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Biomedical subjects

E M Johnson

Publications and source records attributed to E M Johnson.

At least 55 records · Page 3Linked to original sources
PubMed2000

Development of cranial parasympathetic ganglia requires sequential actions of GDNF and neurturin.

The neurotrophic factors that influence the development and function of the parasympathetic branch of the autonomic nervous system are obscure. Recently, neurturin has been found to provide trophic support to neurons of the cranial parasympathetic ganglion. Here we show that GDNF signaling via the RET/GFR(alpha)1 complex is crucial for the development of cranial parasympathetic ganglia including the submandibular, sphenopalatine and otic ganglia. GDNF is required early for proliferation and/or migration of the neuronal precursors for the sphenopalatine and otic ganglia. Neurturin exerts its effect later and is required for further development and maintenance of these neurons. This switch in ligand dependency during development is at least partly governed by the altered expression of GFR(&agr;) receptors, as evidenced by the predominant expression of GFR(&agr;)2 in these neurons after ganglion formation.

Animals↗
PubMed1999

Association of herd composition, stocking rate, and duration of calving season with fecal shedding of Cryptosporidium parvum oocysts in beef herds.

OBJECTIVE: To evaluate the association of herd demographics, parturition variables, stocking rate, and rotational grazing practices with the probability of fecal shedding of Cryptosporidium parvum from beef cow-calf herds in California. DESIGN: Cross-sectional study. SAMPLE POPULATION: 38 beef cow-calf operations. PROCEDURE: Fecal specimens were collected and examined for C parvum oocysts, using immunofluorescent microscopy. Association between various demographic and management factors and the probability of shedding C parvum were statistically evaluated. RESULTS: Adjusted for age and month of collection of a fecal sample, cattle from herds with a high number of young calves (< or = 2 months old) on the day of sample collection, a high stocking rate (No. of cattle/acre/mo), or a longer calving season were more likely to shed C parvum oocysts, compared with cattle from herds with fewer young calves, a lower stocking rate, or a shorter calving season. Cattle from herds with a higher number of older calves (> 2 months old) on the day of sample collection were less likely to shed C parvum oocysts, compared with cattle from herds with fewer older calves. Using our multivariate model, rotational grazing systems or season of onset of calving were not associated with shedding status for C parvum oocysts. CONCLUSIONS AND CLINICAL RELEVANCE: Reproductive management that would result in a shorter calving season and use of a lower stocking rate for cattle may be associated with reduced risk of C parvum shedding. Intensive rotational grazing systems and time of year for onset of calving season apparently have little effect on reducing prevalence of oocyst shedding.

Age Factors↗
PubMed1999

Analysis of the retrograde transport of glial cell line-derived neurotrophic factor (GDNF), neurturin, and persephin suggests that in vivo signaling for the GDNF family is GFRalpha coreceptor-specific.

Neurturin (NRTN) and glial cell line-derived neurotrophic factor (GDNF) are members of a family of trophic factors with similar actions in vitro on certain neuronal classes. Retrograde transport of GDNF and NRTN was compared in peripheral sensory, sympathetic, and motor neurons to determine whether in vivo these factors are transported selectively by different neuronal populations. After sciatic nerve injections, NRTN was transported by sensory neurons of the dorsal root ganglion (DRG). Competition studies demonstrated only limited cross-competition between NRTN and GDNF, indicating selective receptor-mediated transport of these factors. By using immunohistochemistry, we identified two populations of NRTN-transporting DRG neurons: a major population of small, RET-positive, IB4-positive, non-TrkA-expressing neurons that also show the ability to transport GDNF and a minor population of calretinin-expressing neurons that fail to transport GDNF. Spinal motor neurons in the adult showed relatively less ability to transport NRTN than to transport GDNF, although NRTN prevented the cell death of neonatal motor neurons in a manner very similar to GDNF (Yan et al., 1995) and persephin (PSPN) (Milbrandt et al., 1998). Last, NRTN, like GDNF, was not transported to sympathetic neurons of the adult superior cervical ganglion (SCG) after injection into the anterior eye chamber. These data reveal a high degree of functional selectivity of GDNF family receptor-alpha (GFRalpha) coreceptor subtypes for NRTN and GDNF in vivo.

Animals↗
PubMed1999

Multiple actions of neurturin correlate with spatiotemporal patterns of Ret expression in developing chick cranial ganglion neurons.

The neurotrophic effects of neurturin (NRTN) on chick cranial ganglia were evaluated at various embryonic stages in vitro and related to its receptor expression. NRTN promoted the outgrowth and survival of ciliary ganglion neurons at early embryonic (E) stages (E6-E12), trigeminal ganglion neurons at midstages (E9-E16), and vestibular ganglion neurons at late stages (E12-E16). NRTN had no positive effects on cochlear ganglion neurons throughout development. In accordance with the time and order of onset in NRTN responsiveness, Ret protein was first detected in ciliary ganglia at E6, subsequently in trigeminal ganglia at E9, and in vestibular ganglia at E12. Ret was absent in E16 ciliary ganglia as well as in cochlear ganglia at all developmental stages that were tested. Exogenous application of retinoic acid induced NRTN responsiveness and Ret protein expression from E9 vestibular ganglion neurons, suggesting that retinoic acid can regulate Ret protein expression in peripheral sensory neurons in vitro. Ret was confined to the neuron cell body, whereas GFRalpha was localized predominantly in peripheral and central neurite processes. No noticeable change in GFRalpha expression was seen in any cranial ganglia throughout the developmental stages that were tested (E6-E16). These results demonstrate that NRTN exerts neurotrophic effects on different cranial ganglia at different developmental stages and that the onset and offset of NRTN responsiveness are regulated mainly by the spatiotemporal patterns of Ret, but not of GFRalpha receptors. The results also substantiate the recently emerging view that NRTN may be an essential target-derived neurotrophic factor for parasympathetic neurons during development.

Animals↗
PubMed1999

Association of HIV-1 Tat with the cellular protein, Puralpha, is mediated by RNA.

The interaction between two regulatory proteins plays a crucial role in the control of several biological events, including gene transcription. In this report, we demonstrate that the interaction between the cellular sequence-specific single-stranded DNA binding protein Puralpha and the HIV type 1 (HIV-1) Tat protein is mediated by specific ribonucleic acids. The region of Tat that is important for its interaction with Puralpha includes the region demonstrated to bind Tat's viral RNA target, TAR. A 10-nucleotide GC-rich consensus sequence identified in RNAs associated with Puralpha derived from human U-87MG cells plays an important role in the Puralpha:Tat interaction as examined by an in vitro reconstitution assay. Furthermore, expression of the Puralpha-associated RNA in these cells enhances transcriptional activation of the HIV-1 promoter by Tat and Puralpha.

Cyclic AMP Response Element-Binding Protein↗
PubMed1999

Self-association of Puralpha is mediated by RNA.

The 322 amino acid cellular protein, Puralpha, is a sequence-specific single-stranded DNA-binding protein implicated in control of transcription and replication. Previous studies have demonstrated that the interaction between Puralpha and its target DNA sequence results in the formation of multimeric complexes. In this study, we demonstrate that Puralpha can self-associate in the absence of DNA. This self-association, while independent of DNA, is mediated by RNA. Through in vitro studies with bacterially expressed glutathione S-transferase fusion proteins, and the synthetic peptides corresponding to various central regions of Puralpha, the domain which is important for the self-association of Puralpha is localized to acidic leucine-rich repeats. Interestingly, these repeats have previously been shown to interact with the human immunodeficiency virus 1 (HIV-1) Tat protein and in this study we demonstrate that Tat is able to disrupt the self- association of Puralpha. We have recently cloned a Puralpha associated-RNA, PU-RNA, and here we show that PU-RNA can specifically reconstitute the self-association of Puralpha. RNA not only mediates the self-association of Puralpha, but also modulates the ability of Puralpha to interact with its target DNA sequence. Electrophoretic mobility shift assays performed with and without RNase treatment demonstrate that RNA inhibits the interaction between Puralpha and its target DNA sequence. Moreover, we demonstrate that the self-association of Puralpha can be reconstituted by a specific oligonucleotide encompassing the Puralpha binding site. The implications of these findings with respect to Puralpha's role in transcription and replication are discussed.

Amino Acid Sequence↗
PubMed1999

BAX translocation is a critical event in neuronal apoptosis: regulation by neuroprotectants, BCL-2, and caspases.

Members of the BCL-2 family of proteins either promote or repress programmed cell death. Here we report that neonatal sympathetic neurons undergoing apoptosis after nerve growth factor (NGF) deprivation exhibited a protein synthesis-dependent, caspase-independent subcellular redistribution of BAX from cytosol to mitochondria, followed by a loss of mitochondrial cytochrome c and cell death. Treatment with elevated concentrations of the neuroprotectants KCl or cAMP at the time of deprivation prevented BAX translocation and cytochrome c release. However, administration of KCl or cAMP 12 hr after NGF withdrawal acutely prevented loss of mitochondrial cytochrome c, but not redistribution of BAX; rescue with NGF acutely prevented both events. Overexpression of Bcl-2 neither altered the normal subcellular localization of BAX nor prevented its redistribution with deprivation but did inhibit the subsequent release of cytochrome c, caspase activation, and cell death. Bcl-2 overexpression did not prevent cell death induced by cytoplasmic microinjection of cytochrome c into NGF-deprived competent-to-die neurons. These observations suggest that the subcellular redistribution of BAX is a critical event in neuronal apoptosis induced by trophic factor deprivation. BCL-2 acts primarily, if not exclusively, at the level of mitochondria to prevent BAX-mediated cytochrome c release, whereas NGF, KCl, or cAMP may abort the apoptotic program at multiple checkpoints.

Animals↗
PubMed1999

Cell cycle arrest and morphological alterations following microinjection of NIH3T3 cells with Pur alpha.

Levels of Pur alpha, a protein implicated in control of both DNA replication and gene transcription, fluctuate during the cell cycle, being lowest in early S phase and highest just after mitosis. Here we have employed a new video time-lapse technique enabling us to determine the cell cycle position of each cell in an asynchronous culture at a given time and to ask whether introduction of Pur alpha protein at specific times can affect cell cycle progression. Approximately 80% of all NIH3T3 cells injected with Pur alpha were inhibited from passing through mitosis. Cells injected with Pur alpha during S or G2 phases were efficiently blocked with a 4N (G2 phase) DNA level, as determined by quantitative DNA photometry of individual cells. Of the cells injected with Pur alpha during G1 phase, 40% experienced a rapid cell death characterized by extreme cellular fragmentation. Of those G1 injected cells which remained viable, approximately equal numbers were arrested with either 2N or 4N DNA levels. Cells arrested by Pur alpha in G2 phase grew to cover a large surface area. These results link fluctuations in Pur alpha levels to aspects of cell cycle control.

3T3 Cells↗
PubMed1999

Living donors >55 years: to use or not to use?

BACKGROUND: Kidney transplants using older donors are becoming increasingly accepted as a strategy for alleviating the growing donor organ shortage. Most studies to date have shown decreased graft survival associated with the use of older cadaver donors; however, studies on the effect of living donor age on graft survival are less clear-cut. METHODS: We studied the effect of donor age on patient and graft survival after 1126 consecutive cyclosporine-treated primary kidney transplants performed between January 1, 1985 and December 31, 1995. Of these grafts, 598 were from living donors (74 from donors >55 years old) and 528 from cadaver donors (54 from donors >55 years). We calculated actuarial patient survival, graft survival, and death-censored graft survival for recipients of both living donor and cadaver kidneys. Living donors were then further divided by HLA mismatch (0 vs. 1 - 6) and the presence or absence of an acute rejection episode. Multivariate analysis of factors associated with decreased graft survival was performed for recipients of both living and cadaver donor kidneys. Factors included for analysis were donor age >55 years, recipient age >50 years, the presence of diabetes mellitus, HLA mismatch (0 vs. 1 - 6), and the presence of an acute rejection episode. RESULTS: For cadaver kidneys, univariate analysis indicates that both overall (P=0.004) and death-censored (P=0.001) graft survival was significantly better with younger cadaver kidneys. This is supported by our multivariate analysis, which shows that cadaver donor age >55 years is an independent predictor of poor actuarial graft survival (P=0.0003). For living donor kidneys, univariate analysis also indicates that both overall (P=0.045) and death-censored (P=0.005) graft survival was significantly better with younger living donor kidneys. However, in the absence of acute rejection, 10-year death-censored graft survival for patients with older vs. younger living donor kidneys was 93% vs. 94%, whereas in the presence of one or more acute rejection episodes, 10-year death-censored graft survival dropped markedly to 39% with older and 54% with younger living donors. Kidneys from living donors >55 years had significantly better long-term graft survival than cadaver donors >55 years (P=0.012) and had comparable graft survival to younger cadaver donors. In contrast to our univariate analysis, multivariate analysis of our living donor data shows that decreased actuarial living donor death-censored graft survival was significantly associated only with the presence of one or more acute rejection episodes (P<0.0001). Living donor age >55 years was not independently associated with decreased graft survival. CONCLUSIONS: Ours is the largest single-center study of outcome for recipients of kidneys from living donors >55 years. Using univariate analysis, we have shown that graft survival of kidneys from older living donors is significantly better than that of kidneys from older cadaver donors and is comparable to that of kidneys from younger cadaver donors. Using multivariate analysis, we have shown that the presence of one or more acute rejection episodes significantly shortens both cadaver and living donor long-term graft survival. Most significantly, we have shown that, although the use of kidneys from cadaver donors >55 years is associated with significantly decreased long-term graft survival, no such association exists for recipients of kidneys from living donors >55 years. We feel that our data support the continued use of kidneys from older living donors.

Adolescent↗
PubMed1999

Long-term follow-up of living kidney donors: quality of life after donation.

The University of Minnesota has been a strong advocate of living donor kidney transplants. The benefits for living donor recipients have been well documented. The relative low risk of physical complications during donation has also been well documented. Less well understood is the psychosocial risk to donors. Most published reports have indicated an improved sense of well-being and a boost in self-esteem for living kidney donors. However, there have been some reports of depression and disrupted family relationships after donation, even suicide after a recipient's death. To determine the quality of life of our donors, we sent a questionnaire to 979 who had donated a kidney between August 1, 1984, and December 31, 1996. Of the 60% who responded, the vast majority had an excellent quality of life. As a group, they scored higher than the national norm on the SF-36, a standardized quality of life health questionnaire. However, 4% were dissatisfied and regretted the decision to donate. Further, 4% found the experience extremely stressful and 8% very stressful. We used multivariate analysis to identify risk factors for this poor psychosocial outcome and found that relatives other than first degree (odds ratio=3.5, P=0.06) and donors whose recipient died within 1 year of transplant (odds ratio=3.3, P=0.014) were more likely to say they would not donate again if it were possible. Further, donors who had perioperative complications (odds ratio=3.5, P=0.007) and female donors (odds ratio=1.8, P=0.1) were more likely to find the overall experience more stressful. Overall, the results of this study are overwhelmingly positive and have encouraged us to continue living donor kidney transplants.

Adolescent↗
PubMed1999

Generation of neuronal intranuclear inclusions by polyglutamine-GFP: analysis of inclusion clearance and toxicity as a function of polyglutamine length.

Recent evidence suggests that, in huntingtin and many other proteins, polyglutamine repeats are a toxic stimulus in neurodegenerative diseases. To investigate the mechanism by which these repeats may be toxic, we transfected primary rat cerebellar granule neurons with polyglutamine-green fluorescent protein (GFP) fusion constructs containing 19 (Q19-GFP), 35 (Q35-GFP), 56 (Q56-GFP), or 80 (Q80-GFP) glutamine residues. All constructs, except Q19-GFP, aggregated within the nuclei of transfected cells in a length- and time-dependent manner. Although Q35-GFP expression led to the development of several small aggregates per cell, these aggregates were cleared or degraded, and the cells remained viable. In contrast, Q80-GFP expression resulted in one or two large aggregates and induced cell death. Caspase activation was observed after Q80-GFP aggregation, but inhibition of caspases with Boc-aspartyl(OMe)-fluoromethylketone (BAF) only served to delay, not prevent, toxicity. In addition, aggregation and toxicity were not affected by other modulators of neuronal cell death such as genetic deletion of the proapoptotic bcl-2 family member bax or addition of the protein synthesis inhibitor cycloheximide. Lastly, nuclear condensation did not occur as part of the toxicity. These data suggest that polyglutamine-GFP expression is toxic to primary neurons but that the death is distinct from classical apoptosis.

Animals↗
PubMed1999

Regulation of myelin basic protein gene transcription by Sp1 and Puralpha: evidence for association of Sp1 and Puralpha in brain.

Direct interaction between transcription factors may provide a mechanism for the regulatory function of these proteins on transcription of the responsive genes. These interactions may be facilitated if the target DNA sequences for the participant regulatory proteins are overlapped or positioned in close proximity to each other within the promoter of the responsive genes. In earlier studies, we identified a cellular protein, named Puralpha, which upon binding to the MB1 regulatory DNA sequence of the myelin basic protein (MBP) gene, stimulates its transcription in central nervous system (CNS) cells. Here, we provide evidence for binding of the ubiquitous DNA binding transcription factor, Sp1, to the MB1 DNA motif at the region that partially overlaps with the Puralpha binding site. We demonstrate that binding of Puralpha to its target sequence is enhanced by inclusion of Sp1 in the binding reaction. Under this condition, binding of Sp1 to the MB1 regulatory sequence remained fairly unchanged, and no evidence for the formation of Puralpha:MB1:Sp1 was observed. This observation suggests that transient interaction of Puralpha and Sp1 may result in stable association of Puralpha and the MB1 element. In support of this notion, results from immunoprecipitation/Western blot studies have established association of Puralpha and Sp1 in nuclear extracts from mouse brain. Of interest, Puralpha appears to bind to the phosphorylated form of Sp1 which is developmentally regulated and that coincides with the periods when MBP gene expression is at its maximum level. Results from cotransfection studies revealed that ectopic expression of Puralpha and Sp1 synergistically stimulates MBP promoter activity in CNS cells. The importance of these findings in stage-specific expression of MBP during brain development is discussed.

Animals↗
PubMed1999

Neurturin: an autocrine regulator of renal collecting duct development.

Urine is produced in the kidney by excretory nephrons and is drained by a tree-like system of collecting ducts to the ureter. The collecting ducts develop by arborisation of an initially unbranched epithelial rudiment, the ureteric bud, which ramifies through the surrounding mesenchyme and induces the formation of nephrons by mesenchyme-to-epithelial transition. The question of how collecting duct morphogenesis is controlled is an important one, from the points of view of both basic developmental biology and congenital renal pathology (multi- and polycystic renal disease, and some forms of renal agenesis, arise from defective collecting duct development). We report that neurturin, a neurotrophin related to glial cell line-derived neurotrophic factor and expressed in the developing kidney, acts as a collecting duct morphogen in culture. Applied in culture medium, it promotes epithelial branching and can induced branch initiation that has otherwise been blocked by depleting cultured kidneys of their sulfated proteoglycans or by antibody treatments. Applied locally on agarose beads, neurturin induces supernumerary ureteric buds to emerge from the wolffian duct and causes nearby collecting duct branches to distend to an abnormally large diameter. Like its receptors, neurturin is expressed by the developing collecting ducts themselves, suggesting that it forms an autocrine morphoregulatory control loop. This is in marked contrast to previously identified morphogens such as glial cell line derived neurotrophic factor and hepatocyte growth factor, which act in a paracrine manner.

Animals↗
PubMed1999

Expression of neurturin, GDNF, and GDNF family-receptor mRNA in the developing and mature mouse.

The GDNF family of neurotrophic factors currently has four members: neurturin (NRTN), glial cell line-derived neurotrophic factor (GDNF), persephin, and artemin. These proteins are potent survival factors for several populations of central and peripheral neurons. The receptors for these factors are complexes that include the Ret tyrosine kinase receptor and a GPI-linked, ligand-binding component called GDNF family receptor alpha 1-4 (GFRalpha1-4). We have used in situ hybridization to study the mRNA expression of NRTN, GDNF, Ret, GFRalpha1, and GFRalpha2 during embryonic development and in the adult mouse. GDNF receptors were prominently expressed during embryonic development in the nervous system, the urogenital system, the digestive system, the respiratory system, and in developing skin, bone, muscle, and endocrine glands. In some regions, incomplete receptor complexes were expressed suggesting that other, as yet unidentified, receptor components exist or that receptor complexes are formed in trans. NRTN and GDNF were expressed in many trigeminal targets during embryonic development including the nasal epithelium, the teeth, and the whisker follicles. NRTN and GDNF were also expressed in the developing limbs and urogenital system. In the embryo, GDNF factors and receptors were expressed at several sites of mesenchyme/epithelial induction, including the kidney, tooth, and submandibular gland. This expression pattern is consistent with the possibility that the GDNF factors function in inductive processes during embryonic development and with the recently discovered role of NRTN as a necessary trophic factor for the development of some parasympathetic neurons. In the mature animal, receptor expression was more limited than in the embryo. In the adult mouse, NRTN was most prominently expressed in the gut, prostate testicle, and oviduct; GDNF was most prominently expressed in the ovary.

Aging↗
PubMed1999

Gene targeting reveals a critical role for neurturin in the development and maintenance of enteric, sensory, and parasympathetic neurons.

Neurturin (NTN) is a neuronal survival factor that activates the Ret tyrosine kinase in the presence of a GPI-linked coreceptor (either GFR alpha1 or GFR alpha2). Neurturin-deficient (NTN-/-) mice generated by homologous recombination are viable and fertile but have defects in the enteric nervous system, including reduced myenteric plexus innervation density and reduced gastrointestinal motility. Parasympathetic innervation of the lacrimal and submandibular salivary gland is dramatically reduced in NTN-/- mice, indicating that Neurturin is a neurotrophic factor for parasympathetic neurons. GFR alpha2-expressing cells in the trigeminal and dorsal root ganglia are also depleted in NTN-/- mice. The loss of GFR alpha2-expressing neurons, in conjunction with earlier studies, provides strong support for GFR alpha2/Ret receptor complexes as the critical mediators of NTN function in vivo.

Animals↗
PubMed1999

Analysis of a novel mechanism of neuronal toxicity produced by an apolipoprotein E-derived peptide.

The apolipoprotein E (apoE)-derived peptide (141-155)2 has a neurotoxic effect, implying that apoE itself could be a source of toxicity in Alzheimer's disease brain. We characterized the toxicity of this peptide on superior cervical ganglion (SCG) neurons and compared the death with the apoptotic death that occurs after nerve growth factor (NGF) deprivation in these cells. A dose of 10 microM apoE (141-155)2 resulted in the death of approximately 50% of the neurons within 24 h. Nuclear condensation and DNA fragmentation preceded the death. However, most inhibitors of NGF deprivation-induced death, including the caspase inhibitor Boc-aspartyl(O-methyl)fluoromethyl ketone and genetic deletion of bax-/-, had no effect on the toxicity. Inclusion of depolarizing levels of potassium did block the toxicity. Receptor-associated peptide (RAP), an antagonist for apoE receptors, did not protect cells in either SCG or hippocampal cultures. In addition, RAP had no effect on internalization of the apoE peptide. These data support the observation that apoE (141-155)2 is neurotoxic but suggest that the neurotoxicity is distinct from classical apoptosis or necrosis. Furthermore, these results indicate that the toxic effect may occur independently of members of the low-density lipoprotein receptor gene family.

Amino Acid Sequence↗
PubMed1999

In situ immunodetection of neuronal caspase-3 activation in Alzheimer disease.

The mechanism by which cells die in Alzheimer disease (AD) is unknown. Several investigators speculate that much of the cell loss may be due to apoptosis, a highly regulated form of programmed cell death. Caspase-3 is a critical effector of neuronal apoptosis and may be inappropriately activated in AD. To address this possibility, we examined cortical and hippocampal brain sections from AD patients, as well as 2 animal models of AD, for in situ evidence of caspase-3 activation. We report here that senile plaques and neurofibrillary tangles in the AD brain are not associated with caspase-3 activation. Furthermore, amyloid beta (A beta) deposition in the APPsw transgenic mouse model of AD does not result in caspase-3 activation despite the ability of A beta to induce caspase-3 activation and neuronal apoptosis in vitro. AD brain sections do, however, exhibit caspase-3 activation in hippocampal neurons undergoing granulovacuolar degeneration. Our data suggests that caspase-3 does not have a significant role in the widespread neuronal cell death that occurs in AD, but may contribute to the specific loss of hippocampal neurons involved in learning and memory.

Aged↗