Ostrich chick survival presents challenge.
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Animal Nutritional Physiological Phenomena↗
Biomedical subjects
C A Smith
Publications and source records attributed to C A Smith.
Soluble tumor necrosis factor (TNF) receptors are effective therapeutic agents in lethal endotoxemia and function simultaneously as both TNF carriers and TNF antagonists.
Two forms (monomeric or dimeric) of the extracellular, ligand-binding portion of the human p80 cell-surface receptor for TNF were used to antagonize TNF activity in vitro and in vivo. The dimeric sTNFR:Fc molecule was a more potent inhibitor of TNF than the monomeric sTNFR (50 to 1000x), as assessed in vitro by inhibition of TNF binding or bioactivity and in vivo by protection of mice from an otherwise lethal injection of LPS. Surprisingly, the dimeric sTNFR:Fc construct demonstrated a beneficial effect even when administered 3 h after a lethal LPS injection (i.e., after serum TNF levels had peaked and receded). To study the mechanism by which the soluble TNFR functions in vivo, serum TNF levels were examined in mice given LPS in the presence or absence of soluble receptor. Administration of a mortality-reducing dose of sTNFR:Fc ablated the rise in serum TNF bioactivity that normally occurs in response to LPS. However, TNF bioactivity was revealed in these "TNF-negative" serum samples when the L929 bioassay was modified by inclusion of a mAb that blocks the binding of murine TNF to the human soluble TNFReceptor. These results indicate that the absence of direct cytolytic activity in the L929 assay was caused by neutralization of TNF, rather than to an absence of TNF in the serum. Moreover, administration of either monomeric sTNFR or low doses of dimeric sTNFR:Fc actually resulted in increased serum TNF levels compared to mice given LPS but no soluble receptor. However, these "agonistic" doses of soluble receptor did not lead to increased mortality when an LD60 dose of LPS was given. Thus, dimeric sTNFR are effective inhibitors of TNF and under some circumstances function simultaneously as both TNF "carriers" and antagonists of TNF biologic activity.
Animals↗
DNA photolyase repairs the trans-syn cyclobutane thymine dimer.
DNA photolyases catalyze the splitting of the cyclobutane ring joining the two dihydropyrimidines of a pyrimidine dimer by a photoinduced electron-transfer reaction. Previous studies concluded that photolyase repairs only the cis-syn form of the eight stereoisomers of the cyclobutane pyrimidine dimer (Pyr[ ]Pyr). In this study we found that Escherichia coli photolyase binds to the trans-syn-I isomer of T[ ]T with about 10(4)-fold lower affinity than the cis-syn isomer but it repairs it relatively efficiently.
Base Sequence↗
CD30 antigen, a marker for Hodgkin's lymphoma, is a receptor whose ligand defines an emerging family of cytokines with homology to TNF.
CD30 is a surface marker for neoplastic cells of Hodgkin's lymphoma and shows sequence homology to members of the tumor necrosis factor (TNF) receptor superfamily. Using a chimeric probe consisting of the extracellular domain of CD30 fused to truncated immunoglobulin heavy chains, we expression cloned the cDNA cognate from the murine T cell clone 7B9. The encoded protein is a 239 amino acid type II membrane protein whose C-terminal domain shows significant homology to TNF alpha, TNF beta, and the CD40L. Cross-hybridization to an induced peripheral blood T cell cDNA library yielded the human homolog, which is 72% identical at the amino acid level. The recombinant human ligand enhances the proliferation of CD3-activated T cells yet induces differential responses, including cell death, in several CD30+ lymphoma-derived clones. The human and murine genes map to 9q33 and the proximal region of chromosome 4, respectively.
Amino Acid Sequence↗
Preparation and characterization of a set of deoxyoligonucleotide 49-mers containing site-specific cis-syn, trans-syn-I, (6-4), and Dewar photoproducts of thymidylyl(3'-->5')-thymidine.
Deoxyoligonucleotide 49-mers containing a central cis-syn, trans-syn-I, (6-4), or Dewar photoproduct of TpT were constructed for use in repair and replication studies by ligation of shorter photoproduct-containing oligonucleotides. A (6-4) product-containing 6-mer was prepared in 3.4% yield by 254 nm irradiation of d(AATTAA) and converted in nearly quantitative yield to the Dewar isomer by irradiation with Pyrex- and Mylar-filtered medium-pressure mercury arc light. d(CGAATTAAGC) containing a site-specific cis-syn or trans-syn-I dimer was prepared via automated solid-phase DNA synthesis utilizing photoproduct building blocks. The photoproduct-containing 49-mers were characterized by their susceptibility to base cleavage and a number of enzymes routinely used to map the sites of DNA photoproduct formation. 1 M piperidine at 90 degrees C cleaved the Dewar product faster than the (6-4) product, but did not cleave the cyclobutane dimers. The 3'-->5' exonuclease activity of T4 DNA polymerase was completely blocked by all the lesions except the (6-4) product, which it slowly bypassed. T4 endonuclease V did not cleave the (6-4) or Dewar photoproduct, but unexpectedly cleaved the trans-syn-I dimer at most 1% the rate of the cis-syn dimer in double-stranded DNA. The trans-syn-I dimer was cleaved at a 50-fold higher rate in double- than in single-stranded DNA. Escherichia coli photolyase was found to be specific for the cis-syn dimer at low concentrations. Implications of this work to methodology for mapping and quantifying DNA photoproducts are also discussed.
Base Sequence↗
Effect of sequence, adduct type, and opposing lesions on the binding and repair of ultraviolet photodamage by DNA photolyase and (A)BC excinuclease.
The cis,syn and (6-4) products of dipyrimidine sites are the major mutagenic and lethal UV photoproducts in DNA. To investigate their relative susceptibilities to repair and other factors such as sequence context and lesions in the complementary strand that might influence repair efficiencies, we constructed 49-mer duplexes containing site-specific photoproducts of thymidylyl(3',5')thymidine sites at central locations. Using these substrates, we measured the binding of Escherichia coli DNA photolyase to cis,syn dimers in four sequence contexts and to two cis,syn dimers in close proximity and on opposing strands. We found that the sequence within a 10-base pair region had little effect on binding and that two enzyme molecules bound to substrate containing two dimers in the 5'-staggered orientation, but not in the 3'-staggered one. Similarly, the excision of a cis,syn dimer by (A)BC excinuclease was not influenced by the sequence in the immediate vicinity of the dimer, and the enzyme was active on 5'-staggered cis,syn dimers, but not on 3'-staggered ones. Of special significance, we found that (A)BC excinuclease removed the cis,syn, trans,syn-I, (6-4), and Dewar photoproducts at vastly differing relative rates of 1:6:9:9, respectively.
Base Composition↗
Molecular and biological characterization of a ligand for CD27 defines a new family of cytokines with homology to tumor necrosis factor.
CD27 is a surface antigen found on T and B cells that has homology to a family of molecules including the receptors for tumor necrosis factor (TNF) and nerve growth factor. A cDNA encoding a ligand for CD27 was isolated by a direct-expression cloning strategy using a fusion protein composed of the extracellular domain of CD27 linked to the constant domain of a human immunoglobulin G1 molecule as a probe. The predicted protein product is a type II transmembrane protein whose gene maps to 19p13 and that shows homology to TNF and the ligand for CD40. Biological characterization indicates that the cloned ligand induces the proliferation of costimulated T cells and enhances the generation of cytolytic T cells.
Amino Acid Sequence↗
Synthesis and antibody-mediated detection of oligonucleotides containing multiple 2,4-dinitrophenyl reporter groups.
A series of non-nucleoside-based 2,4-dinitrophenyl (DNP) phosphoramidites have been prepared and used in the multiple labelling of oligonucleotides during solid-phase synthesis. The length of spacer arm between the DNP label and the oligonucleotide phosphate backbone, and the number of attached DNP groups have both been varied in order to determine the optimum conditions for anti-DNP antibody binding. Detection using enzyme-linked colorimetric techniques showed sensitivity equivalent to that obtainable using biotinylated oligonucleotides.
2,4-Dinitrophenol↗
Hyperkalemic periodic paralysis presents medical and ethical challenge.
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Animals↗
Analysis of cytokine gene expression in subpopulations of freshly isolated thymocytes and thymic stromal cells using semiquantitative polymerase chain reaction.
Using a semi-quantitative polymerase chain reaction (PCR) technique we have examined the expression of a panel of cytokines during thymus development, localizing the expression to individual components of the thymic stroma and thymocytes at different maturational stages. The expression of interleukin (IL)-7, stem cell factor (SCF), IL-1 alpha and granulocyte-monocyte-colony-stimulating factor (GM-CSF) mRNA was mapped to individual stromal cell types, while the expression of IL-1 alpha and GM-CSF, along with interferon (IFN)-gamma and IL-4 was detected in the lymphoid compartment of fetal day (Fd) 14 thymus. The expression of lymphoid-specific cytokines genes was selectively down-regulated in thymocytes undergoing maturation. CD3-/lo4+8+ cells, representing an intermediate stage of thymocyte maturation, were devoid of cytokine gene expression. Their CD3+ progeny, on the other hand, expressed IFN-gamma mRNA, supporting the notion that positive selection of cells for further maturation induces the reexpression of some cytokine genes. The cytokine profiles of the various stromal components differed. Purified major histocompatibility complex class II+ cortical epithelial cells strongly expressed IL-7 and SCF, but only limited expression of IL-1 alpha and GM-CSF could be detected. Fetal mesenchyme, on the other hand, expressed SCF, IL-1 alpha and GM-CSF but not IL-7. The importance of these cytokine profiles in relation to T cell development is discussed.
Animals↗
Localization of the CYP2D gene locus to human chromosome 22q13.1 by polymerase chain reaction, in situ hybridization, and linkage analysis.
Using a combination of somatic cell hybrids, in situ hybridization, and linkage mapping, we have been able to localize the cytochrome P450 CYP2D6 gene to chromosome 22 in the region q13.1. Linkage analysis, using locus-specific primers, showed a maximum sex-average lod score of 8.12 (theta = 0.00) between the marker pH130 (D22S64) and CYP2D6, of 6.92 (theta = 0.00) between the marker KI839 (D22S95) and CYP2D6, and of 4.80 (theta = 0.036) between the platelet-derived growth factor beta subunit gene (PDGFB) and CYP2D6.
Chromosome Mapping↗
Cloned mouse cerebrovascular endothelial cells that maintain their differentiation markers for factor VIII, low density lipoprotein, and angiotensin-converting enzyme.
This communication describes a relatively novel cell culture technique for the isolation of cerebrovascular endothelial cells from three strains of inbred mice. Cerebrovascular endothelial cells were identified by their morphology, the presence of Factor VIII-related antigen and angiotensin-converting enzyme, and the uptake of acetylated low-density lipoprotein. Cloned cerebrovascular endothelial cells were found to maintain their differentiated state and diploid genotype through 15 serial passages. The morphology and growth characteristics of these cells were found to be altered when cultured on different extracellular matrices. The isolation and cloning methods described are simple and highly reproducible.
Animals↗
Housing stressors, social support and psychological distress.
This paper explores the links between housing stressors, social supports and psychological distress. It was hypothesised that exposure to housing stressors would be significantly related to psychological distress, but that the adverse effects of housing stressors would be moderated by perceptions of social support resources. In other words, the stress/symptom relationship would be attenuated for individuals who believe that they have active and supportive social relationships. The results of a study conducted in the two New Zealand cities of Auckland and Christchurch partially confirmed this hypothesis, indicating that social support plays a role in mitigating the adverse effects of housing stressors. However, this relationship depends on the severity of the housing stressors. Among our respondents, the presence of social support was indeed associated with reduced symptom levels for those exposed to moderate housing stressors. However, among respondents subjected to high levels of housing stressors, social support was not associated with reduced psychological distress, indicating the need for a more specific policy response to the issue of seriously deficient housing.
Adaptation, Psychological↗
Temperature dependence of the phosphorescence quantum yield of various alpha-lactalbumins and of hen egg-white lysozyme.
The radiative quantum yield, phi op, of the triplet state of human alpha-lactalbumin (HLA) has been measured in the temperature range between 6 K and the softening point of the aqueous glass (approximately 150 K). phi op has little temperature dependence below approximately 30 K, but above this it decreases sharply with increasing temperature. The unusual temperature dependence is fitted by a phenomenological two-state model in which the phosphorescence originates primarily from a donor, tryptophan (Trp) 104, and an acceptor, Trp 60, the populations of which are coupled by a thermally activated triplet-triplet energy transfer process. The model assumes that the acceptor (Trp 60) triplet state undergoes radiationless deactivation by a proximal disulfide residue, while the donor (Trp 104) has no such extrinsic quencher. The decrease of phi op with increasing temperature is accounted for by the thermally activated triplet-triplet energy transfer process. The disulfide quenching rate constant itself is assumed to be temperature independent, in accord with recent measurements of simple disulfide quenching in long chain snake venom neurotoxins (Schlyer, B. D., E. Lau, and A. H. Maki. 1992. Biochemistry. 31:4375-4383; Li, Z., A. Bruce, and W. C. Galley. 1992. Biophys. J. 61:1364-1371). We find that the phosphorescence quenching in HLA occurs with an activation energy of 97 cm-1, which we associate with a barrier to the energy transfer process. The data are fit well by the model if we assume a value for the temperature-independent disulfide quenching constant of kQ > 3 s-1 that is consistent with recent measurements on indole-disulfide model systems (Li, Z., A. Bruce, and W. C. Galley. 1992. Biophys. J. 61:1364-1371). Similar results are reported for bovine alpha-lactalbumin (BLA) and for hen egg-white lysozyme (HEWL) that contains the structural equivalents of Trp 104 and Trp 60 of HLA. HLA provides the best agreement with calculations since it is the simplest, lacking Trp 26, a residue not considered in the model, that probably contributes significantly to the phosphorescence of BLA, guinea pig alpha-lactalbumin (GPLA), and HEWL. GPLA, which contains Trp 104 but lacks Trp 60, shows qualitatively less thermally induced phosphorescence quenching than HLA, BLA, and HEWL, thus supporting the postulated quenching model.
Animals↗
In search of the "hot" cognitions: attributions, appraisals, and their relation to emotion.
Two studies examined the hypothesized status of appraisals, relative to attributions, as proximal antecedents of emotion. In Study 1, which looked at 6 emotions (happiness, hope-challenge, anger, guilt, fear-anxiety, and sadness), undergraduates (N = 136) reported on their attributions, appraisals, and emotions during past encounters associated with a variety of situations. In Study 2, which was focused on anger and guilt, undergraduates (N = 120) reported on these same variables in response to experimenter-supplied vignettes that systematically manipulated theoretically relevant attributions. The results of both studies indicated that the emotions were more directly related to appraisals than they were to attributions, and Study 2 provided evidence that appraisal serves as a mediator between attribution and emotional response. These findings lend support to the hypothesized status of appraisal as the most proximal cognitive antecedent of emotion.
Anger↗
Transfer of the ADA gene into bone marrow cells and peripheral blood lymphocytes for the treatment of patients affected by ADA-deficient SCID.
Severe combined immunodeficiency (SCID) caused by deficiency of the enzyme adenosine deaminase (ADA) is the first genetic disorder considered for human somatic cell gene therapy. ADA-SCID patients can be cured by HLA-matched sibling donor bone marrow transplantation. Alternative transplantation strategies as well as enzyme replacement are being tested in those patients who do not have a suitable matched sibling donor. Some ADA-SCID patients may not be candidates for cytoablation due to infectious damage to the lung or liver, or may have a milder phenotype that does not justify the risks associated with haploidentical bone marrow transplantation. Replacement therapy with PEG-ADA has resulted in improvement in growth, a variable increase in the number of peripheral blood lymphocytes, and a decrease in the incidence of severe infections. Another approach to the treatment of severe genetic diseases is now represented by somatic cell gene therapy. We and others have conducted experiments in vitro and in vivo that have documented that T-lymphocytes are suitable vehicles for gene transfer. Although the pluripotent stem cell remains the ideal target cell for somatic cell gene therapy of disorders of the hematopoietic system, the use of T-lymphocytes as gene therapy vehicles is specifically indicated for ADA-deficient patients where they represent the affected cells. Furthermore, the selective engraftment of T-cells only, following bone marrow transplantation, has resulted in reconstitution of cellular and humoral immunity. A model for the functional analysis in vivo of the human immune system has been utilized for the preclinical evaluation of this approach.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine Deaminase↗
The ancestral IncP replication system consisted of contiguous oriV and trfA segments as deduced from a comparison of the nucleotide sequences of diverse IncP plasmids.
In most plasmids which have been studied to date the functions required for plasmid replication are clustered in a 2-3 kb region. However, in all known naturally occurring plasmids of the Escherichia coli incompatibility group P the essential replication functions, oriV, the vegetative replication origin and trfA, which encodes proteins essential to activate oriV, are separated by blocks of DNA consisting of either known genes conferring resistance to antimicrobial agents and/or putative transposable elements. Nucleotide sequence comparisons reported here reveal that these blocks of DNA have inserted at different points into a backbone of DNA common to IncP plasmids. The results indicate that in the common ancestor of present IncP plasmids oriV and trfA must have been contiguous, whilst a rho-independent transcriptional terminator, now lost in IncP alpha plasmids, may have prevented trfA operon transcription from interfering with the activity of oriV.
Bacterial Proteins↗
Effects of specific carotid body and brain hypoxia on respiratory muscle control in the awake goat.
1. We assessed the effects of specific brain hypoxia on the control of inspiratory and expiratory muscle electromyographic (EMG) activities in response to specific carotid body hypoxia in seven awake goats. We used an isolated carotid body perfusion technique that permitted specific, physiological, steady-state stimulation of the carotid bodies or maintenance of normoxia and normocapnia at the carotid bodies while varying the level of systemic, and therefore, brain oxygenation. 2. Isolated brain normocapnic hypoxia of up to 1.5 h duration increased inspired minute ventilation (VI) by means of increases in both tidal volume (VT) and respiratory frequency (fR). Electromyographic activities of both inspiratory and expiratory muscles were augmented as well. These responses were similar to those produced by low levels of whole-body normoxic hypercapnia. We conclude that moderate levels of brain hypoxia (Pa,O2 approximately 40 mmHg) in awake goats caused a net stimulation of ventilatory motor output. 3. Hypoxic stimulation of the carotid bodies alone caused comparable increases in VT and fR, and EMG augmentation of both inspiratory and expiratory muscles whether the brain was hypoxic or normoxic. These responses were quite similar to those obtained over a wide range of whole-body normoxic hypercapnia. We conclude that the integration of carotid body afferent information is not affected by moderate brain hypoxia in awake goats. 4. We found no evidence for an asymmetrical recruitment pattern of inspiratory vs. expiratory muscles in response to carotid body hypoxia or in response to brain hypoxia alone. 5. Our data support the concept that moderate brain hypoxia results in a net stimulation of respiratory motor output. These findings question the significance of 'central hypoxic depression' to the regulation of breathing under physiological levels of hypoxaemia in the awake animal.
Animals↗