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Biomedical subjects

R F Pettersson

Publications and source records attributed to R F Pettersson.

At least 19 recordsLinked to original sources
PubMed1997

Cloning and functional characterization of a subunit of the transporter associated with antigen processing.

The transporter associated with antigen processing (TAP) is essential for the transport of antigenic peptides across the membrane of the endoplasmic reticulum. In addition, TAP interacts with major histocompatibility complex class I heavy chain (HC)/beta2-microglobulin (beta2-m) dimers. We have cloned a cDNA encoding a TAP1/2-associated protein (TAP-A) corresponding in size and biochemical properties to tapasin, which was recently suggested to be involved in class I-TAP interaction (Sadasivan, B., Lehner, P. J., Ortmann, B., Spies, T. & Cresswell, P. (1996) Immunity 5, 103-114). The cDNA encodes a 448-residue-long ORF, including a signal peptide. The protein is predicted to be a type I membrane glycoprotein with a cytoplasmic tail containing a double-lysine motif (-KKKAE-COOH) known to maintain membrane proteins in the endoplasmic reticulum. Immunoprecipitation with anti-TAP1 or anti-TAP-A antisera demonstrated a consistent and stoichiometric association of TAP-A with TAP1/2. Class I HC and beta2-m also were coprecipitated with these antisera, indicating the presence of a pentameric complex. In pulse-chase experiments, class I HC/beta2-m rapidly dissociated from TAP1/2-TAP-A. We propose that TAP is a trimeric complex consisting of TAP1, TAP2, and TAP-A that interacts transiently with class I HC/beta2-m. In peptide-binding assays using cross-linkable peptides and intact microsomes, TAP-A bound peptides only in the presence of ATP whereas binding of peptides to TAP1/2 was ATP-independent. This suggests a direct role of TAP-A in peptide loading onto class I HC/beta2-m dimer.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Processing and membrane topology of the spike proteins G1 and G2 of Uukuniemi virus.

The membrane glycoproteins G1 and G2 of the members of the Bunyaviridae family are synthesized as a precursor from a single open reading frame. Here, we have analyzed the processing and membrane insertion of G1 and G2 of a member of the Phlebovirus genus, Uukuniemi virus. By expressing C-terminally truncated forms of the p10 precursor containing the whole of G1 and decreasing portions of G2, we found that processing in BHK21 cells occurred with an efficiency of about 50% if G1 was followed by 50 residues of G2, while complete processing occurred if 98, 150, or 200 residues of G2 were present. Surprisingly, processing of all truncated G2 forms was less efficient in HeLa cells. Proteinase K treatment of microsomes isolated from infected cells indicated that the C terminus of G1 is exposed on the cytoplasmic face. Using G1 tail peptide antisera, the tail was likewise found by immunofluorescence to be exposed on the cytoplasmic face in streptolysin O-permeabilized cells. By introducing stop codons at various positions of the G1 tail and at the natural cleavage site between G1 and G2 and expressing these mutants in BHK cells, we found that no further processing of the G1 C terminus occurred following cleavage of G2 by the signal peptidase. This was also supported by the finding that an antiserum raised against a peptide corresponding to the region immediately upstream from the G2 signal sequence reacted in immunoblotting with G1 from virions. Finally, we show that both G1 and G2 are palmitylated. Taken together, these results show that processing of p10 of Uukuniemi virus occurs cotranslationally at only one site, i.e., downstream of the internal G2 signal sequence. G1 and G2 are inserted as type I proteins into the lipid bilayer, leaving the G1 tail exposed on the cytoplasmic face of the membrane. Since the G2 tail is only 5 residues long, the G1 tail is likely to be responsible for the interaction with the nucleoproteins during the budding process, in addition to harboring a Golgi localization signal.

Amino Acid Sequence↗
PubMed1997

A retention signal necessary and sufficient for Golgi localization maps to the cytoplasmic tail of a Bunyaviridae (Uukuniemi virus) membrane glycoprotein.

Members of the Bunyaviridae family mature by a budding process in the Golgi complex. The site of maturation is thought to be largely determined by the accumulation of the two spike glycoproteins, G1 and G2, in this organelle. Here we show that the signal for localizing the Uukuniemi virus (a phlebovirus) spike protein complex to the Golgi complex resides in the cytoplasmic tail of G1. We constructed chimeric proteins in which the ectodomain, transmembrane domain (TMD), and cytoplasmic tail (CT) of Uukuniemi virus G1 were exchanged with the corresponding domains of either vesicular stomatitis virus G protein (VSV G), chicken lysozyme, or CD4, all proteins readily transported to the plasma membrane. The chimeras were expressed in HeLa or BHK-21 cells by using either the T7 RNA polymerase-driven vaccinia virus system or the Semliki Forest virus system. The fate of the chimeric proteins was monitored by indirect immunofluorescence, and their localizations were compared by double labeling with markers specific for the Golgi complex. The results showed that the ectodomain and TMD (including the 10 flanking residues on either side of the membrane) of G1 played no apparent role in targeting chimeric proteins to the Golgi complex. Instead, all chimeras containing the CT of G1 were efficiently targeted to the Golgi complex and colocalized with mannosidase II, a Golgi-specific enzyme. Conversely, replacing the CT of G1 with that from VSV G resulted in the efficient transport of the chimeric protein to the cell surface. Progressive deletions of the G1 tail suggested that the Golgi retention signal maps to a region encompassing approximately residues 10 to 50, counting from the proposed border between the TMD and the tail. Both G1 and G2 were found to be acylated, as shown by incorporation of [3H]palmitate into the viral proteins. By mutational analyses of CD4-G1 chimeras, the sites for palmitylation were mapped to two closely spaced cysteine residues in the G1 tail. Changing either or both of these cysteines to alanine had no effect on the targeting of the chimeric protein to the Golgi complex.

Amino Acid Sequence↗
PubMed1996

aFGF, bFGF and NGF differentially regulate neuropeptide expression in dorsal root ganglia after axotomy and induce autotomy.

Using immunohistochemistry and in situ hybridization the in vivo effects of acidic and basic fibroblast growth factor (aFGF, bFGF), and of nerve growth factor (NGF) on the expression of galanin, neuropeptide Y (NPY) and substance P in axotomized dorsal root ganglia (DRGs) were examined. Self-mutilation (autotomy), a supposed pain-related behavior, was investigated after growth factor treatment. One microgram of aFGF, bFGF or NGF was applied directly to the transected sciatic nerve via a capsule. In normal rats 3.2%, 0% and 17.5% of the neuron profiles in the DRGs contained galanin-, NPY- and substance P-like immunoreactivity (LI), respectively. Sciatic nerve transection induced a distinct increase in galanin- and NPY-LIs, but a downregulation of substance P-LI. Thus three days after axotomy 23.5%, 26.9% and 9.8% of the DRG neuron profiles showed immunoreactivity for galanin-, NPY- and substance P-LI, respectively. In vivo administration of aFGF counteracted the axotomy-induced increase in galanin and NPY, whereas bFGF only suppressed NPY upregulation. NGF reversed in the injury-induced decrease in substance P-LI, but had no significant effect on galanin- and NPY-LIs. These results were confirmed by monitoring the mRNA levels for these neuropeptides. Moreover, aFGF was found to induce autotomy in 60% of the rats 3 days after axotomy. NGF produced autotomy in about 30% of the rats. Taken together, the present results suggest (1) that aFGF, bFGF and NGF differentially regulate neuropeptide expression in vivo; (2) that FGFs can inhibit neuropeptide upregulation of some peptides after nerve injury; and (3) that aFGF and NGF may induce pain-related behavior.

Animals↗
PubMed1996

Vascular endothelial growth factor B, a novel growth factor for endothelial cells.

We have isolated and characterized a novel growth factor for endothelial cells, vascular endothelial growth factor B (VEGF-B), with structural similarities to vascular endothelial growth factor (VEGF) and placenta growth factor. VEGF-B was particularly abundant in heart and skeletal muscle and was coexpressed with VEGF in these and other tissues. VEGF-B formed cell-surface-associated disulfide-linked homodimers and heterodimerized with VEGF when coexpressed. Conditioned medium from transfected 293EBNA cells expressing VEGF-B stimulated DNA synthesis in endothelial cells. Our results suggest that VEGF-B has a role in angiogenesis and endothelial cell growth, particularly in muscle.

Amino Acid Sequence↗
PubMed1996

Molecular cloning and expression of a 58-kDa cis-Golgi and intermediate compartment protein.

An abundant 58-kDa (p58) homodimeric and hexameric microsomal membrane protein has been biochemically characterized and localized to tubulo-vesicular elements at the endoplasmic reticulum-Golgi interface and the cis-Golgi cisternae in pancreatic acinar cells (Lahtinen, U., Dahllöf, B., and Saraste, J. (1992) J. Cell Sci. 103, 321-333). Here we report the purification of p58 by two-dimensional gel electrophoresis, and the cloning and sequencing of the rat and part of the Xenopus laevis cDNAs. The rat cDNA encodes a 517-amino acid protein having a putative signal sequence, a transmembrane domain close to the C terminus and a short cytoplasmic tail. The C-terminal tail contains a double-lysine motif (KKFF), known to mediate retrieval of proteins from the Golgi back to the endoplasmic reticulum. The rat p58 sequence was found to be 89% identical with those of ERGIC-53 and MR60, two previously identified human membrane proteins. Strong homology with the frog sequence was also observed indicating high evolutionary conservation. Overexpression of c-Myc-tagged p58 resulted in accumulation of the protein both in the endoplasmic reticulum and in an apparently enlarged Golgi complex, as well as its leakage to the plasma membrane. Immunolocalization using antibodies raised against a lumenal peptide stained the total cellular pool of p58, while anti-tail peptide antibodies detected p58 only in a restricted Golgi region. This suggests that the C-terminal tail of p58 located in the endoplasmic reticulum and transport intermediates is hidden, but becomes exposed when the protein reaches the Golgi complex.

Amino Acid Sequence↗
PubMed1996

Computer-assisted mapping of basic fibroblast growth factor immunoreactive nerve cell populations in the rat brain.

We have performed a mapping of basic fibroblast growth factor (bFGF) immunoreactive (ir) glial and nerve cell populations in the male rat brain using a rabbit antibody raised against a synthetic peptide of bovine bFGF. Regional morphometric and microdensitometric analysis of the bFGF ir neuronal profiles in coronal brain sections was carried out by means of an automatic image analyser. The density and intensity of the bFGF ir glial profiles were subjectively evaluated. The bFGF immunoreactivity (IR) was detected within the cytoplasm of neurons, except within the pyramidal neurons of hippocampal CA2 region, the fasciola cinerea and the indusium griseum, where bFGF IR was present in the nucleus. In contrast, in glial cells bFGF IR was always found in the nucleus. Neuronal and glial IR was no longer observed after absorption of the bFGF antiserum with recombinant bFGF. Basic FGF IR was found in neuronal and glial cell populations throughout the brain as well as in the choroid plexus and in the ependymal cells lining the ventricles. Basic FGF ir nerve cells were found in all layers of both the neocortex and allocortex. Within the caudate putamen and the nucleus accumbens a low density of weak bFGF ir neuronal profiles was detected. The majority of the thalamic nuclei showed medium to high densities of moderate to strong bFGF ir neuronal profiles. All the hypothalamic nuclei, with the exception of the anterior and lateral hypothalamic area and of the ventral hypothalamic nucleus, contained a high density of bFGF ir profiles. The pons and the medulla oblongata were characterized by the presence of a large number of nuclei containing moderate to high densities of strong bFGF ir profiles. The Purkinje cell layer of the cerebellar cortex contained a high density of moderately bFGF ir profiles. A moderate density of strong bFGF ir nerve cell profiles was observed within all the laminae of the spinal cord, except within the II and III laminae where a high density of strongly ir profiles was found. Histogram analysis of total immunoreactivity showed that the distribution of bFGF ir profiles within the telencephalon and mesencephalon tend to be similar with regard to the central tendency and spread. Using Kendall's tau, a significant correlation between intensity and density values was obtained only in the diencephalon. The cytoplasmic bFGF IR found in distinct nerve cell populations all over the rat brain and spinal cord may represent forms of bFGF which can be released from the nerve cells via non-exocytotic mechanisms in view of the absence of an intracellular signal peptide in bFGF. The presence of nuclear bFGF IR within the glial cells all over the central nervous system (CNS) suggests an intracellular function of bFGF, such as the promotion of mitogenesis and/or participation in the transcriptional regulation of various genes.

Animals↗
PubMed1995

Prominent expression of bFGF in dorsal root ganglia after axotomy.

Using quantitative in situ hybridization and immunohistochemistry the expression of acidic and basic fibroblast growth factors (aFGF, bFGF) in dorsal root ganglia (DRGs) was examined. Around 5% of the small neurons expressed bFGF mRNA in normal DRGs. Nerve injury induced a very dramatic and rapid up-regulation in bFGF mRNA levels, and around 80% of all DRG neurons expressed bFGF mRNA 3 days after axotomy. A distinct increase in bFGF-like immunoreactivity (LI) was also detected as early as 15 h after axotomy. The elevation of bFGF mRNA and protein levels declined after 1 week. bFGF mRNA was also up-regulated in non-neuronal cells following axotomy. Normally bFGF-LI was mainly localized in the nuclei of DRG neurons and in some non-neuronal cells. After nerve section, bFGF-LI was in addition found in the cytoplasm, and many more bFGF-positive non-neuronal cells were observed. By means of confocal microscopy analysis of axotomized DRGs, some bFGF-LI could be detected in vesicle-like structures in the cytoplasm as well as in the nucleoli, in addition to the nuclear location. Application of leukaemia inhibitory factor to the transected sciatic nerve significantly increased the number of bFGF-positive neurons, whereas the bFGF-LI in non-neuronal cells was strongly suppressed. About 70% of the normal DRG neurons expressed aFGF mRNA and aFGF-LI. Axotomy produced a moderate increase in aFGF mRNA levels, but no detectable effect on protein levels. Taken together, the results show that bFGF may be involved in the neuronal response to injury and suggest a role in neuronal survival and regeneration in axotomized DRG neurons.

Animals↗
PubMed1995

The site of action of neuronal acidic fibroblast growth factor is the organ of Corti of the rat cochlea.

Here we show that the mature cochlear neurons are a rich source of acidic fibroblast growth factor (aFGF), which is expressed in the neuronal circuitry consisting of afferent and efferent innervation. The site of action of neuronal aFGF is likely to reside in the organ of Corti, where one of the four known FGF receptor (FGFR) tyrosine kinases--namely, FGFR-3 mRNA--is expressed. Following acoustic overstimulation, known to cause damage to the organ of Corti, a rapid up-regulation of FGFR-3 is evident in this sensory epithelium, at both mRNA and protein levels. The present results provide in vivo evidence for aFGF being a sensory neuron-derived, anterogradely transported factor that may exert trophic effects on a peripheral target tissue. In this sensory system, aFGF, rather than being a neurotrophic factor, seems to promote maintenance of the integrity of the organ of Corti. In addition, aFGF, released from the traumatized nerve endings, may be one of the first signals initiating protective recovery and repair processes following damaging auditory stimuli.

Animals↗
PubMed1995

Acidic FGF and FGF receptors are specifically expressed in neurons of developing and adult rat dorsal root ganglia.

Employing complementary technical approaches, we have studied the expression of acidic fibroblast growth factor (aFGF) and FGF receptors in rat dorsal root ganglia. The results clearly showed that within spinal nerves aFGF and two high-affinity FGF receptors, FGFR-1 and FGFR-2, were prominently expressed in neurons, while expression in Schwann cells was undetectable. FGFR-3 and FGFR-4 were not expressed in dorsal root ganglia. Acidic FGF mRNA was detected in the majority of dorsal root ganglion neurons, including all size classes: FGFR-1 and FGFR-2 transcripts were only detected in subpopulations of mainly large and medium size neurons. In subcellular fractionation studies on dorsal root ganglion and spinal root tissue, aFGF was recovered in the soluble fraction and was thus not tightly associated with neuronal membranes. During development FGFR-1 and FGFR-2 mRNAs were found to be present at all stages examined (embryonic days 15-21 and postnatal days 1-120). Acidic FGF mRNA and protein were first detected at embryonic day 18, and their expression then increased progressively up to postnatal levels. In cultures of dorsal root ganglion neurons derived from day 15 embryos, aFGF expression was first detected 3 days after plating. The resulting neuron cultures continued to express aFGF in a Schwann cell-independent manner. In combination, these results indicate that aFGF expression in dorsal root ganglia is initiated and maintained in postmitotic neurons. Furthermore, the data suggest that the physiological function of aFGF in the peripheral nervous system is connected to processes specific to the mature sensory (and motor) system, such as the maintenance and survival of peripheral nerve neurons.

Animals↗
PubMed1995

The membrane glycoprotein G1 of Uukuniemi virus contains a signal for localization to the Golgi complex.

Members of the Bunyaviridae family acquire their envelopes by budding into the Golgi complex (GC). The accumulation of the membrane glycoproteins G1 and G2 in the GC probably determines the site of maturation. Here we have studied the intracellular transport and targeting to the GC of G1 and G2 of Uukuniemi virus, a member of the Phlebovirus genus, and report on their expression from cloned cDNAs either together or separately by using a T7 RNA polymerase-driven vaccinia virus expression system. When G1 and G2 were expressed together from a full-length cDNA as the p110 precursor, both proteins were localized to the Golgi complex, as evidenced by colocalization with the Golgi marker enzyme mannosidase II. Immunofluorescent staining indicated that G1 expressed alone also localized to the GC. However, pulse-chase experiments showed that G1 remained endoglycosidase H sensitive. G2 expressed alone remained associated with the endoplasmic reticulum (ER). G2 could be rescued from the ER and transported to the GC by coexpression with G1 from separate mRNAs. Coexpression also increased the efficiency of G1 transport to the GC. With none of the constructs could the glycoproteins be observed on the cell surface. These results show that efficient export of G1 and G2 from the ER requires coexpression of both proteins, in conformity with our previous results showing that G1 and G2 form heterodimeric complexes in the ER. Since G1 expressed alone is retained in the GC, we conclude that G1 contains a retention signal for localization to the GC. G2 might thus become associated with the GC indirectly via its interaction with G1.

Biological Transport↗
PubMed1994

Temporal and spatial increase of astroglial basic fibroblast growth factor synthesis after 6-hydroxydopamine-induced degeneration of the nigrostriatal dopamine neurons.

The present study investigates the temporal and spatial changes of the cellular expression of basic fibroblast growth factor messenger RNA and immunoreactivity after a 6-hydroxydopamine-induced lesion in the nigrostriatal dopamine system. In situ hybridization revealed a sustained (from 4 h to two weeks) and strong (300-400% of control, at the peak intervals) increase of basic fibroblast growth factor messenger RNA in the pars compacta of the substantia nigra and the ventral tegmental area ipsilateral to the lesion. A short-lasting increase of basic fibroblast growth factor messenger RNA was observed in he ipsilateral pars reticulata of the substantia nigra (from 4-24 h, 300% of control) and neostriatum (24 h, 180% of control) as well as in the ipsilateral and contralateral hippocampus and neocortex (by 4 h, 200% of control). Brightfield microscopy showed an increased number of putative glial cells expressing the basic fibroblast growth factor messenger RNA signal. Basic fibroblast growth factor immunohistochemistry revealed on control brains the protein in the nuclei of glial cells throughout the forebrain and the midbrain and in the nuclei of neurons of the layer II of the retrosplenial granular cortex, the CA2 region of the hippocampus and the fasciola cinereum as well as in the nuclei of ependymal cells. The injection of 6-hydroxydopamine increased basic fibroblast growth factor immunoreactivity in the nuclei of astrocytes only within the ipsilateral substantia nigra and ventral tegmental area. By 2 h after the drug injection, the density of glial basic fibroblast growth factor-immunoreactive profiles was increased in the pars compacta of the substantia nigra and the ventral tegmental area. The density, size and intensity of the astroglial basic fibroblast growth factor immunoreactive nuclei were increased in the entire substantia nigra and the ventral tegmental area at 72 h, and peaked one week after the 6-hydroxydopamine injection. The saline injection promoted a time-dependent increase in the density of the glial basic fibroblast growth factor immunoreactivity but only in the ipsilateral pars compacta of the substantia nigra. In conclusion, the dopamine cell degeneration may give rise to extracellular signals activating the surrounding astroglia, leading to a sustained increased synthesis of astroglial basic fibroblast growth factor, which may exert neuroprotective action and increase repair on the nigrostriatal dopamine system.

Animals↗
PubMed1993

Photochemically induced focal cerebral ischemia in rat: time dependent and global increase in expression of basic fibroblast growth factor mRNA.

Induction of basic fibroblast growth factor (bFGF) mRNA expression was studied in a Rose bengal induced focal cerebral ischemia during a time course of 2, 4, 24, 72 h and 7 days. Focal cerebral ischemia induced by Rose bengal resulted in a global upregulation in bFGF gene expression at the 24 h time-interval. This upregulation in bFGF gene expression was due to an upregulation in glial bFGF expression in most of the areas studied as seen by means of non-radioactive in situ hybridization in combination with immunocytochemistry for glial fibrillary acidic protein. However, in the piriform cortex a putative neuronal upregulation of bFGF could be detected by combination of non-radioactive in situ hybridization, immunohistochemistry for glial fibrillary acidic protein and nuclear staining with Neutral red. Semiquantitative data concerning bFGF mRNA expression were obtained by use of computer-assisted microdensitometry and revealed substantial increases in bFGF mRNA expression in the cingulate cortex, the neostriatum, a 1 mm marginal zone close to the external capsule and the olfactory tubercle at bregma levels 1 to 2 mm rostral to the lesion. No changes in bFGF gene expression were seen in field CA1 of Ammon's horn on the lesioned side and in dentate gyrus at bregma levels between -2.12 to -3.30 mm. We observed significant changes in bFGF upregulation in the caudate putamen, the piriform cortex and the amygdaloid region and the frontoparietal cortex at bregma levels -2.12 to -3.30 mm. These data indicate that photochemically induced focal cerebral ischemia leads to an early and global response in bFGF gene expression, which is due to an upregulation mainly in astrocytes. The observed widespread upregulation of the bFGF gene transcription rostral and caudal to the lesion is suggested to be due in part to neuronal glutaminergic connections between the areas investigated and in part due to increases in extracellular fluid signals (volume transmission).

Animals↗
PubMed1993

GAP-43, aFGF, CCK and alpha- and beta-CGRP in rat spinal motoneurons subjected to axotomy and/or dorsal root severance.

The mRNA levels for growth-associated protein 43 (GAP-43), acidic fibroblast growth factor (aFGF), alpha- and beta-calcitonin gene-related peptide (CGRP), cholecystokinin (CCK) and choline acetyltransferase (ChAT) in rat lumbar spinal motoneurons were studied by in situ hybridization 1, 5 and 21 days and 20 weeks following unilateral peripheral nerve sectioning, ventral rhizotomy or dorsal rhizotomy. Furthermore, CGRP- and aFGF-like immunoreactivities in the ventral horn were studied using immunohistochemistry. One to 21 days after axotomy, GAP-43 and alpha-CGRP mRNAs increased in lesioned motoneurons, while the aFGF mRNA levels were marginally higher in motoneurons on the lesion side as compared to the control side. beta-CGRP, CCK and ChAT mRNA levels, on the other hand, decreased during the short-term response (1-21 days) to axotomy. After ventral rhizotomy, but not peripheral axotomy, there was complete disappearance of aFGF-like immunoreactivity in the ventral root proximal to the lesion. In animals subjected to long-term survival (20 weeks) after peripheral axotomy, the expression of all studied substances had returned to normal levels. Unilateral dorsal rhizotomy did not induce any substantial short- or long-term shifts in the cellular expression of the GAP-43, aFGF, CGRP and CCK peptides or their mRNAs in motoneurons of lesioned segments. These results indicate that peptides/proteins in motoneurons are expressed differentially after axotomy. Whereas alpha-CGRP and GAP-43 are up-regulated, CCK and beta-CGRP become down-regulated and aFGF is largely unaffected.

Animals↗