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Biomedical subjects

K Berlin

Publications and source records attributed to K Berlin.

13 recordsLinked to original sources
PubMed2005

Methylation discriminators in NSCLC identified by a microarray based approach.

Aberrant DNA methylation is a frequent phenomenon in non-small cell lung cancers. We have used a microarray approach to assess the methylation status of 245 CpG positions in 59 candidate genes in 26 squamous cell carcinomas, and 22 adenocarcinomas as well as 26 normal adjacent lung tissue samples from smokers to identify genes that show a distinct methylation status difference between the two different tumour type tissues and normal adjacent tissue. Tumour tissue samples were grouped together and compared to the normal tissue sample group. A multivariate test was performed, taking into account all CpG positions that were analyzed for a particular gene, to calculate p-values for each gene based on the observed methylation difference between the two groups, p-values obtained were corrected for multiple testing. The highest degree of differential DNA methylation in squamous cell carcinoma compared to normal was observed in ARHI, MGMT, GP1bbeta, RARbeta and TMEFF2 genes, while TMEFF2, MGMT and CDKNIC genes differentiated between adenocarcinomas and normal tissue. It is of note that some of the genes for which differential methylation status was observed, have not been previously described in lung cancer. Our results provide compelling evidence that different histological types of lung cancer may be distinguished from normal tissue based on methylation profiles of specific genes.

Adenocarcinoma↗
PubMed2000

Full flexibility genotyping of single nucleotide polymorphisms by the GOOD assay.

Recently a facile method for genotyping single nucleotide polymorphisms (SNPs) using MALDI mass spectrometry, termed the GOOD assay, was developed. It does not require any purification and is performed with simple liquid handling, thermal incubation and cycling steps. Although this method is well suited to automation and high-throughput analysis of SNPs, it did not allow full flexibility due to lack of certain reagents. A complete set of ss-cyanoethyl phosphoramidites is presented herein that give this SNP genotyping method full sequence and multiplex capabilities. Applications to SNP genotyping in the prion protein gene, the ss-2-adrenergic receptor gene and the angiotensin converting enzyme gene using the GOOD assay are demonstrated. Because SNP genotyping technologies are generally very sensitive to varying DNA quality, the GOOD assay has been stabilised and optimised for low quality DNA. A template extraction method is introduced that allows genotyping from tissue that was taken while placing an ear tag on an animal. This dramatically facilitates the application of genotyping to animal agricultural applications, as it demonstrates that expensive and cumbersome DNA extraction procedures prior to genotyping can be avoided.

Animals↗
PubMed2000

A novel procedure for efficient genotyping of single nucleotide polymorphisms.

Due to the surge in interest in using single nucleotide polymorphisms (SNPs) for genotyping a facile and affordable method for this is an absolute necessity. Here we introduce a procedure that combines an easily automatable single tube sample preparation with an efficient high throughput mass spectrometric analysis technique. Known point mutations or single nucleotide polymorphisms are easily analysed by this procedure. It starts with PCR amplification of a short stretch of genomic DNA, for example an exon of a gene containing a SNP. By shrimp alkaline phosphatase digest residual dNTPs are destroyed. Allele-specific products are generated using a special primer, a conditioned set of alpha-S-dNTPs and alpha-S-ddNTPs and a fresh DNA polymerase in a primer extension reaction. Unmodified DNA is removed by 5'-phospho-diesterase digestion and the modified products are alkylated to increase the detection sensitivity in the mass spectrometric analysis. All steps of the preparation are simple additions of solutions and incubations. The procedure operates at the lowest practical sample volumes and in contrast to other genotyping protocols with mass spectrometric detection requires no purification. This reduces the cost and makes it easy to implement. Here it is demonstrated in a version using positive ion detection on described mutations in exon 17 of the amyloid precursor protein gene and in a version using negative ion detection on three SNPs of the granulocyte-macrophage colony stimulating factor gene. Preparation and analysis of SNPs is shown separately and simultaneously, thus demonstrating the multiplexibility of this genotyping procedure. The preparation protocol for genotyping is adapted to the conditions used for the SNP discovery method by denaturing HPLC, thus demonstrating a facile link between protocols for SNP discovery and SNP genotyping. Results corresponded unanimously with the control sequencing. The procedure is useful for high throughput genotyping as it is required for gene identification and pharmacogenomics where large numbers of DNA samples have to be analysed. We have named this procedure the 'GOOD Assay' for SNP analysis.

Amyloid beta-Protein Precursor↗
PubMed1999

Analysis of negatively 'charge tagged' DNA by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

An improvement in detectability and stability of DNA analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) using oligonucleotides modified with a neutralized backbone and a fixed single, positive charge was recently reported. The attachment of the positive charge requires a primary amino group, limiting this approach to accordingly functionalized DNA. The method described here uses backbone neutralized DNA for the same purpose, with a single unmodified phosphate in the DNA backbone carrying the negative charge. Thus, no chemical modification other than neutralizing the remaining charges on the phosphorothioates is required. This is performed in a single methylation step. The enhancement in sensitivity is comparable to that for DNA carrying a single positive charge, interestingly even when using the same non-protonating matrix. The mechanistic implications of these findings regarding the MALDI process are discussed. The DNA derivatization methods presented help to make MALDI-MS of DNA applicable and competitive for genome analysis and medical diagnostics.

DNA↗
PubMed1998

Are porphyrin mixtures favorable photodynamic anticancer drugs? A model study with combinatorial libraries of tetraphenylporphyrins.

Reported here is the preparation of tetraphenylporphyrin libraries via efficient combinatorial solution-phase syntheses, their purification, and preliminary results from a bioorganic study on their uptake in liposome membranes. Libraries with up to 666 components were prepared with substituents including Br, CF3, Cl, CN, CO2Me, Et, F, OAc, and Ph. Further, a first example for the synthesis of more diverse libraries via a "latent libraries" approach is presented. This involves masking polar groups with lipophilic protecting groups. After purification of the latent library, the masking protecting groups are removed in a quantitative reaction that produces the library compounds as the only non-volatile components. Libraries were characterized by laser desorption time-of-flight mass spectrometry, NMR, and UV-vis spectroscopy. In vitro uptake into membranes of small sonicated liposomes was measured, both in terms of total porphyrin incorporation and in terms of structure-incorporation relationships. The latter were determined from isotopically-resolved laser-desorption mass spectra under conditions that yield quantitative results. Smaller libraries showed increased uptake of porphyrins bearing OH and CF3 substituents and lower uptake of ester-, alkyl-, and halide-bearing porphyrins. This structure-dependent selectivity disappears for larger libraries, however, where uniformly high uptake is observed, i.e., at a constant lipid:porphyrin ratio the total porphyrin incorporation is higher for libraries than for single compounds of similar polarity. We propose that the decreased concentration of individual compounds in large libraries is responsible for this effect. Membrane incorporation has previously been shown to correlate with photodynamic activity in vitro and in vivo.16 Therefore, these results may help to explain why photodynamic therapy of tumors, a modern anti-cancer treatment modality, is successfully performed with a complex mixture of porphyrins.

Antineoplastic Agents↗
PubMed1998

Functional status and well-being in patients with glaucoma as measured by the Medical Outcomes Study Short Form-36 questionnaire.

PURPOSE: This study aimed to determine whether patients with glaucoma have different functional status and well-being than patients without glaucoma. DESIGN: Prospective case-control study. PARTICIPANTS: The study population was recruited from 2 university-based glaucoma clinical practices and a university-based general ophthalmology clinic and consisted of 121 patients with open-angle glaucoma, 42 with diagnosis of glaucoma suspect, and 135 with no chronic ocular conditions except cataract. INTERVENTION: Administration of Medical Outcomes Study 36-item short-form survey (SF-36) was performed. Demographic information, medical history, and responses to the SF-36 questionnaire were elicited by an interviewer. Medical record review was performed to obtain clinical examination data and to substantiate the medical and demographic data obtained by the interviewer. MAIN OUTCOME MEASURES: The SF-36 scores by diagnostic group, demographic characteristics, and medical history were examined. Secondary outcome measures were SF-36 scores in patients with glaucoma by visual field impairment and glaucoma medication use. RESULTS: Patients with glaucoma consistently had lower scores, control subjects had higher scores, and glaucoma suspects had scores intermediate between the two groups. After adjusting for the possible influence of all the other covariate factors, glaucoma was found to be a strong predictor of lower SF-36 scores. CONCLUSION: Patients with glaucoma have lower scores, indicating less-functional status, than patients without glaucoma as tested by the SF-36 survey questionnaire.

Adult↗
PubMed1998

Apolipoprotein E isoforms increase intracellular Ca2+ differentially through a omega-agatoxin IVa-sensitive Ca2+-channel.

Apolipoprotein E (apoE) is the major apolipoprotein in the brain and is known for its important role in plasticity and neurodegeneration. We show that apoE dose-dependently increases intracellular free Ca2+ in rat hippocampal astrocytes and neurons. This effect varies with isoforms in the order E4 > E3 > E2. It is insensitive to blockade of action potentials by tetrodotoxin or inhibition of binding of apoE by heparinase, by the LRP ligand lactoferrin and by low density lipoprotein. ApoE evoked Ca2+-increases are blocked in zero [Ca]o and by the Ca-channel antagonists nickel and omega-Agatoxin-IVa but not by nifedipine and omega-Conotoxin-GVIa, demonstrating an isoform-specific activation of P/Q type Ca2+-channels. This novel mechanism is discussed with respect to Alzheimer's disease, that is linked for most cases to the apoE epsilon-allelic variation (epsilon4 > epsilon3 > epsilon2).

Animals↗
PubMed1997

Spectrometrically monitored selection experiments: quantitative laser desorption mass spectrometry of small chemical libraries.

BACKGROUND: Selection experiments involving chemical libraries are routinely used in the pharmaceutical industry for finding and optimizing lead compounds. In principle, almost any process involving a binding event or a reaction could be probed systematically with chemical libraries prepared by combinatorial synthesis. Traditionally, however, the vast majority of library members cannot be monitored during the selection, making a systematic correlation of structure and activity difficult. To interpret selection experiments on the level of all library components, monitoring technologies are required that give a unique and quantitative spectroscopic signal for every compound in a mixture. RESULTS: Quantitative matrix-assisted laser desorption mass spectrometry of libraries of porphyrins and peptide-DNA hybrids consisting of 2-35 compounds is described. Porphyrin libraries were subjected to in vitro selections for liposome incorporation and binding to a protein pocket. It was shown that mesohydroxyphenyl substituted porphyrins, known high activity photosensitizers of tumors, are preferentially incorporated in liposome membranes. A mixture of peptide-DNA hybrids was assayed for the nuclease stability of its components. CONCLUSIONS: Small libraries of non-isobaric compounds can be exhaustively or near-exhaustively monitored by mass spectrometry. Monitored selection experiments can yield detailed structure-activity maps in a single experiment, speeding up drug discovery and the probing of biochemically relevant recognition events. It is proposed that monitored assays for target binding, membrane partitioning, and biostability could be run in parallel, to select drug candidates combining several favorable properties in 'multidimensional' selection experiments.

DNA↗
PubMed1997

Cholecystokinin activates CCKB-receptor-mediated Ca-signaling in hippocampal astrocytes.

Cholecystokinin-8S (CCK-8S) is the most abundant neuropeptide in the mammalian cortex and the limbic system; however, its physiological functions remained largely obscure. We studied effects of CCK on astrocytic Ca signaling, which has met considerable interest as a second messenger in astrocytic-neuronal signaling, by digital ratio-imaging of Fura-2/AM loaded rat and mouse hippocampal astrocytes in dissociated culture. Superfusion of CCK-8S (5-50 nM for 2 min) evoked repetitive Ca increases of several hundred nanomolar in a subpopulation of astrocytes. Mouse astrocytes appeared to be more responsive to CCK than rat cells with respect to the fraction of cells responding as well as to the amplitudes of Ca increases. The Ca responses persisted in the absence of extracellular Ca, indicating that release of Ca from intracellular stores is the primary source of these Ca increases. The CCK-8S-induced Ca increases were blocked by the CCKB receptor antagonist PD135158 (100 nM) but not by the CCKA antagonist lorglumide (100 nM). We surmise that astrocytes might be a major primary target for CCK in the CNS.

Animals↗
PubMed1996

Effects of cholecystokinin on Y, X, and W cells in the dorsal lateral geniculate nucleus of rats.

The role of the cholecystokinergic input to the rat's dorsal lateral geniculate nucleus (dLGN) was studied by examining the effect of iontophoretically administered CCK-8S on the neuronal response to stimulation of the receptive field center. Peristimulus activity was recorded extracellularly from 108 neurons grouped according to the type of receptive field (OFF, ON, or ON-OFF) and classified with respect to their Y, X, or W properties by means of discriminant analysis. CCK affected the response to a center-sized spot of light in two thirds of the neurons investigated. The center response decreased in 50 of 73 CCK-sensitive neurons (69%), predominantly in Y OFF and X OFF center cells (17 of 19). In the remaining 23 cells the center response increased, most consistently (11 of 17) in W ON center cells. Center and surround responses were similarly influenced. Inhibitions and excitations induced by CCK-8S were reproducible, dose dependent, and receptor mediated. The CCKB antagonist PD 135158 reduced the CCK effects in 10 of 14 cells; the CCKA antagonist KL 1001 reduced the CCK effects in 17 of 36 cells. The CCK-induced inhibition was B-receptor specific in 4 of 8 cells, A-receptor specific in 2 of 8 cells, and partially mediated by each of the two types of receptor in the remaining 2 cells. Blocking by the CCKA antagonist was more frequently observed in W cells than in cells with Y or X characteristics. The data show that CCK modifies the activity of dLGN cells in a variable direction depending on the specific cell type (Y, X, W) and response pattern (OFF, ON). The effects of CCK are discussed in relation to proposed functions of the superior collicular input to the dLGN.

Animals↗
PubMed1995

Lead intoxication caused by skeletal disease.

BACKGROUND: Inorganic lead is accumulated in the skeleton, which harbors more than 90% of the body burden of lead. If rapidly mobilized, this pool may constitute a health risk. However, clear evidence of this theory has been lacking. HISTORY: A previously healthy 36-year-old Swedish man with more than 10 years of work-related lead exposure developed headache, musculoskeletal pain, and paresthesia of both arms. Two months after the cessation of exposure, the lead level in his blood (B-Pb) was 5.5 mumol.l-1, and treatment with chelating agents was started. Shortly after the treatment period, he had an accident causing a fracture of the right collum femoris. The B-Pb was fairly stable around 1.5 mumol.l-1 for about two years after the end of exposure (95th percentile 0.6 mumol.l-1 for occupationally unexposed Swedish men). The examination showed that the patient had high skeletal turnover and clearly reduced bone density, as well as signs of tubular dysfunction. He was given the diagnosis idiopathic osteoporosis. His moderately raised bone lead concentration (about 20 micrograms.g wet weight-1; normal level in Sweden 4 micrograms.g-1) can only partly explain the raised B-Pb, remaining for years after the cessation of exposure. Instead, the main explanation is probably the increased skeletal turnover. CONCLUSIONS: A combination of a moderately increased bone lead pool and skeletal disease seems to increase the risk for lead poisoning.

Adult↗
PubMed1995

Cancer incidence and mortality of patients with suspected solvent-related disorders.

OBJECTIVE: The aim of this study was to study the incidence of cancer and deaths from cancer and other diseases among patients referred to the 11 clinics of occupational medicine in Sweden between 1967 and 1987 for examination because of exposure to organic solvents. METHODS: The cohort comprised 5791 persons, 5283 men and 508 women. Information about cancer incidence and causes of death was collected from the Cancer Register of the National Board of Health and Welfare and the National Death Register of Statistics Sweden, respectively. The expected values were calculated from the national death rates and incidence rates of cancer. RESULTS: The overall mortality rate was close to expected, but the mortality rate was decreased for diseases of the circulatory system [standardized mortality ratio (SMR) 0.7, 95% confidence limit (95% CI) 0.5-0.9] and increased for suicide (SMR 2.0, 95% CI 1.2-3.2). The total cancer incidence was slightly elevated [standardized incidence ratio (SIR) 1.2, 95% CI 0.99-1.4], and some specific cancer sites showed an increased incidence, although the lower confidence limits surpassed one. Malignancies of the lymphohematopoietic system and cancer of the uterine cervix had an increased risk (SIR 1.9, 95% CI 1.2-3.2, and SIR 3.7, 95% CI 2.2-6.2, respectively). Patients with presumerably high solvent exposure had an SIR of 1.4 for all malignancies (95% CI 0.9-2.1) and those with presumerably low exposure had an SIR of 1.1 (95% CI 0.9-1.4). CONCLUSIONS: The study showed an increased risk for malignancies of the hematopoietic system and the uterine cervix among patients originally examined with regard to solvent-induced disorders. There was also an increased risk of suicide and a decreased risk of death from diseases of the circulatory system. There was no increased risk for deaths from mental or neurological disorders.

Adult↗