Reinhard Walther

The Apolipoprotein E and β-Fibrinogen G/A-455 Gene Polymorphisms Are Associated With Ischemic Stroke Involving Large-Vessel Disease

The relationship between the apolipoprotein E (apoE) and beta-fibrinogen G/A-455 polymorphisms and cerebrovascular disease (CVD) was examined in the present study. We compared 227 patients with the subtypes of CVD (large-vessel disease, lacunar stroke, cardiac embolism, or undetermined pathomechanisms) with 225 control subjects. The occurrence of apoE isoforms (E2, E3, and E4) and the beta-fibrinogen G/A-455 genotype was determined in these individuals. No differences in apoE polymorphisms or allele frequencies between the CVD patients and control subjects were found. However, analysis of apoE genotypes as a function of stroke subtype revealed that the apoE4 allele was significantly more common in those patients with macroangiopathy-associated CVD. The only CVD risk factor that distinguished patients with the E4 allele from those with other apoE genotypes was elevated cholesterol. No association between the beta-fibrinogen G/A-455 polymorphism and CVD was found. However, homozygosity for the A allele was more common in patients with CVD resulting from large-vessel disease. These data demonstrate that the apoE4 allele and the AA genotype of the beta-fibrinogen G/A-455 polymorphism occur significantly more frequently in patients with CVD resulting from stenosis of large, brain-supplying vessels. Such genetic analyses may further our understanding of the etiology of cerebrovascular disease.

Polymorphisms of the Human Platelet Antigens HPA-1, HPA-2, HPA-3, and HPA-5 on the Platelet Receptors for Fibrinogen (GPIIb/IIIa), von Willebrand Factor (GPIb/IX), and Collagen (GPIa/IIa) Are Not Correlated With an Increased Risk for Stroke

BACKGROUND AND PURPOSE A recent study has described a high incidence of the human platelet antigen (HPA)-1b alloantigen in patients with myocardial infarction. We investigated the distribution of gene polymorphisms of platelet glycoproteins (GPs) in patients with cerebrovascular disease (CVD) and stroke. The polymorphic systems we have studied are HPA-1 and HPA-3 on the fibrinogen receptor (GPIIb/IIIa), HPA-2 on the von Willebrand factor receptor (GPIb/IX), and HPA-5 on one of the platelet collagen receptors (GPIa/IIa). METHODS DNA was isolated from peripheral blood collected from 218 consecutive stroke patients, 165 neurological inpatients without signs of CVD, and 321 healthy blood donors. The genotypes of HPA-1, HPA-2, HPA-3, and HPA-5 were determined by sequence specific primer polymerase chain reactions. RESULTS The calculated allele frequencies were as follows: for CVD patients, HPA-1a/b 0.81/0.19, HPA-2a/b 0.91/0.09, HPA-3a/b 0.61/0.39, and HPA-5a/b 0.92/0.08; for inpatient HPA-1a/b 0.83/0.17, HPA-2a/b 0.91/0.09, HPA-3a/b 0.62/0.3 and HPA-5a/b 0.93/0.07; and for blood donors, HPA-1a 0.85/0.15, HPA-2a/b 0.94/0.06, HPA-3a/b 0.60/0.40, and HPA 5a/b 0.92/0.08. There were no statistically significant difference for the analyzed HPA polymorphism frequencies either between the CVD patients and the non-CVD inpatients or the CVD patients and blood donors. However, the HPA-1b genotype was slightly more frequent in patients (CVD and non-CVD) than in the healthy blood donors. CONCLUSIONS Our results indicate that the HPA-1, HPA-2, HPA-3, and HPA-5 polymorphisms are not associated with an increased risk for stroke.

Apolipoprotein E4 promotes the early deposition of Aβ42 and then Aβ40 in the elderly

Abstract The apolipoprotein Eɛ4 allele (ApoEɛ4) is associated with a selective increase in deposition of the 40-amino acid form of the β-amyloid peptide (Aβ40) in end-stage Alzheimer’s disease. To determine how apoE genotype affects the early events in β-amyloid pathogenesis, we analyzed the medial temporal lobes of 244 elderly persons who were not clinically demented using antibodies selective for the C termini of Aβ40 and Aβ42. We found that: (1) the number of both Aβ42- and Aβ40-positive senile plaques increase with age; (2) Aβ42 appears at younger ages, and in more amyloid deposits, than does Aβ40 in all ApoE groups; (3) when compared at similar ages, older persons with ApoEɛ4 are more likely to have Aβ42- and Aβ40-immunoreactive deposits than are persons without ApoEɛ4; (4) Aβ40-containing plaques arise at least a decade later than do Aβ42 plaques, and are seldom found in the medial temporal lobe of older persons lacking ApoEɛ4; and (5) in the absence of overt Alzheimer’s disease, cerebral amyloid angiopathy is rare in the elderly, but in our sample was significantly augmented in ApoEɛ4 homozygotes. We conclude that ApoEɛ4 hastens the onset of Aβ42 deposition in the senescent brain, which in turn fosters the earlier evolution of fibrillar, Aβ40-positive plaques, thereby increasing the risk of Alzheimer’s disease.

The Tet-On system in transgenic mice: inhibition of the mouse pdx-1 gene activity by antisense RNA expression in pancreatic β-cells

Abstract To elucidate the function of pancreas duodenal homeobox 1 (PDX-1; insulin promoter factor 1/somatostatin transcription factor 1/islet duodenum homeobox 1/insulin upstream factor 1) in differentiated β-cells of adult animals we generated transgenic mice using the Tet-On system. Inducible expression of an antisense RNA should down-regulate the PDX-1 protein. The selective and continuous inhibition of PDX-1 gene expression should impair the expression of PDX-1 dependent β-cell specific genes. A gene switch such as the Tet-On system provides a powerful tool to analyze eukaryotic gene expression and function in transgenic mice. The original Tet system contained two transcriptional units, transactivator and target of control, on two plasmids. We combined the two transcriptional units on a single DNA molecule. The transactivator was placed under control of the mouse insulin promoter. The tet responsive element, driving the gene of interest, was inserted further downstream into the same vector. The tet regulatory system in this approach permitted a tissue-specific and a doxycycline-inducible control of PDX-1 expression in transgenic mice. The expression of glucose transporter 2 and glucokinase was markedly reduced in dox-treated transgenic mice. In contrast, the number of insulin- and amylin-expressing cells was only slightly decreased, whereas the expression of glucagon was increased distinctly in islets of these mice. Furthermore, the exposure to doxycycline resulted in a progressive impairment of glucose tolerance. The characterization of our transgenic mouse model demonstrates the suitability of the Tet-On system for analyzing physiological consequences emerging from a stepwise decrease in a given protein. Using this system we confirmed the essential role of PDX-1 in pancreatic islets and demonstrated that an antisense-mediated PDX-1 deficiency provokes a β-cell dysfunction.

Prohibitin identified by proteomic analysis of prostate biopsies distinguishes hyperplasia and cancer

Prostate cancer (PCA) is the most common type of cancer found in men of western countries and is the leading cancer death next to lung cancer and colorectal cancer. Prostate-specific antigen (PSA) test is an established diagnostic tool for PCA detection, but confirmation of diagnosis by histopathological evaluation of prostate needle biopsies is performed. To define protein expression pattern of prostate biopsies, in the present study we investigated biopsy samples from benign prostate hyperplasia (BPH, n=11) and prostate cancer (PCA, n=12) patients by two-dimensional gel electrophoresis (2-DE) and mass spectrometry to identify potential biomarkers which might distinguish the two clinical situations. 2-DE results revealed 88 protein spots expressed differentially among hyperplasia and cancer groups with statistical significance. Interesting spots were analyzed by MALDI-TOF-MS-MS and 79 different proteins were identified. The important proteins identified included prostatic acid phosphatase precursor, a significant overexpressed protein in PCA, prohibitin, NDRG1 tumor suppressor proteins, heat shock proteins, cytoskeletal proteins, enzymes like DDAH1 and ALDH2. Prohibitin was investigated in detail at mRNA level and protein level using immunohistochemistry on prostatectomized specimens. We found that the level of mRNA for prohibitin correlates with the increased amount of protein indicating involvement of changes at transcriptional level. Furthermore, immunohistochemistry revealed no staining in BPH (n=13), moderate staining in prostate intra-epithelial neoplasia (PIN, n=5) but strong staining in PCA (n=18). Our results demonstrate that protein profiling and mRNA studies can be performed on the same prostate biopsy. Moreover, our study revealed a significant up-regulation of prohibitin in prostate cancer compared to BPH which may be a potential marker to distinguish PCA and BPH. Some of the interesting proteins identified in this approach may serve to develop new targets for PCA diagnosis and treatment.

Identification of Clinically Relevant Protein Targets in Prostate Cancer with 2D-DIGE Coupled Mass Spectrometry and Systems Biology Network Platform

Prostate cancer (PCa) is the most common type of cancer found in men and among the leading causes of cancer death in the western world. In the present study, we compared the individual protein expression patterns from histologically characterized PCa and the surrounding benign tissue obtained by manual micro dissection using highly sensitive two-dimensional differential gel electrophoresis (2D-DIGE) coupled with mass spectrometry. Proteomic data revealed 118 protein spots to be differentially expressed in cancer (n = 24) compared to benign (n = 21) prostate tissue. These spots were analysed by MALDI-TOF-MS/MS and 79 different proteins were identified. Using principal component analysis we could clearly separate tumor and normal tissue and two distinct tumor groups based on the protein expression pattern. By using a systems biology approach, we could map many of these proteins both into major pathways involved in PCa progression as well as into a group of potential diagnostic and/or prognostic markers. Due to complexity of the highly interconnected shortest pathway network, the functional sub networks revealed some of the potential candidate biomarker proteins for further validation. By using a systems biology approach, our study revealed novel proteins and molecular networks with altered expression in PCa. Further functional validation of individual proteins is ongoing and might provide new insights in PCa progression potentially leading to the design of novel diagnostic and therapeutic strategies.

Altered expression of tumor protein D52 regulates apoptosis and migration of prostate cancer cells.

Tumor protein D52 (TPD52) is a protein found to be overexpressed in prostate and breast cancer due to gene amplification. However, its physiological function remains under investigation. In the present study, we investigated the response of the LNCaP human prostate carcinoma cell line to deregulation of TPD52 expression. Proteomic analysis of prostate biopsies showed TPD52 overexpression at the protein level, whereas its transcriptional upregulation was demonstrated by real‐time PCR. Transfection of LNCaP cells with a specific small hairpin RNA giving efficient knockdown of TPD52 resulted in significant cell death of the carcinoma LNCaP cells. As demonstrated by activation of caspases (caspase‐3 and ‐9), and by the loss of mitochondrial membrane potential, cell death occurs due to apoptosis. The disruption of the mitochondrial membrane potential indicates that TPD52 acts upstream of the mitochondrial apoptotic reaction. To study the effect of TPD52 expression on cell proliferation, LNCaP cells were either transfected with enhanced green fluorescence protein‐TPD52 or a specific small hairpin RNA. Enhanced green fluorescence protein‐TPD52 overexpressing cells showed an increased proliferation rate, whereas TPD52‐depleted cells showed the reverse effect. Additionally, we demonstrate that exogenous expression of TPD52 promotes cell migration via αvβ3 integrin in prostate cancer cells through activation of the protein kinase B/Akt signaling pathway. From these results, we conclude that TPD52 plays an important role in various molecular events, particularly in the morphological diversification and dissemination of prostate carcinoma cells, and may be a promising target with respect to developing new therapeutic strategies to treat prostate cancer.

Effects of pharmacological inhibition of NADPH oxidase or iNOS on pro-inflammatory cytokine, palmitic acid or H2O2-induced mouse islet or clonal pancreatic β-cell dysfunction

Various pancreatic β-cell stressors including cytokines and saturated fatty acids are known to induce oxidative stress, which results in metabolic disturbances and a reduction in insulin secretion. However, the key mechanisms underlying dysfunction are unknown. We investigated the effects of prolonged exposure (24 h) to pro-inflammatory cytokines, H(2)O(2) or PA (palmitic acid) on β-cell insulin secretion, ATP, the NADPH oxidase (nicotinamide adenine dinucleotide phosphate oxidase) component p47phox and iNOS (inducible nitric oxide synthase) levels using primary mouse islets or clonal rat BRIN-BD11 β-cells. Addition of a pro-inflammatory cytokine mixture [IL-1β (interleukin-1β), TNF-α (tumour necrosis factor-α) and IFN-γ (interferon-γ)] or H(2)O(2) (at sub-lethal concentrations) inhibited chronic (24 h) levels of insulin release by at least 50% (from islets and BRIN-BD11 cells), while addition of the saturated fatty acid palmitate inhibited acute (20 min) stimulated levels of insulin release from mouse islets. H(2)O(2) decreased ATP levels in the cell line, but elevated p47phox and iNOS levels as did cytokine addition. Similar effects were observed in mouse islets with respect to elevation of p47phox and iNOS levels. Addition of antioxidants SOD (superoxide dismutase), Cat (catalase) and NAC (N-acetylcysteine) attenuated H(2)O(2) or the saturated fatty acid palmitate-dependent effects, but not cytokine-induced dysfunction. However, specific chemical inhibitors of NADPH oxidase and/or iNOS appear to significantly attenuate the effects of cytokines, H(2)O(2) or fatty acids in islets. While pro-inflammatory cytokines are known to increase p47phox and iNOS levels in β-cells, we now report that H(2)O(2) can increase levels of the latter two proteins, suggesting a key role for positive-feedback redox sensitive regulation of β-cell dysfunction.

Human agmatinase is diminished in the clear cell type of renal cell carcinoma

The proteome of RCC was analyzed by 2D PAGE to search for tumor‐associated proteins. Agmatinase, which hydrolyzes agmatine to putrescine and urea, was identified by mass spectrometry and database searches and shown to be downregulated in tumor cells. Additionally, RT‐PCR and Northern blot analyses demonstrated a clearly decreased amount of agmatinase mRNA in tumor cells. The differential expression of agmatinase mRNA was confirmed at the protein level. Western blot analysis showed almost no detectable agmatinase protein in tumor cells compared to corresponding normal renal tissue. Agmatinase mRNA is most abundant in human liver and kidney but expressed to a lesser extent in several other tissues, including skeletal muscle and small intestine. The human agmatinase gene encodes a 352‐residue protein with a putative mitochondrial targeting sequence at the N‐terminus. Using transfection and immunohistochemical studies, we show that agmatinase is localized in the mitochondria. Immunohistochemical studies revealed that agmatinase in the normal kidney is restricted to tubulus epithelial cells, while in tumors staining was low and heterogeneous. Thus, expression of human agmatinase is altered in RCC. We discuss the consequences of these findings in terms of polyamine, NO metabolism and macrophage function.

Proteomic Profiling of Germ Cell Cancer Cells Treated with Aaptamine, a Marine Alkaloid with Antiproliferative Activity

Aaptamine is a marine compound isolated from the sponge Aaptos aaptos showing antiproliferative properties via an undefined mode of action. We analyzed the effects of aaptamine treatment on the proliferation and protein expression of the pluripotent human embryonal carcinoma cell line NT2. Effects on proliferation, cell cycle distribution, and induction of apoptosis were analyzed. At lower concentrations, including the IC50 of 50 μM, aaptamine treatment resulted in a G2/M phase cell cycle arrest, whereas at higher concentrations, induction of apoptosis was seen. Differentially expressed proteins were assessed by 2D-PAGE and mass spectrometry, followed by verification and analysis of protein modifications of the most significantly up- and down-regulated proteins. Aaptamine treatment at the IC50 for 48 h resulted in alteration of 10 proteins, of which five each showed up- and down-regulation. Changes in the 2D map were frequently noticed as a result of post-transcriptional modifications, e.g., of the hypusine modification of the eukaryotic initiation factor 5A (eIF5A). Observed alterations such as increased expression of CRABP2 and hypusination of eIF5A have previously been identified during differentiation of pluripotent cells. For the first time, we describe changes in protein expression caused by aaptamine, providing valuable information regarding the mode of action of this compound.