Do early cyclosporine levels affect the incidence of acute rejection in renal transplant recipients?
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Cadaver↗
Biomedical subjects
E M Johnson
Publications and source records attributed to E M Johnson.
A randomized, double-blind, placebo-controlled study of the safety, tolerance, and preliminary pharmacokinetics of ascending single doses of orally administered sirolimus (rapamycin) in stable renal transplant recipients.
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Administration, Oral↗
Toxic gas generation from plastic mattresses and sudden infant death syndrome.
Microbial generation of toxic gases from antimony, arsenic, or phosphorus in compounds used as fire retardants in cot mattresses has been proposed as a cause of sudden infant death. To test this hypothesis, 23 polyvinyl chloride mattress samples from cot death cases were incubated on malt agar plates until good microbial growth was obtained. Silver nitrate and mercuric chloride test papers were then inserted and the colour reactions recorded. The predominant organism, recovered from all mattresses tested, was not, as claimed in earlier work, the fungus Scopulariopsis brevicaulis, but a mix of common environmental Bacillus spp. Test paper colour changes occurred whenever bacterial growth was present, but these reactions also occurred in control tests in which no mattress material was present on the plates. Chemical and instrumental analyses of exposed test papers showed that the colour reactions were not due to deposits of antimony, arsenic, or phosphorus. Our findings do not support the hypothesis that toxic gases derived from antimony, arsenic, or phosphorus are a cause of sudden infant death. More sulphur was found in test papers exposed in plates containing bacterial growth than in those without such growth. This result suggests that the test paper reactions were due to the generation of sulphur-containing compounds during bacterial growth on the agar medium.
Bacillus↗
Association of human Pur alpha with the retinoblastoma protein, Rb, regulates binding to the single-stranded DNA Pur alpha recognition element.
The retinoblastoma protein, Rb, is detected in extracts of monkey CV-1 cells complexed with Pur alpha, a sequence-specific single-stranded DNA-binding protein implicated in control of gene transcription and DNA replication. These complexes can be immunoextracted from cell lysates using monoclonal antibodies to either Pur alpha or Rb. The Pur alpha-Rb complexes contain a form of Pur alpha with extensive post-synthetic modification, as demonstrated following expression of Pur alpha cDNA fused to a 9-amino acid epitope tag. Human Pur alpha, expressed as a glutathione S-transferase fusion protein, specifically binds to the hypophosphorylated form of Rb with an affinity as high as that of SV40 large T-antigen. In the absence of DNA, glutathione S-transferase-Pur alpha binds to p56RB, an NH2-terminal-truncated Rb protein purified from Escherichia coli, containing the T-antigen binding domain, to form multimeric complexes. The single-stranded DNA Pur alpha recognition element disrupts these complexes. Conversely, high concentrations of p56RB prevent Pur alpha binding to DNA. Through use of a series of deletion mutants, the DNA binding activity of Pur alpha is localized to a series of modular amino acid repeats. Rb binding involves a Pur alpha region with limited homology to the Rb-binding region of SV40 large T-antigen. Binding of Pur alpha to p56RB, the COOH-terminal portion of Rb, is inhibited by a synthetic peptide containing the T-antigen Rb-binding motif.
Amino Acid Sequence↗
Cooperative action of cellular proteins YB-1 and Pur alpha with the tumor antigen of the human JC polyomavirus determines their interaction with the viral lytic control element.
Human JC polyomavirus (JCV) is the etiologic agent of the neurodegenerative disease progressive mulifocal leukoencephalopathy. By using JCV as a model, we investigated the role of the viral early protein tumor antigen (TAg) in the binding of two cellular proteins, Pura alpha and YB-1, to JCV regulatory sequences. Results from band-shift assays with purified YB-1, Pur alpha, and TAg indicated that efficient binding of Pur alpha, a strong activator of early gene transcription, to a single-stranded target sequence corresponding to the viral lytic control element, is diminished in the presence of the late gene activator YB-1, which recognizes the opposite strand of the Pur alpha binding site. Of particular interest was the ability of Pur alpha and TAg to enhance binding of YB-1 to DNA molecules without being associated with this complex. Binding studies using a mutant peptide encompassing the N terminus of YB-1 indicated that the C terminus of YB-1 is important for its DNA binding activity. The ability of Pur alpha and TAg to increase binding of YB-1 to DNA is independent of the YB-1 C terminus. Similarly, results from band-shift assays using Pur alpha variants indicated that two distinct regions of this protein contribute either to its ability to bind DNA or to its ability to enhance YB-1 DNA binding activity. Based on the interaction of Pur alpha, YB-1, and TAg, and their binding to DNA, a model is proposed for the role of these proteins in transcription of viral early and late genes during the lytic cycle.
Animals↗
In vitro replication of plasmids containing human c-myc DNA.
A chromosomal replication initiation zone was previously mapped in cell cultures to the 5' flanking DNA of the human c-myc gene. We have used an in vitro system to examine the replication of a plasmid, pNeo.Myc-2.4, containing 2.4 kb of the c-myc initiation zone. In vitro, pNeo.Myc-2.4 generated high levels of DpnI-resistant DNA above background incorporation into control plasmids. pNeo.Myc-2.4 replicated semiconservatively to produce supercoiled and relaxed plasmid monomers, and replicative intermediates. [32P]dCMP incorporated into pNeo.Myc-2.4 appeared in Okazaki fragments and low molecular weight strands which matured to full length plasmid DNA, whereas [32P]dCMP incorporated into control plasmids appeared as continuous smears on denaturing gels. Other assays also distinguished the processive replication of pNeo.Myc-2.4 from the dispersive labeling of control plasmids. A pNeo.Myc-2.4 replication time course showed a clear preference for initiation within a restriction fragment containing the c-myc DNA. Two-dimensional electrophoresis revealed that a restriction fragment bearing the c-myc origin zone generated an arc characteristic of replicative intermediates containing a central replication bubble, while vector fragments in the plasmid generated arcs of forked intermediates. Replication bubbles visualized by electron microscopy were centered within the replication initiation zone, approximately 1.4 kb upstream of c-myc promoter P1. Okazaki fragments radiolabeled during in vitro replication showed a switch in the asymmetry of template preference within the initiation zone identified by electron microscopy, two-dimensional electrophoresis and early labeling. These data show that bidirectional, semiconservative replication can originate preferentially in vitro in the 5' flanking DNA of the c-myc gene, and that replicative intermediates present at low levels can be distinguished from molecules generated by competing, repair-type processes.
Cell-Free System↗
Suppression of programmed neuronal death by a thapsigargin-induced Ca2+ influx.
Rat sympathetic neurons undergo programmed cell death (PCD) in vitro and in vivo when they are deprived of nerve growth factor (NGF). Chronic depolarization of these neurons in cell culture with elevated concentrations of extracellular potassium ([K+]o) prevents this death. The effect of prolonged depolarization on neuronal survival is thought to be mediated by a rise of intracellular calcium concentration ([Ca2+]i) caused by Ca2+ influx through voltage-gated channels. In this report we investigate the effects of chronic treatment of rat sympathetic neurons with thapsigargin, an inhibitor of intracellular Ca2+ sequestration. In medium containing a normal concentration of extracellular Ca2+ ([Ca2+]o), thapsigargin caused a sustained rise of intracellular Ca2+ concentration and partially blocked death of NGF-deprived cells. Elevating [Ca2+]o in the presence of thapsigargin further increased [Ca2+]i, suggesting that the sustained rise of [Ca2+]i was caused by a thapsigargin-induced Ca2+ influx. This treatment potentiated the effect of thapsigargin on survival. The dihydropyridine Ca2+ channel antagonist, nifedipine, blocked both a sustained elevation of [Ca2+]i and enhanced survival caused by depolarization with elevated [K+]o, suggesting that these effects are mediated by Ca2+ influx through L-type channels. Nifedipine did not block the sustained rise of [Ca2+]i or enhanced survival caused by thapsigargin treatment, indicating that these effects were not mediated by influx of Ca2+ through L-type channels. These results provide additional evidence that increased [Ca2+]i can suppress neuronal PCD and identify a novel method for chronically raising neuronal [Ca2+]i for investigation of this and other Ca(2+)-dependent phenomena.
Adrenergic Fibers↗
Destruction of the cholinergic basal forebrain using immunotoxin to rat NGF receptor: modeling the cholinergic degeneration of Alzheimer's disease.
Degeneration of cholinergic neurons in the basal forebrain (CBF) is a prominent neuropathological feature of Alzheimer's disease and is thought responsible for some cognitive deficits seen in patients. An animal model of pure CBF degeneration would be valuable for analysis of the function of these neurons and testing therapeutic strategies. CBF neurons express receptors for nerve growth factor. In order to selectively destroy these neurons, we developed an immunotoxin using monoclonal antibody (192 IgG) to rat NGF receptor (p75NGFr) armed with the ribosome inactivating protein, saporin. In vitro 192-saporin was highly toxic to neurons expressing p75NGFr. Intraventricular injections of 192-saporin destroyed the CBF and impaired passive avoidance learning. These results indicate that 192-saporin treated rats can be used to model a key feature of Alzheimer's disease and that anti-neuronal immunotoxins are a powerful approach to selective neural lesioning.
Alzheimer Disease↗
Role of BCL-2 in the survival and function of developing and mature sympathetic neurons.
Sympathetic neurons, when placed in culture during the period of naturally occurring cell death, will die by apoptosis when deprived of nerve growth factor (NGF). In this system, the mRNA levels of the BCL-2 family members decrease after NGF deprivation and during apoptosis. Sympathetic neurons from BCL-2-deficient mice died more rapidly after NGF deprivation than neurons from wild-type littermates. Sympathetic neurons of adult animals are relatively independent of NGF for survival. If sympathetic neurons are maintained in vitro for several weeks, loss of acute trophic factor dependence develops with a time course similar to that seen in the intact animal. Examination of neurons from BCL-2-deficient mice showed that BCL-2 expression is not required for the development of trophic factor independence. Therefore, BCL-2 is an important regulator of the survival of sympathetic neurons after NGF deprivation during the period of naturally occurring programmed neuronal death, but BCL-2 is not involved in the development of trophic factor independence in mature sympathetic neurons.
Animals↗
Superoxide dismutase delays neuronal apoptosis: a role for reactive oxygen species in programmed neuronal death.
Sympathetic neurons in culture die by apoptosis when deprived of nerve growth factor (NGF). We used this model of programmed cell death to study the mechanisms that mediate neuronal apoptosis. Cultured sympathetic neurons were injected with copper/zinc superoxide dismutase protein (SOD) or with an expression vector containing an SOD cDNA. In both cases apoptosis was delayed when the neurons were deprived of NGF. The delay was similar to that seen when a bcl-2 expression vector was injected. SOD, injected 8 hr after NGF deprivation, provided no protection, indicating that superoxide production may occur early in response to trophic factor deprivation. We have demonstrated, with a redox-sensitive dye, an increase in reactive oxygen species (ROS) that peaked at 3 hr after sympathetic neurons were deprived of NGF. If NGF was added back to the culture medium after the period of peak ROS generation, apoptosis was completely prevented, suggesting that ROS production serves as an early signal, rather than a toxic agent, to mediate apoptosis.
Animals↗
Molecular genetics of neuronal survival.
The past year has witnessed remarkable progress towards understanding the molecular genetics of neuronal survival. Gene-targeting experiments in mice have confirmed the long-standing idea that the nerve growth factor model of neuronal survival--that is, neuronal dependence on target-derived molecules during a critical period in development--is broadly applicable. Furthermore, a variety of biochemical and genetic techniques applied to both mammals and invertebrates have identified new genes involved in regulating cell survival during development.
Animals↗
Diffusion and partitioning of proteins in charged agarose gels.
The effects of electrostatic interactions on the diffusion and equilibrium partitioning of fluorescein-labeled proteins in charged gels were examined using fluorescence recovery after photobleaching and gel chromatography, respectively. Measurements were made with BSA, ovalbumin, and lactalbumin in SP-Sepharose (6% sulfated agarose), in phosphate buffers at pH 7 and ionic strengths ranging from 0.01 to 1.0 M. Diffusivities in individual gel beads (D) and in the adjacent bulk solution (D infinity) were determined from the spatial Fourier transform of the digitized two-dimensional fluorescence recovery images. Equilibrium partition coefficients (phi) were measured by recirculating protein solutions through a gel chromatography column until equilibrium was reached, and using a mass balance. Diffusion in the gel beads was hindered noticeably, with D/D infinity = 0.4-0.5 in each case. There were no effects of ionic strength on BSA diffusivities, but with the smaller proteins (ovalbumin and lactalbumin) D infinity increased slightly and D decreased at the lowest ionic strength. In contrast to the modest changes in diffusivity, there were marked effects of ionic strength on the partition coefficients of these proteins. We conclude that for diffusion of globular proteins through gel membranes of like charge, electrostatic effects on the effective diffusivity (Deff = phi D) are likely to result primarily from variations in phi with only small contributions from the intramembrane diffusivity.
Animals↗
A 39-kD DNA-binding protein from mouse brain stimulates transcription of myelin basic protein gene in oligodendrocytic cells.
The MB1 regulatory sequence of the myelin basic protein (MBP) gene spanning between nucleotides -14 to -50 with respect to the transcription start site is critical for cell type-specific transcription of the MBP gene, which encodes the major protein component of myelin sheath in cells derived from the central nervous system (CNS). This regulatory sequence has the ability to interact with a developmentally controlled DNA-binding protein from mouse brain that stimulates transcription of MBP promoter in an in vitro system (Haas, S., J. Gordon, and K. Khalili. 1993. Mol. Cell. Biol. 13:3103-3112). Here, we report the purification of a 39-kD protein from mouse brain tissue at the peak of myelination and MBP production that binds to the MB1 regulatory motif. Following partial amino acid sequence analysis, we have identified a complementary DNA encoding a 39-kD DNA-binding protein called pur alpha. Expression of pur alpha cDNA in the prokaryotic and eukaryotic cells resulted in the synthesis of a protein with characteristics similar to the purified brain-derived 39-kD protein in band shift competition assays. Cotransfection of the recombinant pur alpha expressor plasmid with MBP promoter construct indicated that Pur alpha stimulates transcription of the MBP promoter in oligodendrocytic cells, and that the nucleotide sequence required for binding of the 39-kD Pur alpha to DNA within the MB1 region is crucial for this activity. Moreover, transient expression of Pur alpha caused elevation in the level of endogenous MBP RNA in oligodendrocytic cells. Thus, Pur alpha, a sequence-specific DNA-binding protein upon binding to MB1 regulatory region may play a significant role in determining the cell type-specific expression of MBP in brain.
Animals↗
Neuronal apoptosis: current understanding of molecular mechanisms and potential role in ischemic brain injury.
Apoptosis is a rediscovered mechanism of cell death crucial in normal development. Recent exploration of the genetic mechanisms of apoptosis has broadened our insight into the regulation of cell death in development as well as disease states. We present an overview on current understanding of the genetic molecular events in apoptosis in all, or most cell types, with emphasis on events observed in a well-characterized model of neuronal death in vitro. The second part of this article reviews recent studies in in vivo stroke models on the mechanism of cell death relevant to apoptosis after cerebral ischemia. Further delineation of the mechanisms of cell death, especially those that trigger apoptosis, is likely to redirect our approaches in the development of new therapeutic interventions for ischemic stroke.
Animals↗
Emergence of azole drug resistance in Candida species from HIV-infected patients receiving prolonged fluconazole therapy for oral candidosis.
We examined the effect of different fluconazole treatment regimens on the emergence of azole drug resistance among Candida species recovered from the mouths of 54 HIV-infected individuals. Patients were assigned to one of three treatment groups depending on their history of oral candidosis and fluconazole use. Mouthwashes obtained at regular intervals were cultured and isolates identified using standard methods. Antifungal broth micro-dilution tests were performed to determine IC30s of fluconazole and ketoconazole. Sixty-four Candida albicans isolates from 20 patients with no evidence of oral candidosis who had not received fluconazole all had IC30s of < or = 4 mg/L. Thirty-four (83%) of 41 C. albicans isolates from ten patients receiving intermittent, short-term fluconazole treatment for oral candidosis had IC30s of < or = 4 mg/L, but only two isolates (5%) had IC30s > or = 64 mg/L. In contrast, 26 (40%) of 65 C. albicans isolates from 15 patients given long-term fluconazole (50-200 mg/day or 150 mg/week) were classified as resistant having IC30s of fluconazole of > or = 64 mg/L. Ten of these 26 fluconazole-resistant isolates were susceptible to ketoconazole with IC30s of < or = 4 mg/L suggesting azole drug cross-resistance is not inevitable. Tests on multiple colonies from individual isolation plates showed that it was not unusual to obtain differing IC30 values, indicating that a sweep inoculum is essential if resistance is to be detected. Nine (60%) of the 15 patients given long-term fluconazole harboured isolates of C. albicans that were resistant to fluconazole at some time during the study period. All had low CD4 counts and were approaching the final stage of their illness. Three patients on long-term treatment had resistant organisms at the outset of the study; in the remainder, resistant strains emerged during the study period. In six of the nine cases, emergence of resistance in vitro correlated with persistent clinical signs of oral infection. Thirty-six isolates of Candida species other than C. albicans were also recovered from patients receiving long-term fluconazole and 29 (81%) of these had IC30s of > or = 64 mg/L. Our experience with C. albicans in patients with HIV infection, suggests that the long-term azole drug use may be an important factor in the development of fluconazole resistance as such resistance was rare and transient in patients on intermittent short-term treatment.
Antifungal Agents↗
Azole drug resistance in yeasts.
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AIDS-Related Opportunistic Infections↗
Itraconazole susceptibilities of fluconazole susceptible and resistant isolates of five Candida species.
The in-vitro susceptibilities of 1380 isolates of five Candida species were determined in order to establish whether isolates resistant to fluconazole were cross-resistant to itraconazole. IC50 values were determined by a broth microdilution method. 690 Candida albicans isolates, seven Candida glabrata isolates, seven Candida krusei isolates, 120 Candida parapsilosis isolates and 37 Candida tropicalis isolates were susceptible to both fluconazole (IC50 < or = 32 mg/L) and itraconazole (IC50 < or = 4 mg/L). Twenty eight of 160 C. albicans isolates (17.5%), 180 of 293 C. glabrata isolates (61.4%), six of 48 C. krusei isolates (12.5%), and 10 of 18 C. tropicalis isolates (55.5%) resistant to fluconazole (IC50 > or = 64 mg/L) were also resistant to itraconazole (IC50 > or = 8 mg/L). In contrast, drug-specific resistance to itraconazole was not observed in any of the isolates tested. However, the itraconazole IC50s for fluconazole susceptible isolates were lower than those for fluconazole resistant isolates, which suggests that patients who fail fluconazole treatment might require itraconazole at higher dosages than usual.
Antifungal Agents↗
Use of the Pastorex aspergillus antigen latex agglutination test for the diagnosis of invasive aspergillosis.
AIMS: To evaluate the Pastorex aspergillus antigen latex agglutination test for the diagnosis of invasive aspergillosis in patients undergoing liver or bone marrow transplantation. METHODS: Serum samples were taken at least twice weekly post-transplant and tested for Aspergillus antigen. Latex agglutination test results were compared with microbiological examination of respiratory, urine and bile specimens. Serum samples from liver transplant patients were also tested for antibodies to Aspergillus fumigatus by counter immunoelectrophoresis. RESULTS: Eight of the 91 patients studied developed invasive aspergillosis. Positive latex agglutination tests were obtained in eight of 187 (4.3%) serum samples from four of these eight patients. The other four patients with invasive aspergillosis gave consistently negative latex agglutination tests. A positive latex agglutination test was the first indication of invasive aspergillosis in two patients; these patients were already on amphotericin B. Positive latex agglutination tests were the only evidence of invasive aspergillosis in one patient who subsequently died of the infection. False positive latex agglutination tests were obtained in five of 83 (6%) patients with no evidence of invasive aspergillosis and misleading positive cultures seen in nine of 83 (10.8%). No antibodies were detected in three of four liver transplant patients with invasive aspergillosis. Conversely, antibodies were detected in 63 of 262 (24%) serum samples from 43 liver transplant patients with no evidence of invasive aspergillosis; one of these patients had an antibody titre of 1:2 on four separate occasions. CONCLUSIONS: The Pastorex aspergillus antigen latex agglutination test, when used alone, lacks sensitivity and specificity for the early diagnosis of invasive aspergillosis. A diagnosis was made in all patients with invasive aspergillosis when both culture and antigen tests were performed although using these criteria a false positive diagnosis would have been made in 13 of 83 (15.6%) patients. Microbiological and serial serological investigations for antigen should both be performed and the results considered in conjunction with radiological and clinical evidence.
Aspergillosis↗