Biomedical subjects

S G Mueller

Publications and source records attributed to S G Mueller.

18 recordsLinked to original sources

PubMed2006

Measurement of hippocampal subfields and age-related changes with high resolution MRI at 4T.

Histological studies suggest that hippocampal subfields are differently affected by aging and Alzheimer's disease (AD). The aims of this study were: (1) To test if hippocampal subfields can be identified and marked using anatomical landmarks on high resolution MR images obtained on a 4T magnet. (2) To test if age-specific volume changes of subfields can be detected. Forty-two healthy controls (21-85 years) and three AD subjects (76-86 years) were studied with a high resolution T2 weighted fast spin echo sequence. The entorhinal cortex (ERC), subiculum, CA1, CA2 and CA3/4 and dentate were marked. A significant correlation between age and CA1 (r=-0.51, p=0.0002) which was most pronounced in the seventh decade of life was found in healthy controls. In AD subjects, CA1 and subiculum were smaller than in age-matched controls. These preliminary findings suggest that measurement of hippocampal subfields may be helpful to distinguish between normal aging and AD.

Adolescent↗

PubMed2006

Evaluation of treatment effects in Alzheimer's and other neurodegenerative diseases by MRI and MRS.

Neurodegeneration refers to a large clinically and pathologically heterogeneous disease entity associated with slowly progressive neuronal loss in different anatomical and functional systems of the brain. Neurodegenerative diseases often affect cognition, e.g. Alzheimer's disease (AD), dementia with Lewy bodies and vascular dementia, or different aspects of the motor system, e.g., amyotrophic lateral sclerosis, Parkinson's disease and ataxic disorders. Owing to increasing knowledge about the mechanisms leading to neurodegeneration, the development of treatments able to modify the neurodegenerative process becomes possible for the first time. Currently, clinical outcome measures are used to assess the efficacy of such treatments. However, most clinical outcome measures have a low test-retest reliability and thus considerable measurement variance. Therefore, large patient populations and long observation times are needed to detect treatment effects. Furthermore, clinical outcome measures cannot distinguish between symptomatic and disease-modifying treatment effects. Therefore, alternative biomarkers including neuroimaging may take on a more important role in this process. Because MR scanners are widely available and allow for non-invasive detection and quantification of changes in brain structure and metabolism, there is increasing interest in the use of MRI/MRS to monitor objectively treatment effects in clinical trials of neurodegenerative diseases. Particularly volumetric MRI has been used to measure atrophy rates in treatment trials of AD because the relationship between atrophic changes and neuron loss is well established and correlates well with clinical measures. More research is needed to determine the value of other MR modalities, i.e. diffusion, perfusion and functional MRI and MR spectroscopy, for clinical trials with neuroprotective drugs.

Alzheimer Disease↗

PubMed2006

Spectroscopic evidence of hippocampal abnormalities in neocortical epilepsy.

Lesional neocortical epilepsy (NE) can be associated with hippocampal sclerosis or hippocampal spectroscopic abnormalities without atrophy (dual pathology). In this study, magnetic resonance spectroscopic imaging (MRSI) was used to determine the frequency of hippocampal damage/dysfunction in NE with and without structural lesion. Sixteen patients with NE [seven temporal NE (NE-T), nine extratemporal (NE-ET)] and 16 controls were studied with a 2D MRSI sequence (Repetition time/echo time (TR/TE) = 1800/135 ms) covering both hippocampi. Seven NE patients had MR visible lesions (NE-Les), nine had normal MRI (NE-no). In each hippocampus, 12 voxels were uniformly selected. In controls, mean (+/- SD) NAA/(Cr + Cho) values for each voxel were calculated and voxels with NAA/(Cr + Cho) < or = (mean in controls--2SD in controls) were defined as 'pathological' in patients. Eight of 16 NE patients had at least two 'pathological' voxel (mean 2.5, range 2-5) in one hippocampus. Four were NE-Les and four NE-no. Three (43%) NE-T patients, had evidence for hippocampal damage/dysfunction and five (56%) had NE-ET. The ipsilateral hippocampus was affected in six of eight NE patients. Evidence for unilateral hippocampal damage/dysfunction was demonstrated in 50% of the NE patients. The type of NE, i.e. NE-Les or NE-no, NE-T or NE-ET, had no influence on the occurrence of hippocampal damage/dysfunction.

Adolescent↗

PubMed2005

Metabolic characteristics of cortical malformations causing epilepsy.

PURPOSE: Cortical malformations (CMs) are increasingly recognized as the epileptogenic substrate in patients with medically refractory neocortical epilepsy (NE). The aim of this study was to test the hypotheses that: 1. CMs are metabolically heterogeneous. 2. The structurally normal appearing perilesional zone is characterized by similar metabolic abnormalities as the CM. METHODS: Magnetic resonance spectroscopic imaging (MRSI) in combination with tissue segmentation was performed on eight patients with NE and CMs and 19 age matched controls. In controls, NAA, Cr, Cho,NAA/Cr and NAA/Cho of all voxels of a given lobe were expressed as a function of white matter content and thresholds for pathological values determined by calculating the 95% prediction intervals. These thresholds were used to identify metabolically abnormal voxels within the CM and in the perilesional zone. RESULTS: 30% of all voxels in the CMs were abnormal, most frequently because of decreases of NAA or increases of Cho. Abnormal voxels tended to form metabolically heterogeneous clusters interspersed in metabolically normal regions. Furthermore, 15% of all voxels in the perilesional zone were abnormal, the most frequent being decreases of NAA and Cr. CONCLUSION: In CMs metabolically normal regions are interspersed with metabolically heterogeneous abnormal regions. Metabolic abnormalities in the perilesional zone share several characteristics of CMs and might therefore represent areas with microscopic malformations and/or intrinsic epileptogenicity.

Adolescent↗

PubMed2003

Effects of vigabatrin on brain GABA+/Cr signals in focus-distant and focus-near brain regions monitored by 1H-NMR spectroscopy.

The new antiepileptic drug vigabatrin (VGB) increases gamma-aminobutyric acid (GABA) in the brain. We compared GABA+/Cr signals measured focus-near and focus-distant and correlated it with the degree of response to VGB. Brain GABA+/Cr signals were measured in 17 epileptic patients in structurally normal appearing tissue by nuclear proton magnetic resonance (1H-NMR) spectroscopy using a special editing sequence for GABA. In 11 patients the measurements were done in brain areas distant to focus and in six near to focus. Full-responders (seizure reduction of >or=50% at the end of the treatment phase) and partial-responders (seizure reduction of >or=50% at the end of the first month of treatment but <or=50% at end of treatment) had lower GABA+/Cr signals in the hemisphere with the epileptogenic focus and increases of the GABA+/Cr signals with VGB. Non-responders (seizure reduction of <or=50%) had no side difference in the GABA+/Cr signals before treatment and no increase during treatment. These observations were made in structurally normal appearing tissue near to the focus and distant to the focus. A side difference in brain GABA+/Cr signal between the epileptogenic and non-epileptogenic hemisphere before VGB treatment correlates with an improved seizure control under VGB treatment regardless whether the measurement is done focus-near or focus-distant.

Adolescent↗

PubMed2001

Brain glutathione levels in patients with epilepsy measured by in vivo (1)H-MRS.

OBJECTIVE: Glutathione in its reduced form (GSH) is the most important free radical scavenging compound in the mammalian nervous system that prevents membrane lipid peroxidation. It is suspected that epileptic seizures are accompanied by a massive production of reactive oxygen species, i.e., oxidative stress. METHODS: Using an (1)H MRS technique developed at the authors' site, the authors measured glutathione levels in a volume of interest (VOI) of 25 x 25 x 25 mm placed in structurally normal-appearing tissue in the parietooccipital region of each hemispheres in patients with and without active epilepsy, and in a age-matched control group. RESULTS: The GSH/water ratio in patients with epilepsy was significantly reduced in the parietooccipital region of both hemispheres (1.6 +/- 1.0 x 10(-5)) compared to the GSH/water ratio in healthy controls (2.4 +/- 1.1 x 10(-5)). There was no significant difference between the hemisphere with epileptogenic focus and the hemisphere without epileptogenic focus. The GSH/water ratios of the patients without active epilepsy were not different from the GSH/water ratios of patients with active epilepsy. CONCLUSION: The authors found evidence for a widespread impairment of the glutathione system in patients with epilepsy independent from seizure activity.

Adolescent↗

PubMed2001

Influence of pyridoxal 5'-phosphate alone and in combination with vigabatrin on brain GABA measured by 1H-NMR-spectroscopy.

Both iso-forms of the gamma-aminobutyric acid (GABA) synthesising enzyme and also the GABA degrading enzyme need pyridoxal 5'-phosphate (PP) as co-enzyme. The aim of the study was to investigate the influence of PP alone and in combination with various doses of vigabatrin (VGB) on brain GABA levels. In eight healthy subjects 300 mg/d PP and various doses of VGB (range, 1000 mg/d to 4000 mg/d) were given alone or in combination. The GABA+/creatine (Cr) signals in both occipital lobes were measured before treatment, during monotherapy with PP or VGB, and during combination of both using 1H-NMR-spectroscopy (1H-NMRS). PP alone did not change the GABA+/Cr signals. VGB alone increased the GABA+/Cr signals in both hemispheres. The combination PP and low-medium dosed VGB (1000-2000 mg/d) did not increase the GABA+/Cr signals. The effects of the combination of PP and high dosed (3000-4000 mg/d) VGB on the GABA+/Cr signals varied depending on the sequence of the drugs and dose of VGB. PP alone has no effect on the GABA+/Cr signals in healthy volunteers. The combination of PP and low-high dosed VGB had inconsistent effects on the GABA+/Cr signals compared to a VGB monotherapy because PP activates also the GABA-degrading enzyme GABA-transaminase.

Adult↗

PubMed2001

Effects of vigabatrin on brain GABA+/CR signals in patients with epilepsy monitored by 1H-NMR-spectroscopy: responder characteristics.

PURPOSE: Vigabatrin (VGB) is a new antiepileptic drug that increases the human brain gamma-aminobutyric acid (GABA) level by irreversibly inhibiting GABA transaminase. Although some patients respond to VGB with a significant seizure reduction, others do not. The aim of this study was to identify possible responders before or in an early phase of VGB treatment by measuring the GABA and homocarnosine contaminated with macromolecules/creatine and phosphocreatine ratio (GABA+/Cr) signal by means of proton-nuclear magnetic resonance (1H NMR) spectroscopy. METHODS: Measurements were performed immediately before and after a titration period of 1 month (2 g/day during the past 2 weeks). A third measurement followed a maintenance period of 3 months (2 or 3 g/day). In 14 patients with drug-resistant temporal lobe epilepsy and 3 patients with occipital lobe epilepsy, GABA+/Cr was measured in the ipsilateral (i.e., epileptogenic) hemisphere and contralateral (i.e., nonepileptogenic) hemisphere in a volume of 8 cm3. RESULTS: Depending on the therapeutic efficacy of VGB, we defined three groups: (a) full responders (n = 7), (b) nonresponders (n = 7), and (c) partial responders (n = 3). The nonresponders had no significant change in the GABA+/Cr signal during the treatment compared with baseline. The full responders had a significant increase of the GABA+/Cr signal during the whole treatment phase and a lower ipsilateral level at baseline. The partial responders had also a lowered ipsilateral GABA+/Cr signal at baseline and an increase during treatment but a decrease when the seizures started again. CONCLUSIONS: Responders to VGB could be identified by a lower ipsilateral baseline GABA+/Cr signal and a steeper increase during VGB treatment. However, it was not possible to predict the duration of the response (full versus partial responder) with these criteria.

Adolescent↗

PubMed2001

Proton magnetic resonance spectroscopy characteristics of a focal cortical dysgenesis during status epilepticus and in the interictal state.

We report the magnetic resonance imaging and proton magnetic resonance spectroscopic findings ((1)HMRS) in a patient with a focal cortical dysgenesis in the right superior frontal gyrus during intermittent frontal status epilepticus (IFSE) with simple partial seizures, and after she had become seizure free. During the status epilepticus, demonstrated by simultaneous behavioural and electroencephalographic telemetric long-term monitoring with scalp electrodes and ictal SPECT, we performed a single voxel spectroscopy of the dysgenic cortex. The(1)HMRS was repeated after 20 days when the patient's seizures were controlled. The N-acetyl-aspartate concentration in the focal dysgenic cortex was decreased in the interictal state but more during IFSE. The creatine/phosphocreatine concentration was normal in both instances. There was a clear lactate signal during IFSE, which was no longer visible in the interictal state. To our knowledge this is the first report of a(1)HMRS study of a focal cortical dysgenesis during an intermittent status epilepticus. We interpret the observed changes as signs of histopathological changes inherent to a cortical malformation and of an impaired energy metabolism due to the partial status epilepticus.

Adult↗

PubMed1997

Interruption of G protein-coupling in CXCR2 does not alter ligand binding, but eliminates ligand-activation of GTPgamma35S binding, calcium mobilization, and chemotaxis.

CXCR2 is a seven-transmembrane receptor that transduces intracellular signals in response to the chemokines IL-8, MGSA/GRO, and other ELR motif-containing CXC chemokines by coupling to heterotrimeric GTP-binding proteins. In this study, we have mutated two putative G protein-coupling regions of CXCR2 and characterized the effects of these mutations on ligand-activated signal transductions: aspartic acid 89 in the second transmembrane domain and the HRAMR sequence (BBXXB motif, found in the third intracellular loop where B indicates a basic amino acid and X represents any amino acid). The Asp89 was replaced by either asparagine (D89N) or glutamic acid (D89E). For the BBXXB motif, the first two basic amino acids were mutated to two neutral isoleucines (HR-II), or alternatively, two isoleucines were inserted between alanine and methionine (II-insert). When expressed in human embryonic kidney 293 cells, the D89E mutant was localized intracellularly with no detectable cell surface expression. In contrast, D89N, HR-II, and II-insert mutants displayed cell surface expression, with Kd values and expression levels similar to that of the wild-type transfectant. The ability of the mutants to transduce signal was assessed by ligand-stimulated GTPgamma35S binding, mobilization of intracellular free Ca2+, and chemotaxis assays. Both D89N and HR-II mutants signaled similarly to a wild-type receptor in all three assays. However, the II-insert mutant exhibited a loss of ligand-stimulated GTPgamma35S binding, calcium mobilization, and chemotaxis. Unexpectedly, this receptor underwent ligand-induced sequestration comparable to wild-type CXCR2. These data indicate that Asp89 and the basic amino acids in the third intracellular domain do not play essential roles in ligand-induced signal transduction through CXCR2. However, proper secondary structure and orientation of the third intracellular loop of CXCR2 are essential for ligand-mediated signal transduction but not for receptor sequestration.

Calcium↗

PubMed1997

Ligand-induced desensitization of the human CXC chemokine receptor-2 is modulated by multiple serine residues in the carboxyl-terminal domain of the receptor.

We have characterized the ligand-enhanced phosphorylation of the CXC chemokine receptor-2 (CXCR2) in a series of clonal 3ASubE cell lines expressing receptors truncated or mutated in the carboxyl-terminal domain. Truncation of CXCR2 by substitution of a stop codon for Ser-342 (342T) or Ser-331 (331T) results in total loss of melanoma growth stimulatory activity/growth-related protein (MGSA/GRO)-enhanced receptor phosphorylation, which cannot be explained based upon altered ligand binding affinity or receptor number. 3ASubE cells expressing 342T or CXCR2 with mutation of Ser-342, -346, -347, and -348 to alanine (4A) exhibit strong mobilization of Ca2+ in response to ligand (interleukin-8 or MGSA/GRO), with a recovery phase significantly slower than that of cells expressing wild type (WT) CXCR2. In contrast to the WT CXCR2, which is 93% desensitized by 20 nM ligand, the 331T, 342T, and 4A CXCR2 mutants do not undergo significant ligand-induced desensitization, and respond to a second ligand challenge by mobilizing Ca2+. The 3ASubE cells expressing CXCR2 with mutation of Ser-346, -347, and -348 to alanine, or with mutation of only one serine in this domain, continue to be phosphorylated in response to ligand and are 60-70% desensitized following the initial ligand challenge. WT CXCR2 phosphorylation and desensitization occur in <1 min, while receptor sequestration is a much later event (30-60 min). However, mutant receptors that are neither phosphorylated nor desensitized in response to ligand are <10% sequestered 60 min following ligand challenge. These data demonstrate for the first time that ligand binding to CXCR2 results in receptor phosphorylation, desensitization, and sequestration and that serine residues 342 and 346-348 participate in the desensitization and sequestration processes.

Amino Acid Sequence↗

PubMed1995

Activation of protein kinase C enhances the phosphorylation of the type B interleukin-8 receptor and stimulates its degradation in non-hematopoietic cells.

We have previously characterized the stably transfected, clonally selected human placental cell line, 3ASubE P-3, which overexpresses the type B interleukin-8 receptor (IL-8RB) and responds to the chemokine melanoma growth stimulatory activity (MGSA) with enhanced phosphorylation of this receptor. In work described here, we demonstrate that the MGSA-enhanced phosphorylation of this receptor is mediated via a process involving pertussis toxin-sensitive G proteins. Furthermore, treatment of the 3ASubE P-3 cells with either 12-O-tetradecanoylphorbol 13-acetate (TPA) or 1,2-dioctanoyl-sn-glycerol (diC8), two different activators of protein kinase C (PKC), results in a concentration-dependent increase in the phosphorylation of the IL-8RB. Inhibition of PKC, by treatment with staurosporin (50 nM for 2 h), or down-regulation of PKC, by prolonged treatment with TPA (400 nM for 40 h) suppresses the TPA-enhanced receptor phosphorylation, but has no effect on the MGSA-enhanced receptor phosphorylation. These data suggest that the isoforms of PKC that are sensitive to these manipulations may not play a role in mediating the MGSA-enhanced phosphorylation of the IL-8RB. TPA treatment also results in a time-dependent decrease in 125I-MGSA binding to the 3ASubE P-3 cells. A 30-min treatment with 400 nM TPA results in approximately a 50% decrease in binding, whereas a 2-h treatment essentially eliminates specific binding of 125I-MGSA to these cells. The TPA-induced decrease in 125I-MGSA binding is accompanied by enhanced degradation of the IL-8RB, as indicated by Western blot analysis and pulse-chase experiments, suggesting a potential role for PKC as a negative regulator of the IL-8RB. MGSA treatment (50 nM for 2 h) also stimulates receptor degradation in the 3ASubE P-3 cells, indicating that this receptor is down-regulated in response to prolonged exposure to its ligand. In similar studies conducted on the promonocytic cell line, U937, MGSA treatment of the U937 cells resulted in receptor phosphorylation, whereas PKC activation failed to significantly modulate the phosphorylation state of the IL-8RB. Treatment of the U937 cells with MGSA, TPA, or diC8 resulted in a loss of receptor protein present in these cell types. These data imply that MGSA signaling through the IL-8RB is similar in both the non-hematopoietic and hematopoietic cell types, whereas activation of PKC by TPA or diC8 elicits different responses in these two distinct cell types.

Alkaloids↗

PubMed1995

Distributions of melanoma growth stimulatory activity of growth-regulated gene and the interleukin-8 receptor B in human wound repair.

The alpha-chemokines have been implicated as regulators of proliferation and differentiation of normal keratinocytes and as mediators of keratinocyte maturation and migration in inflammatory processes that involve the skin. Using the cutaneous wound repair model, we examined the sites and temporal sequence of the appearance of melanoma growth stimulatory activity or growth-regulated gene (MGSA/GRO;ligand) and the type B interleukin (IL)-8 receptor (IL-8RB) to which MGSA/GRO binds. Human burn tissues (n = 44) representing days 2 to 12 after injury were obtained during surgical debridement, fixed in 4% paraformaldehyde, and embedded in paraffin. Immunolocalizations were performed with polyclonal antisera for both ligand and receptor, as well as a monoclonal antibody for the IL-8 RB. Western blot analysis confirmed the presence of the IL-8 RB in immunoprecipitates of epidermal keratinocyte lysates. In normal skin, MGSA/GRO protein was restricted to sites populated by differentiated keratinocytes (suprabasal compartments, inner root sheath cells, and dermal sweat ducts). MGSA/GRO protein was barely detectable within epithelial margins and islands of burn wounds where the migrating/proliferating keratinocyte populations reside, but staining intensities increased as cells matured into the outer layers. Weak diffuse staining was detected in areas of neutrophilic infiltration (granulation tissue and overlying exudates). By contrast, in normal skin the IL-8 RB was detected in specific locations within epidermal and dermal compartments of healing wounds. In the dermis, polyvalent antibodies detected receptor immunoreactivity most prominently in dermal sweat ducts and endothelium of capillaries, whereas this immunoreactivity was inconspicuous in sections stained with the monoclonal antibody. Receptor immunostaining was noted in migrating/proliferating keratinocytes in epithelial margins and islands but was in the outer layers or in hypertrophic epidermis adjacent to wounds. This same pattern was observed in epidermal appendages such as hair follicles and eccrine sweat ducts. In granulation tissues, IL-8 RB was noted in numerous fibroblasts and in subpopulations of macrophages and smooth muscle. The presence of both MGSA/GRO and its receptor in human burn wounds implicate this cytokine as an autocrine or paracrine mediator of epidermal regeneration in both the inflammatory and proliferative phases of cutaneous wound repair.

Animals↗

PubMed1994

Melanoma growth stimulatory activity enhances the phosphorylation of the class II interleukin-8 receptor in non-hematopoietic cells.

The class II IL-8 receptor (IL-8R) binds both melanoma growth stimulatory activity (MGSA) and IL-8 with high affinity. Reverse transcriptase polymerase chain reaction demonstrates that the class II IL-8R mRNA, which has previously been detected only in cells of hematopoietic lineage, is also expressed in non-hematopoietic cell types shown to respond to MGSA or IL-8. To study the signaling mechanism by MGSA through the class II IL-8R in non-hematopoietic cells, this receptor was overexpressed in the 3ASubE human placental and the 293 human kidney cell lines. Membrane preparations of the class II IL-8R expressing 3ASubE transfectants exhibited a 2.3 +/- 0.2-fold increase in GTP gamma 35S binding, which was sensitive to pertussis toxin, in response to MGSA treatment (0.2 microM). This MGSA response was not observed in cells transfected with the parental expression vector. In vivo phosphorylation studies demonstrated that the class II IL-8R was basally phosphorylated in the untreated transfectants, and MGSA (5 nM) treatment markedly enhanced the phosphorylation of this receptor. The MGSA-induced receptor phosphorylation was both time and concentration dependent and could be mimicked by treatment with the calcium ionophore A23187. Phosphoamino acid analysis indicated that the MGSA-induced receptor phosphorylation was on serine residue(s), suggesting that a serine kinase is activated in response to MGSA binding to the class II IL-8R in non-hematopoietic cells.

Base Sequence↗

PubMed1992

Dopamine receptor activation inhibits estrogen-stimulated transforming growth factor-alpha gene expression and growth in anterior pituitary, but not in uterus.

Transforming growth factor-alpha (TGF alpha) has been localized to the anterior pituitary, specifically to the lactotroph and somatotroph cell populations, by our previous studies. Since pituitary lactotrophs are known to undergo growth in response to estrogens, we have used an estradiol-induced pituitary hyperplasia/adenoma model. Estradiol treatment resulted in induction of TGF alpha mRNA in anterior pituitary, evident by 48 h, preceding actual macroscopic growth, which attained a maximum greater than 500% by 12 weeks. This rapid effect of estradiol also enhanced PRL mRNA, but did not affect other species of mRNA encoding for proenkephalin, D2 receptor mRNA, or hexosaminidase-A. TGF alpha mRNA remained elevated for the duration of rapid pituitary growth. D2 receptor activation by its agonist bromocriptine resulted in marked attenuation of TGF alpha mRNA preceding regression of growth. Coadministration of bromocriptine with estradiol resulted in an involution of pituitary size, indicating the overriding influence of dopamine in spite of a continued estrogenic stimulus. Epidermal growth factor receptor mRNA was not affected by any of these manipulations, suggesting that the receptor was not coregulated in this tissue similarly to TGF alpha. Estradiol also induced uterine TGF alpha mRNA and marked growth of the organ, but TGF alpha in this location was not regulated by dopamine. These results indicate that TGF alpha in the anterior pituitary is rapidly induced by estrogen in a time course preceding the growth of the gland. Estrogen-induced TGF alpha is rapidly attenuated by D2 dopamine receptor activation and is accompanied by a regression of pituitary growth. Interaction between these opposing hormonal/transmitter responses will determine the growth potential of the anterior pituitary.

Aging↗

PubMed1991

Transforming growth factor-beta (TGF beta) inhibits TGF alpha expression in bovine anterior pituitary-derived cells.

Transforming growth factor-beta 1 (TGF beta 1) is a multifunctional regulator of cell growth and differentiation. We report here that TGF beta 1 decreased the proliferation of nontransformed bovine anterior pituitary-derived cells grown in culture. We have previously demonstrated that these cells express both TGF alpha and its receptor [the epidermal growth factor (EGF) receptor] and that expression can be stimulated by phorbol ester (TPA) and EGF. TGF beta 1 treatment over a 2-day period decreased the proliferation of pituitary cells. This decreased growth rate was accompanied by a decrease in the TGF alpha mRNA level. The effect of TGF beta 1 on TGF alpha mRNA down-regulation was both dose dependent (maximal effect observed at 1.0 ng/ml TGF beta 1) and time dependent (minimum of 2-day treatment with TGF beta 1 was required before a decrease in TGF alpha mRNA was observed). Studies on TGF alpha mRNA stability indicated that TGF beta 1 did not alter the TGF alpha mRNA half-life. Treatment of the TGF beta 1 down-regulated cells with EGF resulted in the stimulation of TGF alpha mRNA levels; thus, the TGF beta 1-treated cells remained responsive to EGF. The decreased proliferation in response to TGF beta 1 could be only partially reversed by simultaneous treatment of the cells with EGF (10(-9)M) and TGF beta 1 (3.0 ng/ml). Qualitatively, the TGF beta 1-induced reduction of TGF alpha mRNA content was independent of cell density. TGF beta 1 treatment of the anterior pituitary-derived cells also reduced the levels of c-myc and EGF receptor mRNA. These results represent the first demonstration of the down-regulation of TGF alpha synthesis by a polypeptide growth factor and suggest that TGF beta 1 may be a physiological regulator of TGF alpha production in vivo.

Animals↗

PubMed1990

Transforming growth factor alpha in arterioles: cell surface processing of its precursor by elastases.

Analysis of the transforming growth factor alpha (TGF alpha) cDNA predicts that the mature TGF alpha polypeptide is cleaved from the extracellular domain of its precursor, which is an integral membrane protein. Furthermore, the cleavage sites for the release of this mitogen are compatible with the participation of an elastaselike protease. We have immunohistochemically localized TGF alpha to the vascular smooth muscle cells in the arterioles. To investigate whether polymorphonuclear (PMN) leukocytic elastase, a blood-borne protease, could process the cell surface TGF alpha, NR6 cells were transfected with the rat TGF alpha cDNA. The cDNA encoded the entire open reading frame, and its expression was under the control of the mouse metallothionein I promoter. A cloned transfectant, termed 1B2, synthesized the TGF alpha precursor in a zinc-inducible manner, and the precursor was localized to the cell surface. Western blot (immunoblot) analysis indicated that treatment of the zinc-induced 1B2 cells with either PMN leukocytic or pancreatic elastase resulted in the release of the mature TGF alpha polypeptide. The released TGF alpha was bioactive, as it was capable of both competing with epidermal growth factor for binding to its receptor and stimulating [3H]thymidine incorporation in the mitogenic assay. Formaldehyde fixation of the 1B2 cells eliminated basal release of TGF alpha but allowed normal processing by both PMN leukocytic and pancreatic elastase to occur. However, human cathepsin G, bovine pancreatic alpha 1-chymotrypsin, collagenase, trypsin, subtilisin, and plasmin failed to release any detectable fragments of the TGF alpha precursor from the fixed cells. The location of TGF alpha in the arterioles and ability of PMN leukocytic elastase to process the membrane-bound TGF alpha precursor suggests a novel role for this elastase at the wound site.

Animals↗

PubMed1989

Transforming growth factor-alpha expression in the anterior pituitary gland: regulation by epidermal growth factor and phorbol ester in dispersed cells.

We have previously reported the immunohistochemical localization of transforming growth factor-alpha (TGF alpha) in the intact bovine anterior pituitary gland. Furthermore, we have purified TGF alpha from the conditioned medium of cell cultures derived from the bovine anterior pituitary. We report her the identification of the TGF alpha mRNA from both the intact bovine anterior pituitary gland and the anterior pituitary derived cell cultures. The level of TGF-alpha mRNA in the cell cultures is greater than that present in the intact gland. The TGF-alpha mRNA level increased when the cell cultures were allowed to incubate in their conditioned medium for 3 days, suggesting that a secretory product from the cultured cells is capable of stimulating the accumulation of the TGF-alpha mRNA. 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation of these cells resulted in a 6-fold increase in the level of TGF-alpha secreted into the conditioned medium. TPA appears to stimulate TGF-alpha secretion at the level of gene transcription as TPA treatment also resulted in an increased accumulation of the TGF-alpha mRNA. The epidermal growth factor (EGF) receptor mRNA was examined in these cell cultures and it increased with TPA treatment in an analogous manner to the TGF-alpha mRNA. EGF treatment of the pituitary cells resulted in an increased level of TGF-alpha mRNA which followed the same time course as TPA, maximal stimulation occurred after 8 h of treatment. The magnitude of EGF stimulated TGF-alpha mRNA was not as great as that seen by TPA stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗