Regulation of gene expression by muscarinic acetylcholine receptors.

In the brain, muscarinic acetylcholine receptors (mAChRs) are involved in higher cognitive functions including synaptic plasticity and memory. In Alzheimer's disease (AD) patients the cholinergic nervous system is severely damaged. In order to reinforce the cholinergic system, clinical tests were started to use cholinomimetic drugs to treat AD patients. To identify the genes involved in mAChR signalling, we used a differential display approach and found 11 genes that were readily activated by mAChR with 1 hour of activation. These included the transcription factors Egr-1, Egr-2, Egr-3, c-Jun, Jun-D and Gos-3; the growth regulator hCyr61; the signalling factors NGFi-B (nerve growth factor induced gene-B) and Etr101; the unknown gene Gig-2 (for G-protein-coupled receptor induced gene 2); and the acetylcholinesterase gene (ACHE). Our data show that multiple immediate-early genes are under the control of mAChRs, and they suggest that these genes play important roles in coupling receptor stimulation to long-term neuronal responses. The results also suggest a feedback mechanism where up-regulated ACHE expression and accelerated breakdown of acetylcholine (ACh) at the cholinergic synapses limits increases in cholinergic transmission. Three hours after m1 mAChR activation a different pattern of gene expression was demonstrated. It included the novel genes Gig-3 and Gig-4, as well as the LIM-only protein LM04. Like ACHE, these genes are target genes which may be under the control of the above immediate-early genes. Together, our data show that muscarinic receptors induce a complex and sustained pattern of gene expression that may be involved in the regulation of cholinergic transmission as well as the control of cellular functions in post-synaptic cholinergic target cells. These results may contribute to a better understanding of the effects and side effects of cholinomimetic treatment in AD patients.

Genetic association of a cystatin C gene polymorphism with late-onset Alzheimer disease.

OBJECTIVE: To determine whether the cystatin C gene (CST3) is genetically associated with late-onset Alzheimer disease (AD). DESIGN: A case-control study with 2 independent study populations of patients with AD and age-matched, cognitively normal control subjects. SETTING: The Alzheimer's Disease Research Unit at the University Hospital Hamburg-Eppendorf, Hamburg, Germany, for the initial study (n = 260). For the independent multicenter study (n = 647), an international consortium that included the Massachusetts Alzheimer's Disease Research Center at the Massachusetts General Hospital, Boston; the Scientific Institute for Research and Patient Care, Brescia, Italy; and Alzheimer's research units at the Universities of Basel and Zurich, Switzerland, and Bonn, Goettingen, and Hamburg, Germany. PARTICIPANTS: Five hundred seventeen patients with AD and 390 control subjects. MEASURES: Molecular testing of the KspI polymorphisms in the 5' flanking region and exon 1 of CST3 and the apolipoprotein E (APOE) genotype. Mini-Mental State Examination scores for both patients with AD and control subjects. RESULTS: Homozygosity for haplotype B of CST3 was significantly associated with late-onset AD in both study populations, with an odds ratio of 3.8 (95% confidence interval, 1.56-9.25) in the combined data set; heterozygosity was not associated with an increased risk. The odds ratios for CST3 B/B increased from 2.6 in those younger than 75 years to 8.8 for those aged 75 years and older. The association of CST3 B/B with AD was independent of APOE epsilon4; both genotypes independently reduced disease-free survival. CONCLUSIONS: CST3 is a susceptibility gene for late-onset AD, especially in patients aged 75 years and older. To our knowledge, CST3 B is the first autosomal recessive risk allele in late-onset AD.

Muscarinic acetylcholine receptors induce the expression of the immediate early growth regulatory gene CYR61.

In brain, muscarinic acetylcholine receptors (mAChRs) modulate neuronal functions including long term potentiation and synaptic plasticity in neuronal circuits that are involved in learning and memory formation. To identify mAChR-inducible genes, we used a differential display approach and found that mAChRs rapidly induced transcription of the immediate early gene CYR61 in HEK 293 cells with a maximum expression after 1 h of receptor stimulation. CYR61 is a member of the emerging CCN gene family that includes CYR61/CEF10, CTGF/FISP-12, and NOV; these encode secretory growth regulatory proteins with distinct functions in cell proliferation, migration, adhesion, and survival. We found that CYR61, CTGF, and NOV were expressed throughout the human central nervous system. Stimulation of mAChRs induced CYR61 expression in primary neurons and rat brain where CYR61 mRNA was detected in cortical layers V and VI and in thalamic nuclei. In contrast, CTGF and NOV expression was not altered by mAChRs neither in neuronal tissue culture nor rat brain. Receptor subtype analyses demonstrated that m1 and m3 mAChR subtypes strongly induced CYR61 expression, whereas m2 and m4 mAChRs had only subtle effects. Increased CYR61 expression was coupled to mAChRs by both protein kinase C and elevations of intracellular Ca(2+). Our results establish that CYR61 expression in mammalian brain is under the control of cholinergic neurotransmission; it may thus be involved in cholinergic regulation of synaptic plasticity.

Treatment with the selective muscarinic agonist talsaclidine decreases cerebrospinal fluid levels of total amyloid beta-peptide in patients with Alzheimer's disease.

Brain amyloid load in Alzheimer's disease (AD) is, at least in genetic forms, associated with overproduction of amyloid beta-peptides (A beta). Thus, lowering A beta production is a central therapeutic target in AD and may be achieved by modulating such key enzymes of amyloid precursor protein (APP) processing as beta-, gamma-, and alpha-secretase activities. Talsaclidine is a selective muscarinic M1 agonist that stimulates the nonamyloidogenic alpha-secretase pathway in model systems. Talsaclidine was administered double-blind, placebo-controlled, and randomized to 24 AD patients and cerebrospinal fluid (CSF) levels of total A beta were quantitated before and after 4 weeks of drug treatment. We observed that talsaclidine decreases CSF levels of A beta significantly over time within the treatment group (n = 20) by a median of 16% as well as compared to placebo (n = 4) by a median of 27%. We conclude that treatment with selective M1 agonists may reduce A beta production and may thus be further evaluated as a potential amyloid-lowering therapy of AD.

Identification of genes regulated by muscarinic acetylcholine receptors: application of an improved and statistically comprehensive mRNA differential display technique.

In order to identify genes that are regulated by muscarinic acetylcholine receptors, we developed an mRNA differential display technique (DD) approach. By increasing redundancy and by evaluating optimised reagents and conditions for reverse transcription of total RNA, PCR and separation of PCR products, we generated a DD protocol that yields highly consistent results. A set of 64 distinct random primers was specifically designed in order to approach a statistically comprehensive analysis of all mRNA species in a defined cell population. This modified DD protocol was applied to total RNA of HEK293 cells stably expressing muscarinic m1 acetylcholine receptors and cells stimulated with the receptor agonist carbachol were compared to identical but non-stimulated cells. In 81 of 192 possible PCR experiments, 38 differential bands were identified. Sequence analysis followed by northern blot analyses confirmed differentially expressed genes in 19 of 23 bands analysed. These represented 10 distinct immediate-early genes that were up-regulated by m1AChR activation: Egr-1, Egr-2, Egr-3, NGFi-B, ETR101, c- jun, jun -D, Gos-3 and hcyr61, as well as the unknown gene Gig-2. These data show that this improved DD protocol can be readily applied to reliably identify differentially expressed genes.

Muscarinic acetylcholine receptors activate the acetylcholinesterase gene promoter.

The acetylcholinesterase (AChE) gene promoter contains several overlapping binding sites for Sp1 and Egr-1 transcription factors. Cotransfection experiments and promoter assays showed that Egr-1 can potently activate transcription from the human AChE promoter. Muscarinic acetylcholine receptors (mAChR) rapidly activate, via protein kinase C-mediated signaling, expression of the Egr-1 gene, leading to dramatically increased nuclear concentrations of Egr-1 protein, and to increased binding of Egr-1 to specific DNA recognition sequences. These mAChR-induced increases are followed by increased transcription from the human AChE promoter. In vivo studies with intraventricular infusions of the cholinergic immunotoxin 192 IgG saporin showed more than 80% decrease of AChE activity in cholinergic target areas of the hippocampus and brain cortex. The results are compatible with a combination of decreased AChE activity in degenerating subcortical cholinergic projections, and additional decreases in postsynaptic AChE gene expression. Together our data show that mAChR can activate transcription from the AChE promoter via increased synthesis of Egr-1. The results suggest a feedback mechanism by which the AChE gene is activated by cholinergic neurotransmission, possibly leading to increased formation of AChE protein and accelerated degradation of acetylcholine at cholinergic synapses. This possibility suggests testing of cholinomimetic compounds currently in development for the treatment of Alzheimer's disease for their potential ability to increase AChE gene expression.

Muscarinic acetylcholine receptors activate expression of the EGR gene family of transcription factors.

In order to search for genes that are activated by muscarinic acetylcholine receptors (mAChRs), we used an mRNA differential display approach in HEK293 cells expressing m1AChR. The zinc-finger transcription factor genes Egr-1, Egr-2, and Egr-3 were identified. Northern blot analyses confirmed that mRNA levels of Egr-1, Egr-2, and Egr-3 increased readily after m1AChR stimulation and that a maximum was attained within 50 min. At that time, Egr-4 mRNA was also detectable. Western blots and electromobility shift assays demonstrated synthesis of EGR-1 and EGR-3, as well as binding to DNA recognition sites in response to m1AChR activation. Activation of m1AChR increased transcription from EGR-dependent promoters, including the acetylcholinesterase gene promoter. Activity-dependent regulation of Egr-1 mRNA expression and EGR-1 protein synthesis was also observed in cells expressing m2, m3, or m4AChR subtypes. Increased EGR-1 synthesis was mimicked by phorbol myristate acetate, but not by forskolin, and receptor-stimulated EGR-1 synthesis was partially inhibited by phorbol myristate acetate down-regulation. Together, our results demonstrate that muscarinic receptor signaling activates the EGR transcription factor family and that PKC may be involved in intracellular signaling. The data suggest that transcription of EGR-dependent target genes, including the AChE gene, can be under the control of extracellular and intracellular signals coupled to muscarinic receptors.

Antibody response to keyhole limpet hemocyanin (KLH) treatment in patients with superficial bladder carcinoma.

The dynamics of specific KLH-antibody production after intracutaneous and intravesical instillation was analysed. Nine patients (male, n = 7; female, n = 2, mean age 68.6 years, range 47-75) with primary superficial carcinomas of the bladder were intracutaneously immunized with 1 mg Keyhole limpet hemocyanin (KLH) after the complete resection of the tumors. Treatment was continued for 6 consecutive weeks, monthly for one year and thereafter bimonthly for 2 subsequent years, consisting of 20 mg KLH in 20 ml saline introduced intravesically. The antibodies against KLH in patient sera were determined by means of a specially developed direct enzyme-linked immunosorbent assay (ELISA; according to H. von der Kammer, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany). Blood was taken for antibody-titer examination before treatment and 8 weeks after treatment. The KLH-antibody titer increased significantly (Mann-Whitney-Test P = 0.02) after KLH therapy in bladder cancer patients, however the level varied considerably from patient to patient. 6 of 9 patients (67%) presented increased serum antibody titers to KLH after immunotherapy. 4 patients (44.4%) remained free of tumor during the established follow-up period of 10-45 months (median 30.7 months). One patient without increased antibody titer to KLH was free of tumor, 2 patients however, suffered from tumor recurrence after the KLH course. 2 patients presented with tumor recurrence in spite of increased antibody titers. No evidence of tumor progression occurred in patients with recurrence after KLH therapy. 4 of 5 patients (80%) without tumor recurrence presented with a positive skin test. Of patients with tumor recurrence, 50% had a negative skin test. 44.4% KLH-treated patients had tumor recurrence The recurrence rate was 1.6. The time to recurrence was 8.75 months. KLH instillation did not induce major side effects. Positive skin test reactivity and KLH antibody response were more commonly seen in responding patients (i.e. those who remained tumor free after therapy) than in non-responders. The production of KLH antibodies, apparently is the biological response to the antigen stimulus of KLH.

A monoclonal antibody generated against a recombinant peptide fragment of the B3 domain of carcinoembryonic antigen reacts with intact carcinoembryonic antigen.

The chemical synthesis of a gene coding for a polypeptide of 77 amino acid residues (designated ceaB3) representing a fragment of the CEA-B3 domain of carcinoembryonic antigen (CEA) was achieved. The ceaB3 fragment was cloned into the plasmid pLZPWB1 at the C-terminus of a derivative lacZMF of the lacZ gene, devoid of methionine and cysteine amino acid residues. The fusion protein lacZMF-ceaB3 represented approx. 30% of total proteins expressed after induction. The fusion protein was formed as inclusion bodies. Simple washing steps led to an insoluble fusion protein which was of approx. 80% purity. Another fusion gene was generated by inserting ceaB3 between the malE gene encoding maltose binding protein (mbp) and lacZ alpha of the pmal-c2 vector. Expression of the resulting pmal-c2-ceaB3-lacZ alpha yielded the fusion protein mbp-ceaB3-lacZ alpha with a molecular mass of 57.94 kDa, which was obtained as a soluble protein in almost homogeneous form after affinity chromatography employing amylopectin. Polyclonal sheep anti-CEA antiserum specifically reacted with fusion proteins lacZMF-ceaB3 and mbp-ceaB3-lacZ alpha. A monoclonal antibody CEA/HK2 was generated employing lacZMF-ceaB3 for immunization and CEA for screening purposes. The mAB CEA/HK2 specifically recognized CEA in immunoblots. The described experimental strategy should be generally applicable for generation of fusion proteins. These fusion proteins are suitable for epitope characterization of existing antibodies, production of regiospecific polyclonal or monoclonal antibodies.

Characterization by cDNA cloning of two new human protein kinases. Evidence by sequence comparison of a new family of mammalian protein kinases.

Two new human cDNAs, designated phclk2 and phclk3, which have a high identity to the cDNA of the human protein kinase clk, were characterized. Typical features of hclk2 and hclk3 proteins are non-homologous N-terminal regions and the presence of the C-terminal protein kinase domain, which is characteristic for serine/threonine-type kinases. We also identified the differentially spliced forms phclk2(139) and phclk3(152) with deletions of 88 and 97 nt, respectively, which lead to changes in the open reading frames. hclk2(139) and hclk3(152) proteins do not possess a protein kinase domain and are nearly identical to the N-terminal regions of the above-mentioned protein kinases. We verified that differentially spliced variants also exist for hclk1 as well as for a mouse clk protein kinase. It was shown that shorter and longer alternatively spliced mRNAs co-exist in different human tissues. According to Southern analysis, hclk2 and hclk3 appear to be specified by single copy genes. The genes for hclk2 as well as for hclk3 were localized to human chromosomes 1 and 15, respectively.

A human amyloid precursor-like protein is highly homologous to a mouse sequence-specific DNA-binding protein.

From a cDNA sequence, we have deduced the amino acid sequence for a human amyloid precursor-like protein (APPH) with > 92% identity to the CDEI binding protein (CDEBP) of the mouse and the fragmentary rat protein YWK-II of unknown function. Expression of APPH was found in all tissues examined. A striking homology of APPH to human amyloid precursor protein (APP) was observed. Overall identity accounts for 52.7%. However, there are three domains of APPH with remarkably higher similarities, corresponding to amino acid sequence positions 47-204 (76.6%), 308-567 (67.7%), and 694-763 (69.9%). Using an APPH antiserum, we localized APPH in nuclei of human interphase cells and found an increased synthesis of APPH in mitotic cells. Our results indicate that the highly conserved proteins human APPH, mouse CDEBP, and rat YWK-II are apparently homologues of a CDEI binding protein with indispensible function in mammalian genome segregation.

The gene for the amyloid precursor-like protein APLP2 is assigned to human chromosome 11q23-q25.

The human amyloid precursor-like protein APLP2 is a highly conserved homologue of a sequence-specific DNA-binding mouse protein with a predicted function in the cell cycle. Somatic cell hybrids segregating human chromosomes were used to assign the APLP2 gene to chromosome 11. Fluorescence in situ hybridization confirmed this assignment and further localized the gene to q23-q25.

[Adjuvant immunotherapy with keyhole limpet hemocyanin in category PT 2 N+ and PT 3-4, No-N+, Mo renal cell carcinoma]. / Adjuvante Immuntherapie mit Keyhole Limpet Hemocyanin (KLH) beim Nierenzellkarzinom der Kategorie PT 2 N+ und PT 3-4, No-N+, Mo.

This study was a prospective randomized trial to compare adjuvant immunotherapy with Keyhole Limpet Hemocyanin (KLH) after radical nephrectomy. From January 1983 to December 1988, 50 patients underwent radical nephrectomy for category PT 2 N+ and PT 3-4, No-N+, Mo renal cell carcinoma. Postoperatively 25 patients were given adjuvant treatment with the biological response modifier, Keyhole Limpet Hemocyanin (KLH), and 25 patients were in the control group. In each group 2 patients were lost to follow-up. The mean follow-up time was 55 months. Adjuvant treatment with KLH did not appear to improve the prognosis in renal cell carcinoma patients. The 5-year survival rate was 60% in the KLH group and 56.5% in the control group. Progress was seen in 9/23 in the KLH group, 10/23 in the controls. The median survival in patients showing progress was 27 and 28 months in the two groups, respectively. Studies with a combination of low dose cyclophosphamide and KLH revealed a positive immunostimulating effect, which may provide the rationale for further research concerning different KLH doses, schedules or therapeutic combinations.

Characterization by cDNA cloning of the mRNA of human ribosomal protein L8.

From a lambda UNI-ZAP XR cDNA library derived from poly(A)+RNA of human ovarian granulosa cells a cDNA clone pHG51 was isolated. Sequence analysis showed significant homology to the C-terminal region of rat ribosomal protein L8 cDNA. The 5'-end of the cDNA was completed by PCR with cloned total cDNA. Aligning of DNA sequences from PCR clones with the sequence of the pHG51 insert yielded the full-length cDNA. From the open reading frame of the cDNA an amino acid sequence for a polypeptide of 257 residues was derived, which was identical with rat ribosomal protein L8 and also possessed a high degree of identity to ribosomal proteins L8 of other species. It is therefore assumed that the characterized cDNA represents the mRNA of the human ribosomal protein L8. Southern analysis revealed that human ribosomal protein L8 is specified by multi copy genes.

The complete cDNA coding sequence for the mouse CDEI binding protein.

This work describes the cDNA sequence of the mouse CDEI binding protein (CDEBP), comprising the complete coding sequence. The cDNA encodes a protein of 695 amino acid residues. The derived amino acid sequence displays a sequence identity to human amyloid precursor-like protein (APLP) of > 92%.

The potential use of prostatic secretory protein of 94 amino acid residues (PSP94) as a serum marker for prostatic tumor.

The serum concentrations of prostatic secretory protein of 94 amino acid residues (PSP94) as well as those of prostate-specific antigen (PSA) were determined in 40 patients with established prostatic carcinoma, prior to transurethral resection of the prostate. In a comparison with a control group of healthy men (n = 40) and a group of patients with histologically established benign prostatic hyperplasia (n = 40) no significant differences in PSP94 serum concentrations between the groups were observed. Similarly, correlations of PSP94 serum concentrations with prostatic carcinoma stages or grades were not detected. In contrast, and as expected, PSA behaved as a prostate tumor marker of known sensitivity and specificity. A correlation of PSP94 and PSA concentrations in sera of patients with benign prostatic hyperplasia and/or prostatic carcinoma could not be verified. PSP94 apparently does not fulfill the criteria of a serum marker for monitoring adenomas and/or carcinomas of the prostate.

Clinical evaluation of a new prostate-specific antigen sandwich ELISA which employs four monoclonal antibodies directed at different epitopes of prostate-specific antigen.

A known radioimmunometric prostate-specific antigen (PSA) test based on monoclonal antibodies, as well as a new PSA-ELISA utilizing 4 monoclonal antibodies directed against different epitopes of PSA were compared in a clinical evaluation. For the investigation, collectives of patient sera from patients with independently diagnosed prostatic carcinoma (PCA) as well as benign prostatic hyperplasia (BPH) were employed. The results of the evaluation demonstrated that although the PSA immunoradiometric test and the PSA-ELISA yielded different numerical values for PSA serum concentrations, they possess comparable diagnostic sensitivities as well as specificities. The nonradioactive PSA-ELISA could therefore substitute the PSA-IRMA in a clinical routine diagnostic of PCA.

Characterization by cDNA cloning of the mRNA of a highly basic human protein homologous to the yeast ribosomal protein YL41.

From a cDNA library in lambda gt11 derived from poly(A)+ mRNA of human ovarian granulosa cells, a cDNA clone lambda HG12.1, containing an EcoRI insert of 470 bp, was identified. After subcloning of the insert into pUC18, the clone pHG12.1 was obtained and sequenced. The 5'-region of the insert of pHG12.1 was extended by the polymerase chain reaction (PCR) with cloned total cDNA. Assembly of the PCR fragment with the insert of pHG12.1 yielded clone pHG12. From the first open reading frame of pHG12 the amino acid sequence for a polypeptide of 25 amino acid residues (designated HG12) was derived, which was identical in 22 residues with yeast ribosomal protein YL41. It is therefore assumed that HG12 is the first mammalian homolog of yeast ribosomal protein YL41. Transcription of DNA fragments containing the coding region of pHG12 cloned into BluescriptM13, followed by cell-free translation, yielded a polypeptide with an apparent mol.wt. of 14.5 kDa, much larger than the theoretical mol.wt. (3454 Da). The discrepancy between theoretical and apparent mol.wt. was also observed for yeast ribosomal protein YL41. Southern analysis revealed that HG12 is not specified by a single copy gene. Homology for HG12 specific sequences is observed for bovine, porcine and rat species.

Human elongation factor 1 beta: cDNA and derived amino acid sequence.

From a cDNA library in lambda gt11 derived from poly (A+)RNA of human ovarian granulosa cells a cDNA clone lambda HGP34, containing an EcoRI insert of 829 bp, was identified. After subcloning of the insert into pUC18, the clone pHGP34 was obtained and sequenced. The derived amino acid sequence, corresponding to a protein of 225 amino acids, shows a high degree of homology to elongation factor 1 beta (EF-1 beta) of Artemia salina (57%) and known peptide sequences of Xenopus laevis EF-1 beta (86%). We therefore assume that the protein coded for by pHGP34 represents human EF-1 beta. Northern analysis reveals an EF-1 beta specific mRNA of 900 bp. Southern analysis indicates that EF-1 beta in the human genome, like EF-1 alpha, appears to be specified by more than one gene. A high degree of sequence homology for EF-1 beta specific sequences is observed for bovine, rat and mouse species.