PubMed Health⌕ Search

Biomedical subjects

S R Barnum

Publications and source records attributed to S R Barnum.

At least 19 recordsLinked to original sources
PubMed2000

HSP16.6 is involved in the development of thermotolerance and thylakoid stability in the unicellular cyanobacterium, Synechocystis sp. PCC 6803.

The low molecular weight (LMW) heat shock protein (HSP), HSP16.6, in the unicellular cyanobacterium, Synechocystis sp. PCC 6803, protects cells from elevated temperatures. A 95% reduction in the survival of mutant cells with an inactivated hsp16.6 was observed after exposure for 1 h at 47 degrees C. Wild-type cell survival was reduced to only 41%. HSP16.6 is also involved in the development of thermotolerance. After a sublethal heat shock at 43 degrees C for 1 h and subsequent challenge exposure at 49 degrees C for 40 min, mutant cells did not survive, while 64% of wild-type cells survived. Ultrastructural changes in the integrity of thylakoid membranes of heat-shocked mutant cells also are discussed. These results demonstrate an important protective role for HSP16.6 in the protection of cells and, in particular, thylakoid membrane against thermal stress.

Cell Membrane↗
PubMed2000

Detection of activated complement complex C5b-9 and complement receptor C5a in skin biopsies of patients with systemic sclerosis (scleroderma).

OBJECTIVE: Upregulated matrix synthesis is a hallmark of systemic sclerosis (SSc). There are indications that growth factors such as platelet derived growth factor (PDGF) are involved in proliferative pathways in SSc lesions. As activated complement releases PDGF from endothelial cells, we searched for activated complement and the complement receptor for C5a (C5aR) in skin biopsies of patients with SSc. METHODS: Snap frozen sections of 8 patients with early SSc and 5 patients with longterm SSc were examined. Using monoclonal antibodies against activated complement complex C5b-9 and the C5aR, skin biopsies derived from both clinically involved and non-involved skin were examined by APAAP immunohistochemistry. RESULTS: A pattern of activated complement C5b-9 and the CSaR could be detected in SSc microvasculature. Eleven of the 13 patients (7/8 patients with early SSc) showed positive staining for C5b-9. The CSaR was detected in 6 of the 8 patients with early SSc. In 3 patients with longterm disease, C5aR expression could also be detected in non-involved skin. CONCLUSION: Activated complement and complement receptors could be detected in early and late stages of SSc skin lesions. The presence of complement receptors in non-involved skin may indicate preclinical activation of pathways resulting in growth factor dependent matrix synthesis.

Antigens, CD↗
PubMed1999

Central nervous system-targeted expression of the complement inhibitor sCrry prevents experimental allergic encephalomyelitis.

Although generally thought of as a T cell-driven autoimmune disease, recent studies in experimental allergic encephalomyelitis (EAE), the animal model of multiple sclerosis, suggest a significant role for innate immune mechanisms. To address the possibility that the complement system plays a central role in these diseases, we developed a transgenic mouse with astrocyte-targeted production of a soluble inhibitor of complement activation, complement receptor-related protein y (sCrry). Here, we show that sCrry transgenic mice are either fully protected against EAE or develop significantly delayed clinical signs. These results indicate that complement activation may have an essential role in the pathogenesis of the disease and that complement-mediated events may occur early during the effector phase of EAE. Furthermore, this work underscores the importance of humoral immunity in amplifying a T cell-initiated pathogenic process.

Animals↗
PubMed1999

Human T cells express the C5a receptor and are chemoattracted to C5a.

The anaphylatoxin C5a is a potent mediator of inflammation that exerts a broad range of activity on cells of the myeloid lineage. In this study, we present the first evidence that human T cells express the C5a receptor (C5aR) and are chemotactic to C5a. Using FACS analysis, we found that the C5aR was expressed at a low basal level on unstimulated T cells and was strikingly up-regulated upon PHA stimulation in a time- and dose-dependent manner. CD3+ sorted T cells as well as Jurkat T cells were shown to express C5aR mRNA as assessed by RT-PCR. Moreover, semiquantitative RT-PCR analysis demonstrated that C5aR mRNA was down-regulated in purified T cells upon long-term PHA stimulation. To demonstrate that C5a was biologically active on T cells, we investigated the chemotactic activity of C5a and observed that purified CD3+ T cells are chemotactic to C5a at nanomolar concentrations. Finally, using a combination of in situ hybridization and immunohistochemistry, we showed that the T cells infiltrating the central nervous system during experimental allergic encephalomyelitis express the C5aR mRNA. In summary, these results suggest that C5a exerts direct effects on T cells and could be involved in the trafficking of T cells under physiological and pathological conditions, including inflammatory diseases of the central nervous system.

Animals↗
PubMed1999

Receptor for the C3a anaphylatoxin is expressed by neurons and glial cells.

Little is known about the expression of the receptor for complement anaphylatoxin C3a (C3aR) in the central nervous system (CNS). In this study, we provide the first evidence that neurons are the predominant cell type expressing C3aR in the normal CNS. By using in situ hybridization (ISH) and immunohistochemistry, we found that C3aR is constitutively expressed at high levels in cortical and hippocampal neurons as well as in Purkinje cells. Moreover, we showed that primary culture of human astrocytes and microglia express the C3aR mRNA as assessed by RT-PCR. In situ hybridization performed on rat primary astrocytes confirmed the RT-PCR result demonstrating C3aR expression by astrocytes. In experimental allergic encephalitis (EAE), C3aR expression was elevated on microglia, infiltrating monocyte-macrophage cells and a few astrocytes, whereas neuronal expression remained unchanged during the course of the disease. These data demonstrate that the C3aR is expressed primarily by neurons in the normal CNS and that its neuronal expression is not dramatically upregulated under inflammation. This is in contrast to the increased neuronal expression of the C5aR in several inflammatory CNS conditions. The high constitutive expression of the C3aR by neurons suggests this receptor may play an important role in normal physiological conditions in the CNS.

Anaphylatoxins↗
PubMed1999

Expression of the murine complement regulatory protein crry by glial cells and neurons.

The expression of the murine complement regulatory protein, Crry, in the CNS remains largely unexplored. In this study, we examined murine astrocytes and microglia purified from neonatal brain and sections of adult murine brain for the expression of Crry. Using RT-PCR, immunohistochemistry, in situ hybridization, flow cytometry, and Western blot analysis, we demonstrated that astrocytes and microglia express Crry protein and RNA. Crry expression is greater on microglia than astrocytes and, as determined by Western blot analysis, each cell type expresses a Crry protein of different molecular weight. Interestingly, neuronal expression of Crry was seen only at the RNA level. These data demonstrate Crry expression by astrocytes, microglia, and neurons in the murine CNS and suggest that Crry may play an important role in protecting the CNS against complement-mediated damage.

Animals↗
PubMed1999

Complement anaphylatoxin receptors on neurons: new tricks for old receptors?

Activation of the complement system has been reported in a variety of inflammatory diseases and neurodegenerative processes of the CNS. Recent evidence indicates that complement proteins and receptors are synthesized on or by glial cells and, surprisingly, neurons. Among these proteins are the receptors for the chemotactic and anaphylactic peptides, C5a and C3a, which are the most-potent mediators of complement inflammatory functions. The functions of glial-cell C3a and C5a receptors (C3aR and C5aR) appear to be similar to immune-cell C3aRs and C5aRs. However, little is known about the roles these receptors might have on neurons. Indeed, when compared with glial cells, neurons display a distinct pattern of C3aR and C5aR expression, in either the normal or the inflamed CNS. These findings suggest unique functions for these receptors on neurons.

Anaphylatoxins↗
PubMed1998

Kinetics of anaphylatoxin C5a receptor expression during experimental allergic encephalomyelitis.

In this study, we investigated the expression of the C5aR in spinal cords of Lewis rats with experimental allergic encephalomyelitis (EAE). Using in situ hybridization (ISH) we analyzed the kinetics of C5aR at different time points of EAE (preclinical stage, clinical peak, remission phase). We observed that C5aR mRNA was readily detected in the CNS of EAE rats at all the stages of the disease. Using a combination of ISH and immunohistochemistry, we formally demonstrated that C5aR is strongly expressed on microglial cells and hypertrophic astrocytes during EAE. The potential involvement of C5a receptor in EAE physiopathology is discussed.

Animals↗
PubMed1998

Elevated complement C5a receptor expression on neurons and glia in astrocyte-targeted interleukin-3 transgenic mice.

Evidence from several central nervous system (CNS) inflammatory disease models suggests that intrathecal complement synthesis may contribute to early inflammatory events in the brain. In this study, we examined the expression of the receptor for C5a (C5aR), a potent inflammatory and chemotactic factor, in the brains of transgenic mice with constitutive astrocyte expression of interleukin-3 (IL-3), a hematopoietic and immunomodulatory cytokine. By in situ hybridization, we demonstrated that cells infiltrating the cerebellar meninges, the cerebellum, and demyelinating lesions in the cerebellum were strongly positive for C5aR mRNA. By immunohistochemistry, the infiltrating cells expressing the C5aR were identified as macrophages based on staining with antibodies to the complement receptor type 3 and F4/80, a mouse macrophage-specific marker. In addition, some of the cells in cerebellar lesions were positive for the astrocyte-specific marker, glial fibrillary acidic protein, suggesting that a subpopulation of astrocytes in these lesions express elevated levels of the C5aR. Increased C5aR expression was also observed in cortical neurons in the occipital cortex and in pyramidal neurons in the cornu ammonis and subiculum of the hippocampus, at both the protein and mRNA levels. These data suggest that IL-3 may play an immunomodulatory role in C5aR expression on several cell types in the brain and that increased C5aR expression correlates with the pathology seen in this model. The transgenic mice used in this study provide a useful tool for characterizing the mechanism of regulation of the C5aR expression and for examining the functions of this chemotactic receptor in CNS inflammation.

Animals↗
PubMed1998

A 16.6-kilodalton protein in the Cyanobacterium synechocystis sp. PCC 6803 plays a role in the heat shock response.

The low molecular weight (LMW) heat shock protein (HSP) gene hsp16.6 was identified and cloned from the unicellular cyanobacterium Synechocystis sp. PCC 6803 through comparisons of genomic sequences and conserved gene sequences of the LMW HSPs. Hsp16.6 was isolated using PCR and cloned into the pGEMT plasmid. Hsp16.6 showed a significant increase in transcription after heat shock at 42 degreesC that indicated hsp16.6 was a heat shock gene. To determine the role that hsp16.6 plays in the heat shock response, a mutant Synechocystis cell line was generated. Cell growth and oxygen evolution rates of wild type and mutant cells were compared after heat shock. Results showed significantly decreased cell growth rates and a 40% reduction in oxygen evolution rates in mutants after heat shock treatments. These data indicate a protective role for hsp16.6 in the heat shock response.

Amino Acid Sequence↗
PubMed1998

Transforming growth factor-beta2 regulates C3 secretion in monocytes through a protein kinase C-dependent pathway.

Previously, we reported that TGF-beta2 regulates the C3 gene expression in a dose- and time-dependent manner in monocytes. To extend these studies, we examined the role of PKC in the TGF-beta2-mediated induction of C3 expression by the human monocyte cell line, U937. Treatment of U937 cells with the PKC inhibitors, H7 and calphostin C, suppressed TGF-beta2-mediated induction of C3 protein levels, but not mRNA levels, in a dose-dependent manner. At the highest concentrations of H7 and calphostin C, C3 protein levels were inhibited 50% and 93%, respectively, compared to control levels. Treatment of U937 cells with HA1004, a weak PKC inhibitor used as a control for H7, did not inhibit induction of C3 protein levels. Down-modulating PKC with a prolonged exposure of U937 cells to PMA also suppressed TGF-beta2-mediated C3 protein induction by as much as 82%. Incubating cell extracts isolated from TGF-beta2-treated U937 cells with the PKC substrate, MIBP(4-14), resulted in increased substrate phosphorylation compared to cell extracts isolated from untreated cells. Addition of calphostin C suppressed the increased substrate phosphorylation by TGF-beta2. Furthermore, biosynthetic labeling of U937 cells treated with TGF-beta2 and calphostin C demonstrated an accumulation of C3 protein within cell lysates compared to controls. Collectively, these studies suggest a role for PKC in the secretion of C3 protein during TGF-beta2-mediated regulation of C3 expression in U937 cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1997

Experimental diffuse axonal injury induces enhanced neuronal C5a receptor mRNA expression in rats.

Several studies suggest the involvement of the complement system in the pathophysiology of traumatic brain injury (TBI). Since the intrathecal generation of anaphylatoxin C5a has been shown to mediate inflammatory effects within the central nervous system, we sought to characterize the cellular expression of the mRNA for the C5a receptor (C5aR, CD88) in brains of rats with experimental diffuse axonal injury (DAI) by in situ hybridization. Infiltrating leukocytes expressing C5aR mRNA were seen in meninges and lateral ventricles as early as 4 h after induction of DAI. The number of infiltrating C5aR-positive cells increased gradually up to 24 h after trauma. Within the brain parenchyma, up-regulation of C5aR mRNA expression was first seen in cerebellar Purkinje cells within 8 h. At 24 h after TBI, expression of C5aR mRNA was widespread bilaterally throughout the cortex and cerebellum, the cellular expression being restricted to pyramidal neurons and Purkinje cells. The intensity of C5aR transcript signals on neurons increased further up to 96 h after trauma. Ligand binding of C5a to its receptor on neurons might mediate previously unknown functions, thus possibly leading to neurotoxicity and secondary neuronal damage after TBI.

Animals↗
PubMed1997

TNF-alpha-mediated expression of the receptor for anaphylatoxin C5a on neurons in experimental Listeria meningoencephalitis.

The anaphylatoxin C5a has been implicated in the pathogenesis of bacterial meningitis as a potent mediator of inflammation in the subarachnoid space. We investigated the expression of the receptor for C5a (C5aR) in brains of mice with experimental Listeria monocytogenes (LM) meningoencephalitis. In the course of the disease, infiltrating cells in the meninges and the ventricles were found to express C5aR mRNA and protein. In the brain parenchyma, very low constitutive C5aR expression was seen on pyramidal neurons and Purkinje cells. However, in LM-infected mice, a dramatic increase in C5aR expression occurred on neurons starting 6 h after infection and was maximal between 24 and 36 h. TNF-alpha was identified as an essential mediator of neuronal C5aR expression, since mice lacking the genes for TNF and lymphotoxin-alpha (TNF/lymphotoxin-alpha -/- mice) showed significantly attenuated C5aR expression after LM infection. Furthermore, i.p. injection of recombinant TNF-alpha induced enhanced C5aR expression in the brains of TNF/lymphotoxin-alpha -/- mice and in normal animals even in the absence of a bacterial infection. We also assessed the levels of anaphylatoxin C5a in the cerebrospinal fluid of patients with infectious meningitis. C5a was detected in all patients with bacterial meningitis (n = 9), in 6 of 18 patients with aseptic meningitis, and in 1 of 66 control patients. The finding of TNF-alpha-mediated C5aR expression on neurons in experimental Listeria meningitis and the detection of the ligand, C5a, in the cerebrospinal fluid of human patients with infectious meningitis present new directions in the investigation of the pathophysiologic sequelae leading to secondary brain damage.

Animals↗
PubMed1997

Bacterial meningitis: complement gene expression in the central nervous system.

Inflammation in the subarachnoid space represents the pathological hallmark of bacterial meningitis. The intrathecal accumulation of leukocytes, in response to bacterial pathogens, and the subsequent release of endogenous inflammatory mediators are associated with a breakdown of the blood-brain barrier function and poor prognosis. Complement has been shown to play a major role in the inflammatory response within the intrathecal compartment in bacterial meningitis. In the present review, we provide an outline of the current understanding of the involvement of the complement system in the pathophysiology of bacterial meningitis, and propose future directions of investigation.

Anaphylatoxins↗
PubMed1997

Elevated levels of the complement components C3 and factor B in ventricular cerebrospinal fluid of patients with traumatic brain injury.

Immunological events occurring in the central nervous system (CNS) as a result of head trauma are largely unexplored. We report here that the levels of the alternative pathway complement proteins C3 and factor B are elevated in the cerebrospinal fluid (CSF) of head-injured patients. C3 and factor B indices suggest that changes in C3 and factor B levels in CSF are most likely due to altered blood-brain barrier integrity and not to intrathecal synthesis. These data demonstrate, for the first time, elevated levels of complement proteins in CSF of patients with severe traumatic brain injury. Elevated complement levels in brain injury may contribute to secondary damage.

Adolescent↗
PubMed1997

Evidence for intrathecal synthesis of alternative pathway complement activation proteins in experimental meningitis.

Complement has been shown to contribute to intrathecal inflammation in bacterial meningitis. However, the cellular source of complement in the infected central nervous system has not been determined. In this study, we analyzed protein and mRNA expression of two alternative pathway complement activation proteins, C3 and factor B, in the brains of mice with Listeria monocytogenes meningitis. Complement protein levels were found elevated in the cerebrospinal fluid of infected mice, compared with mock-infected animals. In the course of the disease, enhanced C3 and factor B mRNA expression was detected on pyramidal neurons and Purkinje cells within 6 hours, peaking at 12 hours and then gradually decreasing by 72 hours after infection. In addition, leukocytes infiltrating the subarachnoid space, within 12 to 24 hours, expressed mRNA for C3 and factor B. The cellular infiltration increased dramatically up to 72 hours. Intraperitoneal injection of tumor necrosis factor (TNF)-alpha up-regulated C3 and factor B mRNA expression on neurons in normal mice, suggesting that TNF-alpha may represent one cytokine regulating complement expression in this model of bacterial meningitis. However, additional mediators may be involved in regulation of intrathecal complement expression, as infected mice deficient of TNF/lymphotoxin-alpha genes did not demonstrate attenuated complement expression in the brain.

Animals↗