Generation of transgenic mice with major histocompatibility class II genes.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Biomedical subjects
R Little
Publications and source records attributed to R Little.
A technique for delivery of total body irradiation for bone marrow transplantation in adults and adolescents.
With the increasing use of bone marrow transplantation for cancer, total body irradiation is becoming a more commonplace procedure in many of the larger centers across the country. The technical difficulties in delivering homogenous doses of radiation to the whole body are significant and involve many factors such as creation of a homogeneous, "flat" beam of radiation, and dealing with variations in patient thickness and tissue homogeneity, particularly in the lung. In addition, techniques must be used to safely and efficiently deal with patients who are usually very ill and require long treatment times. Although there is often an advantage in terms of dosimetry to using an AP/PA treatment technique, many institutions use parallel opposed lateral beams because of equipment and facility limitations. A technique has been devised that enables total body irradation to be given by an AP/PA technique using equipment available in many radiotherapy departments. Patients are supported in an upright position during treatment by means of a modified harness attached to the ceiling of the treatment room. Lung compensators are fixed to individually fitted "vests," allowing the patient moderate amounts of movement during treatment while maintaining the position of the compensator relative to the lungs. Thermoluminiscent dosimeter (TLD) dose measurements in a phantom indicate that this system can deliver accurate and homogeneous doses to lung tissue, while allowing a good degree of patient comfort and safety during the long treatment times that are required.
Adolescent↗
Expression and function of mutant Ia antigen in transgenic mice.
Cell surface expression of Ia antigens requires the assembly of alpha and beta heterodimers. We have produced a double transgenic mouse with a wild form Ak alpha gene and a mutant Ak beta (Ak beta MB) gene with d-allele substitution at positions 63 and 65-67. Initial studies indicated that the Ak alpha and Ak beta MB transgenes are not expressed on the surface of lymphoid cells of the transgenic mice. However, when spleen cells were stimulated with LPS prior to FACS analyses, Ak/Ak MB assembly and subsequent surface expression was induced. The tail skins from transgenic founder mice were rejected by the parental mice indicating a role for the mutant antigen on the allograft. In addition, the Ak transgenic mice on H-2q/q background can partially delete V beta 6+ T cells, suggesting the presence of the transgene product in the thymus.
Animals↗
Analysis of the functional domains of biosynthetic threonine deaminase by comparison of the amino acid sequences of three wild-type alleles to the amino acid sequence of biodegradative threonine deaminase.
The nucleotide sequence of the gene, ilvA, for biosynthetic threonine deaminase (Tda) from Salmonella typhimurium was determined. The deduced amino acid sequence was compared with the deduced amino acid sequences of the biosynthetic Tda from Escherichia coli K-12 (ilvA) and Saccharomyces cerevisiae (ILV1) and the biodegradative Tda from E. coli K-12 (tdc). The comparison indicated the presence of two types of blocks of homologous amino acids. The first type of homology is in the N-terminal portion of all four isozymes of Tda and probably indicates amino acids involved in catalysis. The second type of homology is found in the C-terminal portion of the three biosynthetic isozymes and presumably is involved in either (i) the binding or interaction of the allosteric effector isoleucine with the enzyme, or (ii) subunit interactions. The sites of amino acid changes of two E. coli K-12 ilvA alleles with altered response to isoleucine are consistent with the conclusion that the C-terminal portion of biosynthetic Tda is involved in allosteric regulation.
Alleles↗
Incorporation of patient immobilization, tissue compensation and matchline junction technique for three-field breast treatment.
A protocol for the treatment of the intact breast was developed to maximize dose homogeneity and reproducibility. This protocol uses patient and breast immobilization, three-dimensional tissue compensators, and a technique for geometric matching of fields when the supraclavicular area is treated. A series of phantom measurements and analysis of patient port films was performed to evaluate dose homogeneity and reproducibility using this technique, and the potential adverse effect of loss of skin sparing from the immobilization device was investigated. Dose homogeneity throughout the phantom breast was within +/- 6% of the prescribed central axis dose, and homogeneity at the supraclavicular match line was +/- 10%. This represented a significant improvement over techniques not using tissue compensation or geometrically matched fields. Reproducibility of patient treatments was not significantly improved from previous non-immobilized treatment techniques, but there was no loss of skin sparing from the device, and other advantages of immobilization were observed. Details of the protocol are discussed together with changes that are currently being made to improve the results obtained thus far.
Breast Neoplasms↗
Partial purification and properties of human brain aldehyde dehydrogenases.
Acetaldehyde and biogenic aldehydes were used as substrates to investigate the subcellular distribution of aldehyde dehydrogenase activity in autopsied human brain. With 10 microM acetaldehyde as substrate, over 50% of the total activity was found in the mitochondrial fraction and 38% was associated with the cytosol. However, with 4 microM 3,4-dihydroxyphenylacetaldehyde and 10 microM indoleacetaldehyde as substrates, 40-50% of the total activity was found in the soluble fraction, the mitochondrial fraction accounting for only 15-30% of the total activity. These data suggested the presence of distinct aldehyde dehydrogenase isozymes in the different compartments. The mitochondrial and cytosolic fractions were, therefore, subjected to salt fractionation and ion-exchange chromatography to purify further the isozymes present in both fractions. The kinetic data on the partially purified isozymes revealed the presence of a low Km isozyme in both the mitochondria and the cytosol, with Km values for acetaldehyde of 1.7 microM and 10.2 microM, respectively. However, the cytosolic isozyme exhibited lower Km values for the biogenic aldehydes. Both isozymes were activated by Mg2+ and Ca2+ in phosphate buffers (pH 7.4). Also, high Km isozymes were found in the mitochondria and in the microsomes.
Aldehyde Dehydrogenase↗
Transcription of ribosomal component genes and lac in a relA+/relA pair of Escherichia coli strains.
To determine the stringent response, a repression of gene activity during amino acid starvation assumed to be mediated by the effector necleotide guanosine tetraphosphate (ppGpp), of metabolically regulated constitutive genes, we measured the transcription of ribosomal protein genes, the constitutive lac operon, and stable RNA genes in a variety of growth media and after amino acid starvation in a relA+/relA pair of Escherichia coli B/r strains. For rRNA and tRNA (stable RNA) it has previously been shown that the distinction between stringent control and growth rate control is unfounded, as the function describing the stable RNA gene activities at different concentrations of guanosine tetraphosphate is independent of growth conditions (exponential growth or amino acid starvation) and of the relA allele present. Here, the results indicated that the stringent responses of ribosomal protein genes and lac differ from their metabolic control during exponential growth in different media. This can be explained by polarity and RNA polymerase sink effects during amino acid starvation which are irrelevant for stable RNA genes but which are superimposed on mRNA gene activities.
Amino Acids↗
Disseminated histoplasmosis in patients with the acquired immune deficiency syndrome.
The ongoing epidemic of acquired immune deficiency syndrome (AIDS) has affected homosexual men, intravenous (IV) drug abusers, Haitians, hemophiliacs, and others. Defects in cell-mediated immunity place these patients at risk for opportunistic infections. We recently saw three men from Alabama with disseminated infection due to Histoplasma capsulatum. Two of these men were homosexual and the other was an IV drug abuser. These three patients had evidence of depressed cellular immunity consistent with a diagnosis of AIDS. Infection caused by organisms indigenous to certain geographic areas of the United States may become more common in patients with AIDS as the epidemic continues.
Acquired Immunodeficiency Syndrome↗
rpoB mutation in Escherichia coli alters control of ribosome synthesis by guanosine tetraphosphate.
An isogenic pair of relA+ and relA strains of Escherichia coli B/r with a mutation in the RNA polymerase subunit gene rpoB (Rifr) was isolated in which the relationship between guanosine tetraphosphate (ppGpp) concentration and stable RNA (rRNA, tRNA) gene activity was altered. The RNA polymerase in the rpoB strains was found to be about 20-fold more sensitive to ppGpp with respect to its stable RNA promoter activity than was the wild-type enzyme. The existence of such mutants is consistent with the idea that ppGpp interacts with the RNA polymerase enzyme and thereby alters its promoter selectivity, i.e., reduces its affinity for the stable RNA promoters. Under most conditions, the rpoB mutants had a reduced rate of growth and about a 10-fold-reduced intracellular concentration of ppGpp compared with the rpoB wild-type strains. The reduction of the level of ppGpp in the rpoB mutants during exponential growth was presumably a reflection of an indirect effect of the rpoB mutation on the control of relA-independent ppGpp metabolism.
DNA-Directed RNA Polymerases↗
Physiological characterization of Escherichia coli rpoB mutants with abnormal control of ribosome synthesis.
We have previously reported the isolation of Escherichia coli rpoB mutants in which the control of ribosome synthesis by the nucleotide effector guanosine tetraphosphate (ppGpp) is altered, owing to a 20-fold increased sensitivity of the mutant RNA polymerases to ppGpp. In these mutants, the level of ppGpp during exponential growth is decreased about 10-fold, relative to that of rpoB+ wild-type strains, such that a near normal partitioning of RNA polymerase occurs with respect to stable RNA (rRNA and tRNA) gene activity. Here, the physiological effects of two different rpoB alleles in a relA+ and relA background were analyzed in greater detail by comparison with their isogenic rpoB+ wild-type parents. For a given growth medium, the rpoB mutations were found to affect four parameters which resulted in a reduction of growth rate. The results reinforce a previous conclusion that a key element in control of the bacterial growth rate is a mutual relationship between control of ribosome synthesis by ppGpp and control of relA-independent ppGpp metabolism by the concentration and function of ribosomes.
DNA-Directed RNA Polymerases↗
Isolation of rel mutants of Escherichia coli B/r.
A method that relies on the biological effect of near-UV (340-nm) irradiation is described by which large numbers of independent rel mutants of Escherichia coli B/r may be rapidly isolated.
Escherichia coli↗
Quantitation of guanosine 5',3'-bisdiphosphate in extracts from bacterial cells by ion-pair reverse-phase high-performance liquid chromatography.
Explore the source record for details and available documents.
Amino Acids↗
Temperature dependence of RNA synthesis parameters in Escherichia coli.
For Escherichia coli B/r growing in glucose minimal medium, the following parameters of RNA synthesis remained invariant between 20 and 40 degrees C: RNA polymerase concentration (RNA polymerase/mass), rRNA and tRNA concentration (RNA/mass), RNA polymerase activity (fraction of total RNA polymerase actively engaged in RNA chain elongation), and stable RNA synthesis relative to total RNA synthesis. The following parameters increased 3.4-fold over the same temperature range: rRNA chain elongation rate, guanosine tetraphosphate (ppGpp) concentration, and culture growth rate. Above 40 degrees C, the changes became more complex, and the growth rate began to decrease. The observation that most RNA synthesis parameters are temperature invariant despite the increase of ppGpp suggests that the mechanism of RNA synthesis control by ppGpp, assumed to involve an interaction of RNA polymerase wtih ppGpp, is itself temperature dependent such that, with increasing temperature, higher concentrations of ppGpp are required to affect the RNA polymerase.
Bacterial Proteins↗
Control of rRNA and tRNA syntheses in Escherichia coli by guanosine tetraphosphate.
The expression of stable RNA (rRNA and tRNA) genes and the concentration of guanosine tetraphosphate (ppGpp) were measured in an isogenic pair of relA+ and relA derivatives of Escherichia coli B/r. The cells were either growing exponentially at different rates or subject to amino acid starvation when they were measured. The specific stable RNA gene activity (rs/rt, the rate of rRNA and tRNA synthesis relative to the total instantaneous rate of RNA synthesis) was found to decrease from 1.0 at a ppGpp concentration of 0 (extrapolated value) to 0.24 at saturating concentrations of ppGpp (above 100 pmoles per optical density at 460 nm unit of cell mass). The same relationship between the rs/rt ratio and ppGpp concentration was obtained independent of the physiological state of the bacteria (i.e., independent of the growth rate or of amino acid starvation) and independent of the relA allele. It can be concluded that ppGpp is an effector for stable RNA gene control and that stable RNA genes are not controlled by factors other than the ppGpp-mediated system. The results were shown to be qualitatively and quantitatively consistent with data on in vitro rRNA gene control by ppGpp, and they were interpreted in the light of reported ideas derived from those in vitro experiments.
Escherichia coli↗
Control of RNA synthesis in Escherichia coli after a shift to higher temperature.
Parameters of RNA synthesis were measured after a temperature upshift in a pair of Escherichia coli B/r strains that are isogenic except for having relA and relA+ loci, to examine the cause for a reported anomaly in the correlation between guanosine tetraphosphate (ppGpp) and stable RNA (rRNA, tRNA) synthesis under such conditions. Two main results were: (i) the specific stable RNA gene activity (stable RNA per total RNA synthesis) correlated in the conventionally expected fashion with the level of ppGpp but was obscured by a nonspecific increase in the RNA chain elongation rate due to the higher temperature; (ii) the temperature upshift caused a transient reduction in the RNA polymerase activity (transcribing per total enzyme) that accounts for the previously observed oscillating RNA synthesis rate after a temperature shift.
Bacterial Proteins↗
Insertions of transposon Tn5 into ribosomal protein PNA polymerase operons.
The genetic organization and interrelationships between the two ribosomal protein transcription units (the L11 and L10 operons) from near 89 min on the Escherichia coli chromosome were studied by using insertional mutations generated by the kanamycin-resistant transposable element Tn5. The polar effects of Tn5 insertions on the expression of the L11, L1, L10, and L12 ribosomal protein genes and the beta RNA polymerase subunit gene were examined (i) by the level of beta-galactosidase activity generated from L10-lacZ and beta-lacZ gene fusions, (ii) by direct sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins specified by plasmid ribosomal protein genes in UV-irradiated maxicells, and (iii) by urea-polyacrylamide gel electrophoresis of plasmid- and chromosome-specified L12 protein. The results confirmed the organization of these genes into two transcription units as follows: PL11, rplK (L11), rplA (L1), PL10, rplJ (L10), rplL (L12), rpoB (beta). . .; they also localized the position of the PL10 promoter within an 80-nucleotide region near the end of the L1 gene. The results also support the idea that the translational regulatory proteins for the L11 and L10 operons are L1 and L10, respectively, and that the expression of the L12 gene is closely linked to L10 gene expression.
Bacterial Proteins↗
The analysis of stimulus probability inside and outside the focus of attention, as reflected by the auditory N1 and P3 components.
Explore the source record for details and available documents.
Attention↗
Transcriptional and post-transcriptional control of ribosomal protein and ribonucleic acid polymerase genes.
A partial restriction of ribonucleic acid (RNA) polymerase activity has been used to dissociate the coordinate synthesis of ribosomal proteins and subunits of RNA polymerase and to identify transcriptional and post-transcriptional control signals which regulate the expression of these component genes. Within the beta operon [which has the genetic organization: promoter (p beta), rplJ (L10), r;lL (L7/L12), attenuator, rpoB (beta), rpoC (beta'), terminator], the restriction caused a disproportionate increase between proximal and distal gene transcriptions; the transcriptional intensities of the proximal ribosomal protein genes and the distal RNA polymerase genes were elevated about two- and fourfold, respectively. Transcription within the operon containing four ribosomal protein genes and the RNA polymerase alpha gene was also enhanced, whereas transcription within operons containing only ribosomal protein genes was virtually unaffected by the restriction. It was thus concluded that the mechanisms controlling transcription initiation or attenuation or both in operons containing RNA polymerase subunit genes are coupled to the global rate of RNA synthesis. By introducing the composite ColE1 plasmid pJC701 carrying the proximal portion of the L10 operon, including the beta subunit gene, it was possible to achieve a 10- and a 30-fold range in the transcriptional intensities of the genes specifying L10 and L7/L12 and beta, respectively. Under these conditions, the relative synthesis rates of L7/L12 and beta protein varied by less than 2-fold and by about 15-fold, respectively. These observations corroborate the existence of a post-transcriptional mechanism which severely restricts translation of excess L7/L12 and L10 ribosomal protein messenger RNA; this mechanism is probably important in maintaining the balanced synthesis of ribosome components under conditions in which their messenger RNA levels are dissociated. Furthermore, the observed reduction in the translation efficiency of beta subunit messenger RNA may be related to an inhibitory effect caused by accumulation of RNA polymerase assembly intermediates.
Bacterial Proteins↗