Biomedical subjects

Alfred Bach

Publications and source records attributed to Alfred Bach.

13 recordsLinked to original sources

PubMed2006

Monitoring regulated protein-protein interactions using split TEV.

Signaling cascades integrate extracellular stimuli primarily through regulated protein-protein interactions (PPIs). Intracellular signal transduction strictly depends on PPIs occurring at the membrane and in the cytosol. To monitor constitutive and regulated protein interactions within living mammalian cells, we have developed a biological assay termed split TEV. We engineered inactive fragments of the NIa protease from the tobacco etch virus (TEV protease) that regain activity only when coexpressed as fusion constructs with interacting proteins. Functional reconstitution of TEV protease fragments can be monitored with 'proteolysis-only' reporters, which can be previously silent fluorescent and luminescent reporter proteins. Additionally, proteolytically cleavable inactive transcription factors can be combined with any downstream reporter gene of choice to yield 'transcription-coupled' reporter systems. Thus, split TEV combines the advantages of split enzyme- and reporter gene-mediated assays, and provides full flexibility with regard to the final readout. In a first biological application, we monitored neuregulin-induced ErbB2/ErbB4 receptor tyrosine kinase heterodimerization.

Cell Physiological Phenomena↗

PubMed2006

An extended window of opportunity for G-CSF treatment in cerebral ischemia.

BACKGROUND: Granulocyte-colony stimulating factor (G-CSF) is known as a powerful regulator of white blood cell proliferation and differentiation in mammals. We, and others, have shown that G-CSF is effective in treating cerebral ischemia in rodents, both relating to infarct size as well as functional recovery. G-CSF and its receptor are expressed by neurons, and G-CSF regulates apoptosis and neurogenesis, providing a rational basis for its beneficial short- and long-term actions in ischemia. In addition, G-CSF may contribute to re-endothelialisation and arteriogenesis in the vasculature of the ischemic penumbra. In addition to these trophic effects, G-CSF is a potent neuroprotective factor reliably reducing infarct size in different stroke models. RESULTS: Here, we have further delayed treatment and studied effects of G-CSF on infarct volume in the middle cerebral artery occlusion (MCAO) model and functional outcome in the cortical photothrombotic model. In the MCAO model, we applied a single dose of 60 microg/kg bodyweight G-CSF in rats 4 h after onset of ischemia. Infarct volume was determined 24 h after onset of ischemia. In the rat photothrombotic model, we applied 10 microg/kg bodyweight G-CSF daily for a period of 10 days starting either 24 or 72 h after induction of ischemia. G-CSF both decreased acute infarct volume in the MCAO model, and improved recovery in the photothrombotic model at delayed timepoints. CONCLUSION: These data further strengthen G-CSF's profile as a unique candidate stroke drug, and provide an experimental basis for application of G-CSF in the post-stroke recovery phase.

Animals↗

PubMed2006

Granulocyte-colony stimulating factor is neuroprotective in a model of Parkinson's disease.

We have recently shown that the hematopoietic Granulocyte-Colony Stimulating Factor (G-CSF) is neuroprotective in rodent stroke models, and that this action appears to be mediated via a neuronal G-CSF receptor. Here, we report that the G-CSF receptor is expressed in rodent dopaminergic substantia nigra neurons, suggesting that G-CSF might be neuroprotective for dopaminergic neurons and a candidate molecule for the treatment of Parkinson's disease. Thus, we investigated protective effects of G-CSF in 1-methyl-4-phenylpyridinium (MPP+)-challenged PC12 cells and primary neuronal midbrain cultures, as well as in the mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease. Substantial protection was found against MPP+-induced dopaminergic cell death in vitro. Moreover, subcutaneous application of G-CSF at a dose of 40 microg/Kg body weight daily over 13 days rescued dopaminergic substantia nigra neurons from MPTP-induced death in aged mice, as shown by quantification of tyrosine hydroxylase-positive substantia nigra cells. Using HPLC, a corresponding reduction in striatal dopamine depletion after MPTP application was observed in G-CSF-treated mice. Thus our data suggest that G-CSF is a novel therapeutic opportunity for the treatment of Parkinson's disease, because it is well-tolerated and already approved for the treatment of neutropenic conditions in humans.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2006

Reduced oxidative damage in ALS by high-dose enteral melatonin treatment.

Amyotrophic lateral sclerosis (ALS) is the collective term for a fatal motoneuron disease of different etiologies, with oxidative stress as a common molecular denominator of disease progression. Melatonin is an amphiphilic molecule with a unique spectrum of antioxidative effects not conveyed by classical antioxidants. In preparation of a possible future clinical trial, we explored the potential of melatonin as neuroprotective compound and antioxidant in: (1) cultured motoneuronal cells (NSC-34), (2) a genetic mouse model of ALS (SOD1(G93A)-transgenic mice), and (3) a group of 31 patients with sporadic ALS. We found that melatonin attenuates glutamate-induced cell death of cultured motoneurons. In SOD1(G93A)-transgenic mice, high-dose oral melatonin delayed disease progression and extended survival. In a clinical safety study, chronic high-dose (300 mg/day) rectal melatonin was well tolerated during an observation period of up to 2 yr. Importantly, circulating serum protein carbonyls, which provide a surrogate marker for oxidative stress, were elevated in ALS patients, but were normalized to control values by melatonin treatment. This combination of preclinical effectiveness and proven safety in humans suggests that high-dose melatonin is suitable for clinical trials aimed at neuroprotection through antioxidation in ALS.

Adult↗

PubMed2005

The hematopoietic factor G-CSF is a neuronal ligand that counteracts programmed cell death and drives neurogenesis.

G-CSF is a potent hematopoietic factor that enhances survival and drives differentiation of myeloid lineage cells, resulting in the generation of neutrophilic granulocytes. Here, we show that G-CSF passes the intact blood-brain barrier and reduces infarct volume in 2 different rat models of acute stroke. G-CSF displays strong anti-apoptotic activity in mature neurons and activates multiple cell survival pathways. Both G-CSF and its receptor are widely expressed by neurons in the CNS, and their expression is induced by ischemia, which suggests an autocrine protective signaling mechanism. Surprisingly, the G-CSF receptor was also expressed by adult neural stem cells, and G-CSF induced neuronal differentiation in vitro. G-CSF markedly improved long-term behavioral outcome after cortical ischemia, while stimulating neural progenitor response in vivo, providing a link to functional recovery. Thus, G-CSF is an endogenous ligand in the CNS that has a dual activity beneficial both in counteracting acute neuronal degeneration and contributing to long-term plasticity after cerebral ischemia. We therefore propose G-CSF as a potential new drug for stroke and neurodegenerative diseases.

Animals↗

PubMed2004

TorsinA, the gene linked to early-onset dystonia, is upregulated by the dopaminergic toxin MPTP in mice.

Early-onset torsion dystonias are caused by a mutation in TorsinA, a protein widely expressed in the nervous system. Here we report the cloning of the murine TorsinA cDNA and a mRNA in situ hybridization analysis of the expression patterns of TorsinA over developmental periods relevant to the etiology of early-onset dystonias. Several studies have demonstrated a functional involvement of the nigrostriatal dopaminergic system in pathological mechanisms underlying dystonia. In this study, we show that the expression of TorsinA is significantly increased in the brain within hours of treatment with the dopaminergic toxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice, suggesting that the TorsinA gene is regulated by cellular stress. These results provide insights into the pathophysiology of early-onset dystonia and strengthen links between the dopaminergic system and dystonia.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2004

Identification of regulated genes during permanent focal cerebral ischaemia: characterization of the protein kinase 9b5/MARKL1/MARK4.

Cerebral ischaemia induces transcriptional changes in a number of pathophysiologically important genes. Here we have systematically studied gene expression changes after 90 min and 24 h of permanent focal ischaemia in the mouse by an advanced fragment display technique (restriction-mediated differential display). We identified 56 transcriptionally altered genes, many of which provide novel hints to ischaemic pathophysiology. Particularly interesting were two pro-apoptotic genes (Grim19 and Tdag51), whose role in cerebral ischaemia and neuronal cell death has not been recognized so far. Among the unknown sequences, we identified a gene that was rapidly and transiently up-regulated. The encoded protein displayed high homology to the MARK family of serine-threonine protein kinases and has recently been described as MARKL1/MARK4. Here we demonstrate that this protein is a functional protein kinase with the ability to specifically phosphorylate a cognate peptide substrate for the AMP-kinase family. Upon overexpression in heterologous cells, the functional wild-type protein, but not its kinase-dead mutant, led to decreased cell viability. We conclude that the up-regulation of this kinase during focal ischaemia may represent an interesting new target for pharmacological intervention.

Alternative Splicing↗

PubMed2004

Restriction-mediated differential display (RMDD) identifies pip92 as a pro-apoptotic gene product induced during focal cerebral ischemia.

Studies of gene expression changes after cerebral ischemia can provide novel insight into ischemic pathophysiology. Here we describe application of restriction-mediated differential display to screening for differentially expressed genes after focal cerebral ischemia. This method combines the nonredundant generation of biotin-labeled fragment sets with the excellent resolution of direct blotting electrophoresis, reliable fragment recovery, and a novel clone selection strategy. Using the filament model in mouse with 90 minutes MCA occlusion followed by 2, 6, and 20 hours reperfusion, we have compared gene expression in sham-operated animals to both the ipsi- and contralateral forebrain hemisphere of ischemic mice. Our screening method has resulted in the identification of 70 genes differentially regulated after transient middle cerebral artery occlusion (MCAO), several of which represent unknown clones. We have identified many of the previously published regulated genes, lending high credibility to our method. Surprisingly, we detected a high degree of correspondent regulation of genes in the nonischemic hemisphere. A high percentage of genes coding for proteins in the respiratory chain was found to be up-regulated after ischemia, potentially representing a new mechanism involved in counteracting energy failure or radical generation in cerebral ischemia. One particularly interesting gene, whose upregulation by ischemia has not been described before, is pip92; this gene shows a rapid and long-lasting induction after cerebral ischemia. Here we demonstrate that pip92 induces cell death in primary neurons and displays several hallmarks of pro-apoptotic activity upon overexpression, supporting the notion that we have identified a novel pathophysiological player in cerebral ischemia. In summary, restriction-mediated differential display has proven its suitability for screening complex samples such as brain to reliably identify regulated genes, which can uncover novel pathophysiological mechanisms.

Animals↗

PubMed2003

TorsinA protects against oxidative stress in COS-1 and PC12 cells.

Dystonia is a highly frequent movement disorder, the pathogenesis of which remains unclear. The cloning of TorsinA, the gene responsible for early-onset dystonia, was a major breakthrough. However, the function of this protein remains unclear. By sequence homology, TorsinA belongs to the ATPases associated with diverse cellular activities-family, many of whose members are chaperones and/or proteases. We report here that in an in vitro model for oxidative stress, H2O2 treatment, overexpression of TorsinA was protective against cell death. COS-1 cells overexpressing TorsinA demonstrated drastically reduced terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling-staining following exposure to H2O2. Furthermore, transfection with TorsinA significantly increased survival of PC12 after H2O2 treatment. To our knowledge, this is the first demonstration that TorsinA protects against oxidative stress. We speculate that a loss of this cellular function in mutant TorsinA may be linked to the pathogenesis of early-onset dystonia.

Animals↗

PubMed2002

DNA repair capacity after gamma-irradiation and expression profiles of DNA repair genes in resting and proliferating human peripheral blood lymphocytes.

DNA repair plays an important role in maintaining genomic integrity, and deficiencies in repair function are known to promote cancer development. Several studies have used the individual capacity to repair DNA damage in peripheral blood lymphocytes (PBLs) as a cancer risk marker. As the cell's ability to remove DNA damage may be correlated with proliferative activity, it is an important question whether quiescent or dividing cells should be used in such studies. The aim of our study was to compare DNA repair capacity and expression profiles of 70 known DNA repair genes, both in resting and phytohemagglutinin (PHA) stimulated human PBLs. Using the comet assay, gamma-radiation-induced DNA damage and repair in lymphocytes was analyzed. No difference, neither in the rate of radiation-induced DNA damage nor in DNA repair capacity between PHA-stimulated and non-stimulated PBLs was observed. Stimulated cells, however, showed significantly elevated values for background damage. Transcriptional profiles of repair genes were analyzed using cDNA arrays. Hybridization experiments were performed with mRNA isolated from both unstimulated and PHA-stimulated PBLs. More than 70% of all evaluated genes had constant expression levels. Twelve genes responded with a more than two-fold increase of transcripts to the mitogenic stimulus. Most of the up-regulated repair enzymes are also known to play a role in DNA replication. In conclusion, the data presented here suggest that all repair proteins needed for the repair of gamma-irradiation induced DNA-damage, that can be detected by the alkaline comet assay, are already present in G0 cells at sufficient amounts and do not need to be induced once lymphocytes are stimulated to start cycling. Our results thus do not support a general increase in DNA repair activity of PBLs by PHA stimulation, and the use of stimulated PBLs in molecular epidemiological studies on DNA repair of gamma-irradiation induced DNA damage seems not to be mandatory.

Comet Assay↗

PubMed2002

Expression of calnexin reflects paneth cell differentiation and function.

It has been suggested that the behavior and function of Paneth cells in metaplasia are different from those found in normal intestinal mucosa. In this study, we investigated whether calnexin, a protein involved in secretory pathways, might be associated with differentiation and function of Paneth cells in normal small intestine, in complete intestinal metaplasia of the stomach, and in Paneth cell-rich adenomas. Differentiation and function of Paneth cells was monitored by Ki67, lysozyme, and morphologic features. Using a newly established monoclonal antibody, we found that calnexin is regularly synthesized by Paneth cells of normal small intestine. In these cells, the staining intensity of calnexin was inversely correlated with their content of secretory granules (lysozyme). In contrast, Paneth cells of intestinal metaplasia and Paneth cell-rich adenomas showed a reduced immunostaining of both calnexin and lysozyme. Moreover, these Paneth cells synthesized the proliferation marker Ki67, a phenomenon that was never observed in Paneth cells of normal small intestine. In vitro experiments using CaCo2 cells showed that the expression of calnexin is not directly affected by the induction of mitosis. In conclusion, calnexin probably reflects the status of Paneth cell differentiation and function. The results do not necessarily indicate that calnexin has a function in Paneth cell proliferation.

Adenoma↗

PubMed2002

Yellow pages to the transcriptome.

Transcriptomics has become an important tool for the large-scale analysis of biological processes. This review aims to provide sufficient criteria to make an appropriate choice among the variety of 'closed' systems, represented by DNA microarrays, and 'open' systems like fragment display, tag sequencing and subtractive hybridization, depending on the biological system under investigation. The most important technologies currently available are presented, their strengths and weaknesses are discussed and companies active in the field are listed. The potential of transcriptomics in the pharmaceutical research and development process is highlighted by applications in oncology, research on neurological diseases, and predictive toxicology. Finally, a prognosis for future developments of the technologies is given.

Animals↗

PubMed2002

Transcriptomics in predictive toxicology.

Once again, genomics is about to change drug development. Following its major impact on target discovery and assay development, which increased the number of compounds at early stages of the process, genomics is now zeroing in on the prediction of potential toxicological problems of compounds. Toxicogenomics is the analysis of toxicological processes at the transcriptome level of a target organ or cell. By simultaneously monitoring the effect of a compound on the transcription levels of hundreds to thousands of genes, toxicogenomics can provide an enormous amount of data. This data bears information on the way in which compounds act at the molecular level, reaching far beyond the mere conclusion of whether or not a particular toxicological outcome is elicited. By compiling transcription profiles for well-known toxicants, we are beginning to learn how to analyze this novel type of data in the context of mechanistic and predictive toxicology.

Animals↗