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F Kirchhoff

Publications and source records attributed to F Kirchhoff.

At least 55 records · Page 3Linked to original sources
PubMed1998

Alternative splicing of mouse IL-15 is due to the use of an internal splice site in exon 5.

IL-15 is a pleiotropic cytokine modulating growth and differentiation of several hematopoietic cell types. Recently, we have demonstrated that mouse microglial cells, the brain macrophages, express both IL-15 and IL-15/IL-2 receptors. Based on single-cell RT-PCR data, we describe here an alternatively spliced IL-15 mRNA variant found in a small subpopulation of mouse microglia (5%, 3 out of 60 cells expressing IL-15 transcripts). PCR cycle sequencing of this larger transcript revealed the mouse homologue of the alternatively spliced exon A as it is known from the human IL-15 gene. Analysis of the corresponding mouse IL-15 gene region shows that the larger IL-15 transcript contains an yet unidentified 5' sequence of exon 5 while the shorter transcript uses an internal splice acceptor site. The mouse exon 5A segment has a length of 136 nt (17 nt longer than the human exon A). It contains five in-frame stop codons at its 5' end and a new translation initiation site at its 3' end. This new start site is surrounded by a favourable Kozak consensus sequence suggesting a more efficient translation rate. Further translational control by stem-loop binding factors is inferred by a predicted RNA stem-loop structure around the start site. Insertion of exon 5A would lead to an IL-15 polypeptide with a shortened leader sequence of 26 amino acids, as compared to the 48 amino acid leader sequence encoded by the transcript lacking exon 5A. Thus, the final IL-15 protein of the two splice variants is identical; different leader sequences could, however, lead to differences in the intracellular sorting, processing and/or secretion of IL-15.

Alternative Splicing↗
PubMed1998

Gastrin/cholecystokinin type B receptors in the kidney: molecular, pharmacological, functional characterization, and localization.

BACKGROUND: Gastrin/cholecystokinin type B receptors (CCKBRs) can be found on parietal cells and smooth muscle cells and are the predominant brain CCK receptors. Recent cloning studies indicate that this is receptor type might also be expressed in the kidney. MATERIALS AND METHODS: We used Northern blot analysis in guinea pig. kidney and reverse transcriptase polymerase chain reaction (RT-PCR) in several murine kidney cells lines to evaluate this organ for the expression of the CCKBRs. The receptor was pharmacologically characterized by displacement experiments using [125I]-BH-CCKs and various agonists and antagonists. Polyclonal antibodies vs. the CCKBRs were raised in chicken, and immunohistochemistry on tissue sections was used to localize the receptor within the organ. The effect of gastrin on renal cell growth was measured using proximal tubulus (MCT) cells, which were cultured with gastrin (10-9 M) for 24-72 h. Cell counts and [3H]-thymidine incorporation experiments were performed. RESULTS: CCKBR transcripts can be detected in kidney RNA (tubules > glomeruli > interstitium). RT-PCR revealed CCKBR transcripts in proximal tubules (MCT cells) and in mesangium (MMC). The medullary thick ascending limb of Henle's loop and several control tissues such as liver and muscle were negative. Displacement experiments using [125I]-BH-CCK and various agonists and antagonists identified binding sites with typical CCKBR pharmacology. CCKBRs were localized in the proximal tubulus, distal collecting ducts and mesangium cells. Treatment of rested MCT cells with gastrin 17-1 induced cell proliferation and [3H]-thymidine incorporation by at least 40% compared with normal growth (P < 0.05). CONCLUSION: These results show for the first time that CCKBRs are present in selected areas of the kidney, and strongly confirm our previous observation that this organ expresses binding sites for [125I]-gastrin. Furthermore, gastrin might act as a growth factor in the kidney.

Animals↗
PubMed1998

Single-cell characterization of endothelin system gene expression in the cerebellum in situ.

To evaluate the expression of components of the endothelin (ET) system in single Purkinje neurons and Bergmann glial cells in situ, patch-clamp recording was combined with a multiplex RT-PCR approach. Cerebellar slices were rapidly isolated from 20- to 28-day-old mice. Cells were characterized morphologically and electrophysiologically and cell contents were aspirated and immediately reverse-transcribed. The cDNA was used as a template in a multiplex PCR reaction containing primers specific for ET-1, ET-2, and ET-3, ET-converting enzyme 1 (ECE-1) and ECE-2, and ETA and ETB receptors. The resulting PCR products were used as templates in a second PCR reaction containing only one pair of nested primers. Specific single bands were obtained from positive cells, which was confirmed by DNA sequencing of the PCR products. Of the 25 Purkinje neurons assayed, 84% were positive for ECE-1 mRNA and 68% for ECE-2 mRNA. No ET and ETA receptor mRNAs were detected, and only one cell was positive for ETB receptor mRNA. In Bergmann glial cells, ETB receptor mRNA was predominant. A total of 68% of the 25 cells assayed were positive. Sixteen percent were positive for ETA receptor mRNA, 8% for ECE-1 mRNA, and 12% for ECE-2 mRNA. Again, no ET mRNAs were detected. These results confirm the role of the ETB receptor in Bergmann glial cells and provide evidence for expression of ECE-1 and ECE-2 in Purkinje neurons.

Animals↗
PubMed1998

Sequences just upstream of the simian immunodeficiency virus core enhancer allow efficient replication in the absence of NF-kappaB and Sp1 binding elements.

Large deletions of the upstream U3 sequences in the long terminal repeats (LTRs) of human immunodeficiency virus and simian immunodeficiency virus (SIV) accumulate in vivo in the absence of an intact nef gene. In the SIV U3 region, about 65 bp just upstream of the single NF-kappaB binding site always remained intact, and some evidence for a novel enhancer element in this region exists. We analyzed the transcriptional and replicative capacities of SIVmac239 mutants containing deletions or mutations in these upstream U3 sequences and/or the NF-kappaB and Sp1 binding sites. Even in the absence of 400 bp of upstream U3 sequences, the NF-kappaB site and all four Sp1 binding sites, the SIV promoter maintained about 15% of the wild-type LTR activity and was fully responsive to Tat activation in transient reporter assays. The effects of these deletions on virus production after transfection of COS-1 cells with full-length proviral constructs were much greater. Deletion of the upstream U3 sequences had no significant influence on viral replication when either the single NF-kappaB site or the Sp1 binding sites were intact. In contrast, the 26 bp of sequence located immediately upstream of the NF-kappaB site was essential for efficient replication when all core enhancer elements were deleted. A purine-rich site in this region binds specifically to the transcription factor Elf-1, a member of the ets proto-oncogene-encoded family. Our results indicate a high degree of functional redundancy in the SIVmac U3 region. Furthermore, we defined a novel regulatory element located immediately upstream of the NF-kappaB binding site that allows efficient viral replication in the absence of the entire core enhancer region.

Animals↗
PubMed1997

Mouse brain microglia express interleukin-15 and its multimeric receptor complex functionally coupled to Janus kinase activity.

The cytokine, interleukin (IL)-15, and the T cell growth factor, IL-2, exhibit a similar spectrum of immune effects and share the IL-2 receptor (IL-2R) subunits IL-2Rbeta and IL-2Rgamma for signaling in hematopoietic cells. Numerous neuroregulatory activities of IL-2 have been suggested, but its expression in the normal central nervous system (CNS) is apparently very low and regionally restricted. We show by RNA and protein detection that IL-15, its specific receptor molecule, IL-15Ralpha, and the signal-transducing receptor subunits, IL-2Rbeta and IL-2Rgamma, are constitutively present in various regions of the developing and adult mouse brain. We further demonstrate, also at the single-cell level, that IL-15 and the components for IL-15Ralpha/IL-2Rbetagamma receptors are expressed by microglia. Tyrosine phosphorylation data are presented showing that IL-15 signaling in microglia involves Janus kinase 1 activity. At doses of 0.1-10 ng/ml, IL-15 affected functional properties of these cells, such as the production of nitric oxide, and supported their growth in culture, suggestive of a role as an autocrine growth factor. Microglial IL-15 could thus play a pivotal role in the CNS and may participate in certain CNS and neuroendocrine functions previously ascribed to IL-2.

Animals↗
PubMed1997

Endothelin-induced calcium signaling in cultured mouse microglial cells is mediated through ETB receptors.

Microglial cells are the intrinsic immunocompetent cells of the central nervous system, which are activated by brain tissue damage. In this paper we investigated the ability of endothelins (ETs), which are potent vasoconstrictors, to induce intracellular calcium signals in cultured microglia cells. Both endothelin-1 and endothelin-3 increased intracellular Ca2+ concentration ([Ca2+]i). These [Ca2+]i transients were mimicked by BQ3020, an ETB receptor agonist and blocked by BQ788, a selective ETB antagonist, respectively. The calcium signals induced by the endothelins persisted in Ca(2+)-free media. Transcripts encoding the ETB receptor were detected in purified microglial cultures and cDNA fragments derived from ETB receptor mRNA were amplified from 9% of electrophysiologically characterized microglial cells by the use of single-cell RT-PCR.

Animals↗
PubMed1997

Activity of human immunodeficiency virus type 1 promoter/TAR regions and tat1 genes derived from individuals with different rates of disease progression.

Different rates of disease progression may be associated with different human immunodeficiency virus type 1 (HIV-1) promoter and/or transactivator activities. We therefore analyzed the sequences and activities of the first exon of Tat, tat1, and the promoter/trans-acting responsive (TAR) regions amplified directly from peripheral blood mononuclear cells obtained from five long-term nonprogressors and eight progressing HIV-1-infected individuals. The majority of tat1 alleles and promoter/TAR regions from all patients were intact and showed comparable activities in transient reporter assays. A substantial number of point mutations and some length variations were observed in the promoter/TAR region. In a single nonprogressor, the Sp1 binding site 3 was consistently altered and the transcriptional activity in the presence of Tat was diminished. Some LTR clones from a rapid progressor contained a fourth Sp1 binding site, which was associated with an elevated basal promoter activity. These data suggest that defects in the promoter/TAR region or tat1 are rare and that different promoter/transactivator activities are not commonly associated with different progression rates.

Alleles↗
PubMed1997

Bergmann glial cells in situ express endothelinB receptors linked to cytoplasmic calcium signals.

The endothelin (ET) isoforms ET-1, ET-2 and ET-3 applied at 100 nM triggered a transient increase in [Ca2+]i in Bergmann glial cells in cerebellar slices acutely isolated from 20-25 day-old mice. The intracellular calcium concentration ([Ca2+]i) was monitored using Fura-2-based [Ca2+]i microfluorimetry. The ET-triggered [Ca2+]i transients were mimicked by ETB receptor agonist BQ-3020 and were inhibited by ETB receptor antagonist BQ-788. ET elevated [Ca2+]i in Ca(2+)-free extracellular solution and the ET-triggered [Ca2+]i elevation was blocked by 500 nM thapsigargin indicating that the [Ca2+]i was released from InsP3-sensitive intracellular pools. The ET-triggered [Ca2+]i increase in Ca(2+)-free solution was shorter in duration. Restoration of normal extracellular [Ca2+] briefly after the ET application induced a second [Ca2+]i increase indicating the presence of a secondary Ca2+ influx which prolongs the Ca2+ signal. Pre-application of 100 microM ATP or 10 microM noradrenaline blocked the ET response suggesting the involvement of a common Ca2+ depot. The expression of ETB receptor mRNAs in Bergmann glial cells was revealed by single-cell RT-PCR. The mRNA was also found in Purkinje neurones, but no Ca2+ signalling was triggered by ET. We conclude that Bergmann glial cells are endowed with functional ETB receptors which induce the generation of intracellular [Ca2+]i signals by activation of Ca2+ release from InsP3-sensitive intracellular stores followed by a secondary Ca2+ influx.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1997

Expression of myelin-associated glycoprotein transcripts in murine oligodendrocytes.

The recognition molecule myelin-associated glycoprotein is expressed by oligodendrocytes, the myelinating cells of the central nervous system. The myelin-associated glycoprotein gene gives rise to two alternatively spliced transcript variants ("early" and "late" message) which are developmentally regulated. In this study, using mice, we investigated whether both transcripts can be expressed in an individual oligodendrocyte or whether different oligodendrocyte populations exist expressing either one or the other myelin-associated glycoprotein messenger RNA. For this purpose the cytoplasmic RNA content of single oligodendrocytes derived either from cultures of embryonic mouse brain or from the corpus callosum murine slice preparation was harvested during patch-clamping in the whole-cell recording mode by applying negative pressure to the patch pipette. After reverse transcription, cDNA fragments were amplified by the polymerase chain reaction and analysed by agarose gel electrophoresis and restriction enzyme maps. Expression of myelin-associated glycoprotein transcripts could first be detected in those oligodendrocytes which already had acquired a more mature developmental stage. This stage could electrophysiologically be characterized by the dominance of passive K+ currents. In addition to oligodendrocytes expressing only the late or the early transcript, many cells were found expressing simultaneously both transcripts with varying levels. The myelin-associated glycoprotein transcript expression is therefore found to be developmentally regulated at a stage when oligodendrocytes have already acquired the channel properties of the adult.

Amino Acid Sequence↗
PubMed1997

Association of simian immunodeficiency virus Nef with cellular serine/threonine kinases is dispensable for the development of AIDS in rhesus macaques.

The nef gene of simian immunodeficiency virus (SIV) is essential for high viral load and induction of AIDS in rhesus monkeys. A mutant form of the SIVmac239 Nef, which contains changes in a putative SH3-binding domain (amino acids 104 and 107 have been changed from PxxP to AxxA), does not associate with cellular serine/threonine kinases, but is fully active in CD4 downregulation and associates with the cellular tyrosine kinase Src. Infection of two rhesus macaques with SIVmac239 containing the mutant AxxA-Nef caused AIDS and rapid death in both animals. No reversions were observed in the majority of nef sequences analyzed from different time points during infection and from lymphatic tissues at the time of death. Our findings indicate that the putative SH3-ligand domain in SIVmac Nef and the association with cellular serine/threonine kinases are not important for efficient replication and pathogenicity of SIVmac in rhesus macaques.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Epidermal growth factor is a motility factor for microglial cells in vitro: evidence for EGF receptor expression.

Epidermal growth factor (EGF) and its receptor are present in the central nervous system and modulate a variety of neural functions. Here we show that microglial cells, the brain-intrinsic macrophages, express the receptor for EGF and migrate in response to EGF. Transcripts encoding the EGF receptor could be detected in purified microglial cultures obtained from newborn mouse cortex. More specifically, cDNA fragments derived from EGF receptor mRNA could be amplified from 21% of electrophysiologically characterized microglial cells by the use of a single-cell reverse transcription-polymerase chain reaction method. Expression of the protein was confirmed on rat microglia by flow cytometry. EGF dose-dependently stimulated chemotactic migration, as revealed with a microchemotaxis assay. The dose-response curve peaked-at 10 ng/ml EGF, reaching a 3-fold increase in migration over the unstimulated control; migration was about half of that induced by complement 5a (10 nM), a previously described microglial chemoattractant. Chequerboard analysis showed that EGF-induced motility was composed of both chemotaxis and chemokinesis. In contrast to its pronounced effect on cell motility, EGF (0.01-10 ng/ml) was not a mitotic signal for microglia, as shown by lack of bromodeoxyuridine incorporation. Acute and chronic pathological processes within the brain stimulate the synthesis and release of immunoregulators and growth factors (including EGF) that play a major role in the brain's response to injury. EGF may serve as a paracrine factor to direct microglial cells to the lesion site. Moreover, since EGF is secreted by activated microglia themselves in vivo, it may act as an autocrine modulator of microglial cell function.

Animals↗
PubMed1997

Simian immunodeficiency virus variants with differential T-cell and macrophage tropism use CCR5 and an unidentified cofactor expressed in CEMx174 cells for efficient entry.

The recent identification of coreceptors that mediate efficient entry of human immunodeficiency virus type 1 (HIV-1) suggests new therapeutic and preventive strategies. We analyzed simian immunodeficiency virus (SIV) entry cofactors to investigate whether the macaque SIV model can be used as an experimental model to evaluate these strategies. Similar to primary HIV-1 isolates, a well-characterized molecular clone, SIVmac239, which replicates poorly but efficiently enters into rhesus alveolar macrophages and an envelope variant, SIVmac239/316Env, with an approximately 1,000-fold-higher replicative capacity in macrophages used the beta-chemokine receptor CCR5 for efficient entry. The transmembrane portion of 316Env allowed low-level entry into cells expressing CCR1, CCR2B, and CCR3. A single amino acid substitution in the V3 loop of SIVmac239/316Env, 321P-->S, impaired the ability to enter into the T-B hybrid cell line CEMx174 but had relatively little effect on entry into primary cells and HOS.CD4 cells expressing CCR5. Although CEMx174 cells do not express CCR5, most SIVmac variants entered this hybrid cell line efficiently but did not enter the parental T-cell line CEM. It seems likely that CEMx174 cells express an as-yet-unidentified, perhaps B-cell-derived cofactor which allows efficient entry of SIVmac.

Animals↗
PubMed1996

Expression of glycine receptor subunits in glial cells of the rat spinal cord.

We previously demonstrated that the inhibitory neurotransmitter glycine induced membrane currents in glial cells from rat spinal cord. In this present study, the patch-clamp technique was combined with the reverse transcription-mediated PCR to analyze the glycine receptor-subunit expression in individual glial cells of rats age 3-18 days. Using the patch-clamp technique in the whole-cell configuration, glial cells were identified by their membrane current pattern and tested for responsiveness to glycine. Subsequently, the cytoplasm was harvested followed by reverse transcription of total cytoplasmic RNA. Subunit-specific cDNA fragments were amplified and analyzed by agarose gel electrophoresis, Southern blotting, and sequencing. In all cell types investigated, transcripts of the alpha1 subunit, but not of alpha 2 or alpha 3 subunits, were detected. In addition, about one-half the glial cells analyzed contained beta-subunit mRNA. These results illustrate that glial cells of rat spinal cord express functional glycine receptors in contrast to cultured glial cells. Glial cells are in intimate contact with synaptic regions making it likely that these nonneuronal receptors may be activated during glycinergic transmission and may trigger yet unknown responses in the glial cells.

Age Factors↗
PubMed1996

High frequency of defective nef alleles in a long-term survivor with nonprogressive human immunodeficiency virus type 1 infection.

A large number of nef alleles were obtained from peripheral blood mononuclear cells (PBMC) of four long-term nonprogressing survivors of human immunodeficiency virus type 1 (HIV-1) infection and from five individuals with progressive HIV-1 infection. These primary nef alleles were characterized by DNA sequence analysis and for their ability to downregulate CD4 surface expression. Intact nef open reading frames that directed the expression of Nef protein were recovered from all of the individuals. Most of the Nef proteins derived from three of four individuals with nonprogressive infection and from all five individuals with progressive infection were functional as judged by their ability to induce a decrease in surface CD4 expression. In contrast, one individual with nonprogressive HIV-1 infection yielded an unusually high frequency of disrupted nef open reading frames and Nef proteins defective for CD4 downregulation. Approximately 70% of the nef clones obtained from the PBMC of this individual at eight time points over a 12-year period were disrupted or defective for CD4 downregulation. While functional Nef proteins were demonstrated early in the course of infection (1983), functional nef alleles have surprisingly not come to predominate over time in PBMC DNA in this individual.

Alleles↗
PubMed1995

Identification of V3 mutations that can compensate for inactivating mutations in C4 of simian immunodeficiency virus.

A valine to isoleucine substitution at position 322 within variable region 3 (V3) of envelope of simian immunodeficiency virus was previously shown to compensate for an inactivating valine to glycine mutation at position 448 in constant region 4 (C4) (Morrison et al., Virology 195, 167-174, 1993). Cloned DNA fragments with inactivating C4 mutations were combined with complex mixtures of mutant V3 sequences, and full length genomes were transfected into COS-1 cells. By cocultivating transfected cells with CEM x 174 cells, we were able to identify two additional compensatory V3-C4 combinations. Changing 334 proline to leucine compensated for an inactivating 428 asparagine to lysine mutation and changing 324 isoleucine to leucine compensated for an inactivating 448 valine to glycine mutation. The double mutants replicated efficiently in CEM x 174 cells, rhesus monkey peripheral blood mononuclear cells, and the continuously growing rhesus monkey T cell line 221. Surprisingly, the 324 I-->L and 33 P-->L mutations by themselves impaired SIVmac239 wild-type replication in CEM x 174 cells. These results confirm the cooperation between V3 and C4 sequences and they define additional specific residues participating in this cooperation.

Amino Acid Sequence↗
PubMed1995

Effects of mutations in constant regions 3 and 4 of envelope of simian immunodeficiency virus.

Twenty-six mutant forms of simian immunodeficiency virus strain mac239 were constructed with changes in constant region 4 (C4) of env. Twenty-four of these had a single amino acid change, one had changes in two amino acids, and one had a deletion of eight amino acids. The effects of these mutations on viral replication, gp160 processing, and binding of env protein to soluble CD4 receptor were analyzed. The C4 region was relatively sensitive to sequence changes since only 11 of the 26 mutants replicated appreciably. Eight of the 15 mutants that were replication incompetent exhibited grossly defective processing of the gp160 env precursor; these mutations likely resulted in global effects on gp160 structure. Six of the replication incompetent mutants exhibited normal or near normal gp160 processing and binding of env protein to sCD4 and thus were probably blocked at some step subsequent to binding of virus to its CD4 receptor. Only one of the C4 mutations, 441W-->R, resulted in greatly decreased binding to sCD4 while retaining normal processing of gp160. The equivalent residue in HIV-1 has similarly been shown previously to be important for binding of HIV-1 to the CD4 receptor. Since a W-->S mutation at position 441 in C4 of SIVmac239 affected both gp160 processing and sCD4 binding, it is not clear whether the 441 tryptophan is actually important for contacting CD4 or for maintaining an appropriate configuration. mutations within a highly conserved GGDPE sequence in C3 of SIVmac239 specifically affected CD4 binding, which is also similar to previous findings with HIV-1. These results demonstrate similar sequence requirements in SIVmac and HIV-1 env for binding C4, but they raise doubts as to whether C4 sequences are directly involved in the binding.

Amino Acid Sequence↗