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T Goldmann

Publications and source records attributed to T Goldmann.

At least 19 recordsLinked to original sources
PubMed2006

Impact of extraction conditions on the content of acrylamide in model systems and food.

It has recently been suggested that the analytical methods that have been developed to date for the determination of acrylamide (AA) may underestimate the concentration of AA in certain foods, because significantly higher results were obtained upon extraction of the food matrix under alkaline conditions. The present study employs food (potato, rye) and chemical model systems to better understand the tentative release of AA under high pH extraction conditions. The experimental design is based on the generation of AA in an environment containing an AA-isotopomer, and by comparing the ratio of AA, respectively the AA-isotopomer, after extraction at pHs 7 and 12. The results show that the additional AA released is not due to improved extractability of AA from the food matrix, and should therefore be regarded as an extraction artefact. Strongly alkaline conditions seem to induce net formation of AA from water-soluble precursors formed during thermolysis.

Acrylamide↗
PubMed2006

The HOPE technique opens up a multitude of new possibilities in pathology.

Fixation of tissues with formalin results in well-preserved morphology but to a high degree leads to degradation of nucleic acids, which substantially constricts the spectrum of applicable molecular techniques. The novel HOPE-fixative with subsequent paraffin embedding, as an alternative to formalin, has been shown to result in a morphological preservation comparable to formalin-fixed, paraffin-embedded specimens. Due to a similar workflow like in formalin-fixation and paraffin embedding, the HOPE technique can be successfully established within any pathological institute. We have shown that DNA, RNA and proteins are protected in HOPE-fixed, paraffin-embedded tissues for at least eight years. Moreover, we described procedures which permit successful application of all common molecular techniques such as in situ hybridization targeting either DNA or RNA, immunohistochemistry without antigen retrieval and for formalin-refractory antigens, PCR, RT-PCR, Western blot, Northern blot, and transcription microarrays to HOPE-fixed, paraffin-embedded tissues. Furthermore, HOPE-fixed tissues can be used for the construction of tissue microarrays for enhanced high-throughput analyses on the molecular level. Using the HOPE technique as its crucial methodological base, ex vivo model systems could be established, e.g. for the simulation of early events in human infections and detection of chemotherapy resistances in human cancer. In addition to tissues, cell-culture preparations have been prepared utilizing the HOPE technique, which were then successfully applied to in situ hybridization targeting mRNA or immunocytochemistry with excellent preservation of morphological details. Taken together, the HOPE technique to date represents an alternative fixation that is, in contrary to other procedures, scientifically broadly analyzed. Therefore new possibilities are opened up especially within the rapidly growing field of molecular pathology.

Fixatives↗
PubMed2005

Mechanisms of acrylamide formation: Maillard-induced transformation of asparagine.

The formation of acrylamide (AA) from L-asparagine was studied in Maillard model systems under pyrolysis conditions. While the early Maillard intermediate N-glucosylasparagine generated approximately 2.4 mmol/mol AA, the Amadori compound was a less efficient precursor (0.1 mmol/mol). Reaction with alpha-dicarbonyls resulted in relatively low AA amounts (0.2-0.5 mmol/mol), suggesting that the Strecker aldehyde pathway is of limited relevance. Similarly, the Strecker alcohol 3-hydroxypropanamide generated low amounts of AA (0.2 mmol/mol). On the other hand, hydroxyacetone afforded more than 4 mmol/mol AA, indicating that alpha-hydroxycarbonyls are more efficient than alpha-dicarbonyls in transforming asparagine into AA. The experimental results are consistent with the reaction mechanism proposed, i.e. (i) Strecker-type degradation of the Schiff base leading to azomethine ylides, followed by (ii) beta-elimination of the decarboxylated Amadori compound to release AA. The functional group in beta-position on both sides of the nitrogen atom is crucial. Rearrangement of the azomethine ylide to the decarboxylated Amadori compound is the key step, which is favored if the carbonyl moiety contains a hydroxyl group in beta-position to the N-atom. The beta-elimination step in the amino acid moiety was demonstrated by reacting under pyrolysis conditions decarboxylated model Amadori compounds obtained by synthesis.

Acetone↗
PubMed2005

Improved detection of mycobacterial DNA by PCR in formalin-fixed, paraffin-embedded tissues using thin sections.

PCR is a unique methodology allowing for the sensitive detection of mycobacterial DNA-sequences in cases in which no fresh material can be obtained for classic analyses. Despite the limitations of this technique, for example the less satisfactory quality of DNA from paraffin-embedded specimens and the high effort necessary to control contamination, PCR still represents a useful additional tool for routine diagnostic examinations of mycobacterial infections. Fragmentation of the DNA extracted from formalin-fixed, paraffin-embedded samples on the one hand and the rigid cell wall of mycobacteria on the other hand are obstacles to detecting the DNA of these microorganisms by PCR. Here, we describe a simple mechanical procedure that allows us to improve the detection of mycobacterial DNA with the use of thin (1 microm) sections instead of thicker sections. This could be explained by a gentle, mechanical opening of the acid fast mycobacterial cell wall. Thus, even the application of heat/cold shock treatments is not necessary. This inexpensive fast procedure can also be used for the detection of other infectious agents.

DNA, Bacterial↗
PubMed2005

Phenotype and genotype associations of lung carcinoma with atypical adenomatoid hyperplasia, squamous cell dysplasia, and chromosome alterations in non-neoplastic bronchial mucosa.

The frequency of preneoplastic lesions of the lung and bronchial mucosa as well as potential genotype alterations in spatial relationship to pulmonary malignancies still need intensive investigations in order to understand the occurrence and manifestation of lung cancer in detail. To investigate the contemporary manifestation of lung cancer precursor lesions, peripheral (non-neoplastic) lung parenchyma and bronchial mucosa of operated lung carcinomas were analyzed at distinct distances (1, 2, 3, and 4 cm) from the tumor boundary for pre-neoplastic lesions--atypical adenomatoid hyperplasia (AAH) and squamous cell dysplasia (SCD), in 150 surgical specimens. Short-term tissue cultures of additional 55 primary and secondary lung tumors and their surrounding non-neoplastic bronchial mucosa were performed at the same distances in order to search for chromosome alterations, i.e. genotype aberrations. In phenotype observations, atypical adenomatoid hyperplasia was noted in 19/150 (13%) cases, and squamous cell dysplasia in 46/150 (31%) cases. The degree of cellular atypia decreased with increasing distance from the tumor boundary in both AAH and SCM. AAH was observed more frequently in adenocarcinomas, SCQ more frequently in squamous cell carcinomas. In genotype observations, the average number of abnormal metaphases measured 4.5/10 high power fields (HPF) in primary lung carcinomas, and only 2/10 in metastases. Data indicate that the so-called preneoplastic lesions in the lung are not completely tumor-precursor lesions, but, in addition, induced by the tumor itself.

Bronchi↗
PubMed2004

HOPE technique enables Western blot analysis from paraffin-embedded tissues.

In contrast to the spectrum of biochemical analyses of fresh material, that of archived specimens is widely restricted. Fixation of specimens with formalin, the most commonly used fixative, usually prevents further molecular analysis, since it leads to degradation of nucleic acids and denaturation of the antigenic determinants of proteins. To overcome these problems, the Hepes-glutamic acid buffer mediated Organic solvent Protection Effect (HOPE)-fixation technique has been developed, which preserves nucleic acids and antigenic determinants of proteins, thus expanding the applicability of immunohistochemical methods. In this study, we investigated whether HOPE-fixed tissue can be analyzed by Western blotting. Furthermore, a comparison with conventionally fixed and frozen material was made. The specimens used were tumor-free and obtained from lobectomies for lung cancer. All four antibodies tested, i.e., antibodies specific for focal adhesion kinase, surfactant protein A, PI-3-kinase, and IKKalpha, worked well if used for immunoblotting of HOPE-fixed and frozen tissue. By contrast, these antibodies showed no or only very weak specific binding if formalin-fixed specimens were analyzed. Our findings show that HOPE fixation maintains the antigenicity of proteins better than formalin fixation. The possibility for performing Western blotting with archived paraffin-embedded specimens extends the options for diagnostic and scientific analyses of fixed tissues.

Blotting, Western↗
PubMed2004

[Transcriptional activity of surfactant-apoproteins A1 and A2 in non small cell lung carcinomas and tumor-free lung tissues].

UNLABELLED: Objective of this study was to investigate the balance of surfactant-apoprotein A1 and A2 (SP-A1 and SP-A2) at transcriptional level in tumor and tumor-free lung-tissues of patients with non-small-cell lung-carcinomas. MATERIALS AND METHODS: Nonfixed snap-frozen tumor and tumor-free sections of lungs, excised from 21 patients with adenocarcinomas of the lung and 6 patients with squamous cell carcinomas (control group) were analyzed. This was accomplished by RT-PCR using a SP-A1/SP-A2 consensus sequence, followed by enzymatic restriction. RESULTS: Tumor-containing tissues of adenocarcinomas showed higher SP-A1/SP-A2 ratios than the tumor free tissues. There was a significant difference in expression of SP-A1/SP-A2-mRNA in intra-individual tumor and tumor-free lung-tissues of adenocarcinoma patients (p = 0,05). In cases of squamous cell carcinomas this was not observed. CONCLUSIONS: A novel method to investigate the transcriptional activity of surfactant-apoproteins A1 and A2 in snap-frozen lung tissues was established. It was shown, that adenocarcinoma-tissues display higher SP-A1/SP-A2 than the corresponding tumor-free tissues and that the variation of SP-A-mRNA expression rises in cases of higher tumor-grading.

Adenocarcinoma↗
PubMed2003

The classification and discrimination of glass fragments using non destructive energy dispersive X-ray microfluorescence.

Frequency of analytical characteristics is best estimated on glass recovered at random. However, as such data were not available to us, we decided to use control windows for this estimation. In order to use such a database, one has to establish that the recovered fragment comes from a window. Therefore, elemental analysis was used both for classification and discrimination of glass fragments. Several articles have been published on the subject, but most methods alter the glass sample. The use of non destructive energy dispersive X-ray microfluorescence (microXRF) for the analysis of small glass fragments has been evaluated in this context. The refractive index (RI) has also been measured in order to evaluate the complementarity of techniques. Classification of fragments has been achieved using Fisher's linear discriminant analysis (LDA) and neural networks (NN). Discrimination was based on Hotelling's T2 test. Only pairs that were not differentiated by RI followed by the Welch test were studied. The results show that neural network and linear discriminant analysis using qualitative and semi-quantitative data establishes a classification of glass specimens with a high degree of reliability. For discrimination, 119 windows collected from crime scene were compared: using RI it was possible to distinguish 6892 pairs. Out of 129 remaining pairs, 112 were distinguished by microXRF.

Journal Article↗
PubMed2003

[Progressive paralysis caused by radiation-induced cervical malignant peripheral nerve sheath tumor].

HISTORY AND DIAGNOSIS: A 59-year old engineer was admitted to the hospital because of pain in his right collar region and the onset of incomplete paresis of the right arm. Magnetic resonance tomography displayed an advanced tumour arising from the right paravertebral soft tissue. Histological examination revealed a malignant peripheral nerve sheath tumor (MPNST). Thirteen years before admission, the patient had a right-sided tumor-tonsillectomy of a squamous cell carcinoma and local radiation of a cystic squamous cell carcinoma in the ipsilateral cervical soft tissue. CLINICAL COURSE AND THERAPY: In the following course, progressive neurological symptoms occurred including beginning paraplegia, right phrenic paralysis and a severe concomitant pain syndrome. Due to the location and advanced tumor state, surgical treatment was not performed and palliative chemotherapy remained ineffective. Three months later the patient died due to rapidly progressive neurological failure. CONCLUSION: MPNST represents a rare entity which has been related to postoperative radiation. Unusual neurological symptoms in anatomical regions of former radiation should therefore include neurogenic secondary malignancies in the differential diagnosis for early surgical intervention.

Arm↗
PubMed2003

HOPE-fixation enables improved PCR-based detection and differentiation of Mycobacterium tuberculosis complex in paraffin-embedded tissues.

Standard PCR-based detection of mycobacterial DNA in paraffin-embedded specimens may lack sufficient sensitivity because of the degradation of nucleic acids caused by routinely used formalin fixation. Therefore, we set up an approach that aimed at improving the results by applying the novel HOPE-fixative in PCR-detection of mycobacteria in paraffin-embedded tissues. Comparison of PCR-results using DNA extracted from either HOPE- or formalin-fixed specimens in BCG-infected SCID-mice revealed a more than 100fold enhanced sensitivity for the HOPE-fixed material. Owing to the preservation of DNA from degradation in HOPE-fixed tissues, even differentiation within the M. tuberculosis complex was possible by spoligotyping. We therefore conclude that the HOPE-fixative is a useful tool for molecular pathology that enhances the sensitivity of PCR-based methods for the detection of pathogens in paraffin-embedded tissues compared to formalin-fixation. Owing to the better preserved DNA, improved differentiation of mycobacteria from archived materials is possible. These results promise new and a substantially wider range of possibilities in the field of molecular pathology.

Animals↗
PubMed2003

Alveolar macrophages are the main source for tumour necrosis factor-alpha in patients with sarcoidosis.

Tumour necrosis factor (TNF)-alpha is known to play a major role in the formation of noncaseating granuloma, a hallmark of sarcoidosis. The main cellular source in situ is still ambiguous. Serial sections of transbronchial biopsies from 14 patients with and 12 without sarcoidosis were studied, using immunohistochemistry (IHC), for TNF-alpha, T-cells (CD3), macrophages (CD68), and epithelial cells (MNF116). TNF-alpha spontaneously released (sr-TNF-alpha) by freshly isolated bronchoalveolar lavage cells, isolated from the same patients and cultured without any stimulus over a 24-h period was measured using an enzyme-linked immunosorbent assay. IHC revealed colocalisation of TNF-alpha with CD68 cells only. Cases with TNF-alpha tissue immunoreactivity exhibited higher sr-TNF-alpha (1,667 +/- 504 pg x mL(-1)) than cases without tissue immunoreactivity (211 +/- 60 pg x mL(-1)). In an explorative approach, a subgroup of patients could be identified and characterised by the presence of alveolar macrophage aggregates. It was found that sr-TNF-alpha was highest in this subgroup (2,700 +/- 769 pg x mL(-1)) compared with patients with normal histology (221 +/- 61 pg x mL(-1)) or with prominent granuloma (460 +/- 137 pg x mL(-1)), whereas in most clinical parameters this subgroup was intermediate. The findings from this study strongly corroborate the view that alveolar macrophages are the main cellular source for tumour necrosis factor-alpha in the initial phase of sarcoidosis. The authors suggest that in these patients, aggregates of alveolar macrophages may represent at least predecessors to granulomas if not granulomas in statu nascendi.

Adult↗
PubMed2003

Techniques of DNA-studies on prehispanic ectoparasites (Pulex sp., Pulicidae, Siphonaptera) from Animal mummies of the Chiribaya culture, southern Peru.

During a paleoparasitological survey of several animal mummies (Cavia aperea f. porcellus and Canis familiaris) from Chiribaya Baja, an archaeological site in Southern Peru, an unexpected find was made. In the well preserved fur, large numbers of mummified fleas (Pulex simulans/irritans) that parasitized the animals during life were encountered. Due to the relative recent event of the host mummification and the outstanding preservation of the fleas, an attempt for the retrieval of DNA was made. A DNA extraction and sequencing protocol for archaeological ectoparasitic remains has been established, taking additional studies for tissue and protein preservation into account. Tissue preservation was assessed with transmission electron microscopy and the protein preservation was tested through the racemisation ratios of aspartic acid. Regions of the 28S rDNA gene were successfully amplified and sequenced. Further research perspectives are outlined.

Animals↗
PubMed2002

Assessment of transcriptional gene activity in situ by application of HOPE-fixed, paraffin-embedded tissues.

We report the use of HOPE-fixation (HOPE = Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) for specimens utilized for in situ hybridization targeting mRNA. For this purpose, an optimized protocol was developed and repeatedly tested on HOPE-fixed lung specimens. We observed that neither pretreatment, permeabilizing the cells, nor prehybridization is necessary to generate signals. After deparaffinizing, the random primed digoxigenin-labeled probes are directly hybridized together with yeast tRNA for blocking unspecific signals. Detection was performed using anti digoxigenin antibodies conjugated with alkaline phosphatase and new-fuchsine or NBT/BCIP as substrates. The results were verified by RT-PCR and adequate negative controls. Signals for human surfactant protein-A and interferon-gamma-inducible protein-10 developed rapidly within 10 min, accompanied by high signal intensities comparable to those observed in immunohistochemistry. Signal enhancement by biotinyl-tyramide, although giving suitable results as well, did not lead to higher signal intensities, and thus was not necessary in conjunction with the probes tested so far. These experiments were performed with material stored under appropriate conditions (at +4 degrees C) up to five years. To sum up, these initial results, obtained with the novel HOPE-fixative, are promising as regards the enhancement of the capabilities of in situ hybridization in the future.

Cells, Cultured↗
PubMed2002

Pattern of NOS2 and NOS3 mRNA expression in human A549 cells and primary cultured AEC II.

The human alveolar type II epithelium-like cell line A549 expresses nitric oxide synthase type 2 (NOS2), but not NOS3, and produces nitric oxide (NO) upon appropriate stimulation. However, relatively little is known regarding the NOS2 and NOS3 expression of type II human alveolar epithelial cells (AEC II) in primary culture. We detected NOS3 mRNA in freshly isolated AEC II and after 24 h of culture. NOS3 mRNA levels were much higher in AEC II cultured for 24 h with or without interferon-gamma, interleukin-1beta, and tumor necrosis factor-alpha, compared with freshly isolated cells. Cytokine stimulation did not change the NOS3 mRNA expression level in AEC II compared with unstimulated cells. NOS3 protein expression was verified by Western blot, and measuring nitrate/nitrite revealed that the protein is active. In contrast, neither NOS2 mRNA nor protein could be detected in freshly isolated, unstimulated or cytokine-stimulated human AEC II in 24- or 72-h primary cultures, whereas A549 cells expressed NOS2 message and protein upon stimulation with proinflammatory cytokines. In situ hybridization confirmed that AEC II express NOS3, but not NOS2 mRNA in vivo. These data demonstrate that there are significant differences between primary AEC II and A549 cells in NOS mRNA expression pattern.

Adenocarcinoma↗
PubMed2002

Human alveolar epithelial cells induce nitric oxide synthase-2 expression in alveolar macrophages.

It was hypothesized that cell-to-cell interaction between human alveolar macrophages (AM) and alveolar epithelium, might be an important factor leading to nitric oxide synthase-2 (NOS2) messenger ribonucleic acid (mRNA) and protein expression by constituent cells of the alveolar wall and/or AM. NOS2 mRNA and the protein expression patterns of human AM and alveolar epithelial cells type II (AEC-II) isolated from normal parts of lung resections of patients with pulmonary malignancies were determined. In addition, NOS2 mRNA expression in human AM co-cultured with autologous AEC-II in the presence of pro-inflammatory cytokines interleukin (IL)-1beta, tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma or lipopolysaccharide (LPS) was investigated. The effect of human surfactant protein-A (SP-A) on IFN-gamma-mediated NOS2 mRNA expression in human AM was also studied. Neither NOS2 mRNA nor protein could be detected in freshly isolated, unstimulated or cytokine-stimulated AEC-II. In contrast, freshly isolated AM from bronchoalveolar lavage or lung tissue samples expressed immunoreactivity for NOS2 protein, but no NOS2 mRNA could be detected by reverse transcriptase polymerase chain reaction. All stimuli tested failed to induce NOS2 mRNA expression in human AM in vitro. Only AM-AEC-II co-culture in the presence of IFN-gamma led to NOS2 mRNA and protein expression. In situ hybridization of NOS2 mRNA on lung tissue explants and immunohistochemical staining of cytospin preparations of AM-AEC-II co-cultures demonstrated that NOS2 is expressed in AM but not in AEC-II. This co-culture effect could not be reproduced by substitution of AEC-II with SP-A. These data give evidence of a regulatory network controlling human nitric oxide synthase-2 expression in the lower respiratory tract.

Blotting, Western↗
PubMed2002

Ventilation-induced activation of the mitogen-activated protein kinase pathway.

Mechanical ventilation of patients can be a life-saving treatment, but also imposes additional stress on the lung. Mitogen-activated protein kinases (MAPK) represent a family of protein kinases that become phosphorylated and activated by many different forms of stress. Using Western blot analysis, the present study analysed the effects of high distending pressure ventilation on the activation of the MAPK extracellular signal-related kinases (ERK)-1/2, c-Jun amino-terminal kinases (JNK) and p38 kinase, and on the MAPK-activated transcription factors c-Jun, ETS-like protein (Elk)-1 and activating transcription factor (ATF)-2. In adult rats, ventilation with high pressure (45/10 peak inspiratory pressure/positive end-expiratory pressure in cmH2O) for 30 or 60 min did not affect arterial oxygenation, but resulted in enhanced phosphorylation of ERK-1/2, JNK, c-Jun, Elk-1 and ATF-2 compared to normally ventilated (13/3) rats. The activation of ERK-1/2 and JNK was located to cells resembling alveolar type II cells. In addition, high pressure ventilation enhanced phosphorylation of the inhibitor of nuclear factor (NF)-kappaB and nuclear translocation of the transcription factor NF-kappaB. In isolated perfused mouse lungs, the MAPK/ERK kinase inhibitor U0126 prevented ventilation-induced activation of ERK-1/2 and Elk-1, but had no effect on ventilation-induced cytokine release. The present authors conclude that mechanical ventilation triggers specific signalling pathways, such as the mitogen-activated protein kinase and the nuclear factor-kappaB pathways, which may contribute to pulmonary inflammation and proliferation.

Analysis of Variance↗
PubMed2001

Cost-effective gel documentation using a web-cam.

In search for a cost effective gel documentation system applicable for different fields of molecular biology, we analyzed the capabilities of a cheap CCD-camera originally designed to capture images for transmission through the internet (web-cam) with regard to gel documentation. The camera was connected to a personal computer by universal serial bus (USB) and used for the documentation of DNA separated on agarose gels and stained by ethidium-bromide using the software provided with the camera. The web-cam provided digital images of sufficient quality for routine documentation and combined the low set-up costs of a Polaroid system with the low running costs of video capture systems, hence is ideal as a start-up system and as augmentation to existing equipment.

Computers↗