aggregation of tubulin into spiral-like or circular structures, even in the absence of MAPs. These results suggest that in the absence of MAPs, the carboxy-terminal domain of tubulin may inhibit vinblastine-induced polymerization of tubulin into spiral-like structures.
Biopolymers↗
Proteolytic modification of tubulin.
Explore the source record for details and available documents.
Animals↗
A method for visualizing the acrosome by light microscopy.
A silver staining technique applied to squash preparations of material previously fixed in 3:1 ethanol:acetic acid produces differential staining of the acrosomal region of spermatids during spermiogenesis in orthopteroid species. The method includes treatment with saline sodium citrate solution for 15 min at 60 C, and staining with 50% aqueous silver nitrate adjusted to pH 2.9 with formic acid.
Acrosome↗
The localization of tau proteins on the microtubule surface.
The localization of porcine brain tau factor on in vitro assembled microtubules has been carried out by immunoelectron microscopy, using affinity-purified antibodies and protein A-gold particles. A parallel experiment was done using antibody against microtubule associated protein 2 (MAP2) and also a double labelling experiment using both antibodies with different sized gold particles. Our results indicate that, within the limits of resolution imposed by immunolabelling, the distribution patterns of tau factor and MAP2 on the microtubule are indistinguishable.
Animals↗
Surprisingly rich projection from locus coeruleus to the olfactory bulb in the rat.
The brainstem nucleus, locus coeruleus (LC), is the major, if not the sole, source of noradrenergic (NE) innervation of the telencephalon. It is generally held that LC neurons project diffusely to the entire neuroaxis and this had been the basis for theories that postulate 'general' functions (sleep, attention, learning, etc.) for LC. We report that at least 40% of all LC neurons project to the olfactory bulb; the projection is almost 10 times greater than to any other part of the cerebral cortex. This unsuspectedly rich LC-olfactory bulb connection is consistent with current theories which implicate LC neurons in heightened sensory vigilance and trophic regulation of connectional development and plasticity.
Animals↗
Differential association of the different brain microtubule proteins in different in vitro assembly conditions.
Microtubule protein was assembled in vitro for different time periods and at different protein concentrations near and far from the critical concentration needed for assembly. When the isolated polymers were characterized by electrophoresis, a higher association of high-molecular-weight microtubule-associated proteins, particularly of the larger microtubule-associated protein, was found in the polymers assembled close to the critical concentration, while tau factor polypeptides are predominantly present in those polymers assembled at higher protein concentration. Also, a higher proportion of high-molecular-weight microtubule associated proteins MAP1 and MAP2 was observed in microtubules assembled for short time periods, compared with those obtained after the monomer-polymer equilibrium was reached. When the assembled protein was further characterized by isoelectric focusing, no differences were found in the proportion of the different isotubulins present in the polymers assembled under different conditions tested.
Animals↗