Evaluation of family therapy for disturbed children.
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Child↗
Biomedical subjects
L Simpson
Publications and source records attributed to L Simpson.
Specific cleavage of kinetoplast minicircle DNA from Leishmania tarentolae by mung bean nuclease and identification of several additional minicircle sequence classes.
Multiple sequence classes of kinetoplast minicircle DNA from Leishmania tarentolae were cleaved by mung bean nuclease in the presence of formamide, yielding unit length linear molecules which retained the anomalous electrophoretic mobility in acrylamide characteristic of minicircle DNA. No specific cleavage site sequence common to all minicircle sequence classes was apparent, although the main region of nuclease cleavage was localized approximately 350 bp from the unique SmaI restriction site of the conserved region found in all minicircle sequence classes. Covalent closure of the minicircle substrate was not a requirement for cleavage, as linearized network-derived or cloned minicircles were also cleaved by mung bean nuclease at similar locations. The partial sequences of several new minicircle sequence classes released from the network by mung bean nuclease are also reported.
Animals↗
Impaired drug uptake in methotrexate resistant Crithidia fasciculata without changes in dihydrofolate reductase activity or gene amplification.
Crithidia fasciculata cells grown in defined medium are sensitive to methotrexate (MTX), an inhibitor of dihydrofolate reductase (DHFR). When cells are challenged with 2-5 microM MTX, cell division ceases after 3-4 divisions and the cells become rounded and immotile for approximately 60 h, with a 40% decrease in cell viability occurring during this period. The cells then recover normal morphology and cell division resumes. Cells which undergo this treatment can be transferred directly into high levels of the drug (1-2 mM). The resistance phenotype is stable in the absence of the drug. Resistance correlates with impaired uptake of [3H]MTX, which in wild-type cells is taken up by a carrier-mediated process. There is no indication of gene amplification at the DNA level or at the level of DHFR activity, as occurs in the case of MTX-resistant Leishmania major. Several lines of MTX-resistant L. major which show gene amplification also exhibit impaired uptake of [3H]MTX.
Animals↗
Kinetoplast DNA in trypanosomid flagellates.
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Animals↗
Introduction of plasmid DNA into the trypanosomatid protozoan Crithidia fasciculata.
Crithidia fasciculata cells were treated with a plasmid (pDK96) containing pBR322 sequences, a Leishmania tarentolae maxicircle autonomously replicating sequence, and the bacterial gene for aminoglycoside 3' phosphotransferase I inserted between the yeast alcohol dehydrogenase 1 promotor and terminator sequences. Resistant colonies were selected on agar plates containing paromomycin and screened for vector DNA by hybridization. Approximately 1% of the resistant colonies contained detectable vector DNA, which was present as extrachromosomal closed circular molecules ranging in copy number from 1 to 160 per cell. The plasmids could be recovered from Escherichia coli transformed to ampicillin resistance with Crithidia total cell DNA. Most of the recovered plasmids were a deleted product of pDK96, which lacked the maxicircle autonomously replicating sequence and contained a unique fragment of Crithidia nuclear DNA present at a low copy number in the wild-type genome. The plasmid DNA in resistant Crithidia was unstable even under selective conditions and was lost within 30 cell divisions.
Ampicillin↗
Mapping and 5' end determination of kinetoplast maxicircle gene transcripts from Leishmania tarentolae.
Transcripts for six Leishmania tarentolae maxicircle structural genes (cytochrome oxidase subunits I, II and III, cytochrome b, human mitochondrial unidentified reading frames 4 and 5) and several unidentified open reading frames were mapped, and the locations of the 5' ends determined by primer runoff analysis. All genes studied here are transcribed from the same strand as the 12S and 9S ribosomal RNAs except for the cytochrome oxidase subunit I gene. In two cases (ORF3 and ORF4, ORF5 and ORF6), a single transcript covers two contiguous overlapping reading frames. The 5' ends of the RNAs are located 20-64 nt from the putative translation initiation codons. Primary transcripts from a mitochondrial RNA preparation were 5' end-labeled with guanylyltransferase and alpha -32P-GTP; the major labeled species comigrated with the 12S and 9S mitochondrial rRNAs, and in addition there were at least four higher molecular weight labeled species.
Animals↗
Effect of tryptophan derivatives on the phase properties of bilayers.
Binding of several tryptophan derivatives and tryptophan-containing peptides to bilayers is examined by monitoring fluorescence enhancement as a function of lipid concentration. The thermodynamic and spectral parameters of the solutes in the bilayers of vesicles and liposomes do not exhibit any anomalous dependence upon the gel or the liquid-crystalline phase state of the bilayer. Effects of these solutes on the phase-transition profiles of the bilayers of liposomes and vesicles are examined, and the lowering of the phase-transition temperature is correlated with the mole fraction of the solute in the bilayer. The partition coefficients do not change at the main phase-transition temperature. These observations contradict the thermodynamic explanation of the solute-induced lowering of the phase-transition temperature which is based on the Van't Hoff relationship for distribution of the solute in the two coexisting phases at the phase-transition temperature. It is postulated that solute molecules bound to defect sites in bilayers modulate the phase properties of bilayers. These defect sites are induced in the gel phase of bilayers of liposomes above the subtransition temperature.
Chemical Phenomena↗
The divergent region of the Leishmania tarentolae kinetoplast maxicircle DNA contains a diverse set of repetitive sequences.
A 2.76 kb segment of the 12 kb divergent region of the Leishmania tarentolae kinetoplast maxicircle DNA consists almost entirely of repeated sequences. The repeats can be grouped into six families, some of which are present throughout the remainder of the divergent region. The repeats are oriented in a head-to-tail fashion with the three simplest repeats clustered into large arrays. A 47 bp palindrome and two copies of a "supercluster" of three different types of repeats are also present in the sequenced region. A sequence change in the divergent region is described for a clonal strain of L. tarentolae which was passaged continuously for several years. The repetitive sequences found in the divergent region appear to be appropriate substrates for the presumed deletion/insertion/recombination events occurring in this rapidly evolving portion of the maxicircle.
Animals↗
Primary sequence and partial secondary structure of the 12S kinetoplast (mitochondrial) ribosomal RNA from Leishmania tarentolae: conservation of peptidyl-transferase structural elements.
The sequence of the 1173 nt 12S kinetoplast ribosomal RNA from Leishmania tarentolae was determined from the maxicircle DNA sequence, and the 5' and 3' ends localized by primer runoff and S1 nuclease protection experiments. The gene was shown to be free of introns by S1 nuclease analysis. A partial secondary structure model of the 12S RNA molecule is presented which is equivalent in certain respects to the corresponding portions of the Escherichia coli 23S ribosomal RNA model. Domain II of the E. coli model is completely missing in the kinetoplast model with the exception of several phylogenetically conserved stems and one loop. There is a striking conservation of the functionally important peptidyl-transferase region except for the deletion of a few stems and loops. The 12S RNA is the smallest large subunit ribosomal RNA described to date.
Animals↗
Central nervous regulation of body temperature in vertebrates: comparative aspects.
Thermal fluctuations affect, and are responded to by, nearly all forms of life. The basic vertebrate template has guided and shaped the ways that animals in this subphylum cope with thermal challenges. This has led to a situation where there are major similarities in the neuronal mechanisms which sense temperature and control the responses to temperature change in all vertebrates, from fish to mammals. The PO/AH is the most important single integration site for temperature regulation and (except for birds) is also important in the sensing of core temperature. Other portions of the brainstem as well as the spinal cord are also involved in thermal control and can sense, integrate, and produce appropriate efferent signals to varying degrees. Peripheral thermal input to the hypothalamic areas is via the brainstem reticular areas. A number of studies has related the thermal response characteristics of CNS single neurons to the thermoregulatory output of intact animals. These studies have been performed on neurons in whole animal, brain slice, and tissue culture preparation. These neurophysiological studies of central neurons are informative, but are sometimes difficult to interpret because of the chronic lack of definite criteria to differentiate generalized thermal sensitivity from thermal sensitivity utilized for regulating body temperature. Recent neuroanatomical work has illustrated that many areas previously implicated in the thermoregulatory network (such as the septum, various hypothalamic nuclei, the midbrain reticular formation, and the midbrain raphé nuclei) receive direct projections from PO/AH neurons. When compared, the neurophysiological and neuroanatomical characteristics of the preoptic nucleus and anterior hypothalamic area are similar, but not identical. The broad differences in the responses that vertebrates utilize to deal with thermal change is largely determined by the respiratory medium (water or air) and whether metabolic energy (endothermy) or ambient temperature (ectothermy) is of primary importance in the determination of internal temperature. A number of physiological systems are perturbed in water breathing ectotherms when the ambient temperature is altered. In these vertebrates long-term acclimation is very important and has a major effect on temperature selection. Air breathing ectotherms are less adversely affected by temperature change; long-term thermal acclimation is less important and has little effect upon temperature selection; large thermal changes are often initiated by these animals. Endotherms rely on insulation and a high, variable metabolic rate to maintain a constant internal temperature.(ABSTRACT TRUNCATED AT 400 WORDS)
Amphibians↗
A minimal ribosomal RNA: sequence and secondary structure of the 9S kinetoplast ribosomal RNA from Leishmania tarentolae.
The portion of the Leishmania tarentolae kinetoplast maxicircle DNA encoding the 9S RNA gene was sequenced, and the 5' and 3' ends of the transcript were determined. A secondary structure for the 9S RNA was determined based on the Escherichia coli 16S model. The 610-nucleotide 9S RNA exhibits a minimal secondary structure in which all four domains of the E. coli 16S structure are preserved. Within domains, however, some stems and loops have been greatly reduced or eliminated entirely. It is presumed that these reduced domains represent the minimal essential small ribosomal RNA secondary structures necessary for a functional ribosome. Alignment of the L. tarentolae 9S rRNA sequence with the published Trypanosoma brucei 9S rRNA sequence shows a nucleotide similarity of 84% and a transversion/transition ratio of 1.66.
Animals↗
High resolution CT scanning of the bronchial tree. CT bronchography: technique & clinical application.
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Adolescent↗
Trypanosoma brucei: differentiation of in vitro-grown bloodstream trypomastigotes into procyclic forms.
Trypanosoma brucei strain 366D trypomastigotes grown at 37 degrees C in the presence of a human fibroblast cell line formed foci underneath the feeder cells whereas trypanosomes grown in the presence of a human epithelial cell line grew only in the culture supernatant. A culture system was developed to study the differentiation of bloodstream trypomastigotes grown in the epithelial cell system into procyclic trypomastigotes at 27 degrees C. The morphological differentiation into the procyclic form was complete by 48 h. Cell division did not occur until 30-40 h after transfer to 27 degrees C. Various characteristics of this system were examined, including the effect of the feeder layer, the type of medium, the presence of the metabolites cis-aconitate and citrate, the preadaptation period, and the trypanosome cell concentration. The respiration of the recently differentiated procyclic cells was less sensitive to inhibition by CN- than that of established procyclic forms, implying a delayed appearance of complete mitochondrial oxidative pathways. This trypanosome differentiation system has the advantage that the animal host is not needed and the entire process is carried out in in vitro culture.
Aconitic Acid↗
Decubitus ulcer care: a comparative study.
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Adult↗
Sequences of six genes and several open reading frames in the kinetoplast maxicircle DNA of Leishmania tarentolae.
The DNA sequence of approximately 80% of the transcribed region of the kinetoplast maxicircle DNA of Leishmania tarentolae was obtained, and structural genes were localized by comparison of the translated amino acid sequences with those of known mitochondrial genes from other organisms. By this method, the genes for cytochrome oxidase subunits I, II, and III, cytochrome b, and human mitochondrial unidentified reading frames 4 and 5 were identified. By comparing the amino acid sequences of the putative L. tarentolae genes with those of known genes, we conclude that TGA codes for tryptophan, as in most other mitochondrial systems. This is the only apparent change from the universal genetic code. The six identified structural genes show various degrees of divergence from the homologous genes in other species, with cytochrome oxidase subunit I being the most conserved and cytochrome oxidase subunit III being the least conserved. A comparison of the cytochrome b genes from L. tarentolae and Trypanosoma brucei showed that the ratio of transversions to transitions is 1:1, suggesting that these species diverged from each other more than 80 X 10(6) years ago. Several as yet unidentified open reading frames were also present in the maxicircle sequence. These data confirm that maxicircle DNA has a coding potential which typifies other mitochondrial systems.
Amino Acid Sequence↗
Autonomous replication sequences in the maxicircle kinetoplast DNA of Leishmania tarentolae.
Four fragments from the maxicircle DNA of Leishmania tarentolae cloned into the selectable Saccharomyces cerevisiae shuttle vector, YIp5, exhibited autonomous replicating sequence (ars) activity. Two of the fragments (pSK120, pSK152) produced large yeast transformant colonies and two (pSK30, pSK150) produced small colonies. All yeast transformants contained extrachromosomal self replicating YIp5 hybrid plasmids as shown by mitotic instability in non selective medium and by the transformation of Escherichia coli with yeast minilysates and recovery of the plasmid from the transformed bacteria. The copy numbers of pSK30, pSK150 and pSK152 in the transformed yeast were approximately the same as that of the YRp12 control, which contains the yeast arsl element; the copy number of pSK120, however, was at least 10 fold lower. A 1.87 kb subfragment of the pSK120 fragment also showed strong ars activity. The entire DNA sequences of the pSK120, pSK152 and pSK150 fragments are known, and several yeast 11 mer consensus ars sequences are present within each fragment. In addition there is a sequence (Lt ars 189) within the pSK152 subclone that has 78% similarity with a 189 nt sequence of an ars element from the Crithidia fasciculata maxicircle (Cf ars 189), implying an evolutionary conservation of this putative origin of replication in at least two different kinetoplastid species. The relative positions of the Lt ars 189 sequence in the L. tarentolae maxicircle map and the Cf ars 189 sequence in the C. fasciculata map with respect to the 9 and 12 S ribosomal genes are similar, implying an overall conservation of gene order in this portion of the transcribed regions of these two species and perhaps in all kinetoplastid species.
Animals↗
Sequence heterogeneity and anomalous electrophoretic mobility of kinetoplast minicircle DNA from Leishmania tarentolae.
Several unit-length minicircles from the kinetoplast DNA of Leishmania tarentolae were cloned into pBR322 and into M13 phage vectors. The complete nucleotide sequences of three different partially homologous minicircles were obtained. The molecules contained a region of approx. 80% sequence homology extending for 160-270 bp and a region unique to each minicircle . A 14-mer was found to be conserved in all kinetoplast minicircle sequences reported to date. The frequency distributions of various minicircle sequence classes in L. tarentolae were obtained by quantitative gel electrophoresis and by examination of the "T ladder" patterns of minicircles randomly cloned into M13 at several sites. By these methods we could assign approx. 50% of the total minicircle DNA into a minimum of five sequence classes. A sequence-dependent polyacrylamide gel migration abnormality was observed with several minicircle fragments both cloned and uncloned. The abnormality was dependent on the presence of a portion of the conserved region of the minicircle .
Animals↗
Phorbol esters inhibit murine B cell differentiation to immunoglobulin secretion but not proliferation.
Bacterial lipopolysaccharide (LPS) induces resting B cells to proliferate and to secrete IgM. We have found that addition of phorbol esters (PE) such as phorbol myristate acetate (PMA) to murine B cells specifically inhibits LPS-induced IgM secretion but not proliferation. PMA is extremely potent, with half-maximal inhibition occurring at about 10 pM. The effect on B cells appears to be due to interaction with PE receptors, because a series of PE have similar potencies for tumor promotion, binding to receptors, and inhibition of IgM secretion. PE also inhibit IgM secretion induced by T cell-derived lymphokines and LPS-induced IgG secretion. Results of these studies suggest that the protein kinase C, with which PE interact, plays an important role in the regulation of B cell differentiation and may also provide a powerful tool for dissecting molecular events involved in induction of Ig secretion.
Animals↗