Nicotinamide adenine dinucleotide a unique compound for theoretical and synthetic model studies: chirality as source for high stereospecificity

A dynamic model is given for the hydride transfer of the redox couple NAD+-NADH with model systems and quantum chemical calculations.     

In vitro evidence for senescent multinucleated melanocytes as a source for tumor-initiating cells

Oncogenic signaling in melanocytes results in oncogene-induced senescence (OIS), a stable cell-cycle arrest frequently characterized by a bi- or multinuclear phenotype that is considered as a barrier to cancer progression. However, the long-sustained conviction that senescence is a truly irreversible process has recently been challenged. Still, it is not known whether cells driven into OIS can progress to cancer and thereby pose a potential threat. Here, we show that prolonged expression of the melanoma oncogene N-RAS^61K in pigment cells overcomes OIS by triggering the emergence of tumor-initiating mononucleated stem-like cells from senescent cells. This progeny is dedifferentiated, highly proliferative, anoikis-resistant and induces fast growing, metastatic tumors. Our data describe that differentiated cells, which are driven into senescence by an oncogene, use this senescence state as trigger for tumor transformation, giving rise to highly aggressive tumor-initiating cells. These observations provide the first experimental in vitro evidence for the evasion of OIS on the cellular level and ensuing transformation.Cellular senescence is characterized by cell-cycle arrest and alterations in cell shape and metabolism, and can be triggered either by the sequential loss of telomeres or by numerous forms of cellular stress, for example, UV irradiation, oxidative stress or aberrant oncogenic signaling (premature senescence). In particular, oncogene-induced senescence (OIS), driven for example by activated RAS or BRAF, is an anti-cancer protection mechanism that prevents tumor generation despite the presence of oncogenic mutations. For instance, human nevi exhibit enhanced MAPK signaling caused by activating mutations in B-RAF or N-RAS. They display classical characteristics of senescence,¹ and remain benign in the large majority of cases. However, nevi are also supposed to give rise to a quarter of all melanomas.²Along the same lines, oncogenic RAS clearly triggers OIS in different cell types in vivo,^{3, 4, 5, 6} but activated RAS is detected in up to 30% of human cancers.^{7, 8} This indicates that senescence bypass is a key feature of cancer development. The fact that many premalignant tissues with tumorigenic potential display features of senescence has led to the concept that OIS precedes transformation, and tumors arise from senescent tissue.^{1, 5, 6, 9} However, as OIS was long considered to be irreversible, it was not clear how this transformation process can take place. Recently, there has been accumulating evidence that OIS can be reversed under certain circumstances on the cellular level. Specifically, H-RAS^12V induces senescence in fibroblasts, which is caused by ribonucleotide reductase (RRM2) suppression and accompanying dNTP reduction.¹⁰ Interestingly, the forced re-expression of RRM2 is able to overcome senescence. Still, is not known whether a senescent primary cell might give rise to cancer. While the later steps of tumor progression are fairly well understood, early events in tumorigenesis such as the transition of a benign senescent lesion to a tumor are still enigmatic.Here, we reveal that long-term NRAS^61K activation in melanocytes triggers a strong senescent phenotype characterized by multinucleation, which then is followed by the post-senescence generation of tumor-initiating cells with stem cell-like properties. The results demonstrate that senescence in melanocytes can be overcome on the cellular level and can also be a source for malignant cancer cells.     

The dried corncob as a source of DNA for PCR analysis

We developed a procedure to isolate DNA from dried corncobs. This DNA was amplified successfully by PCR, producing well-defined bands in response to specific primers. The SSR patterns between cob and leaf DNA of the same inbred line were found to be identical, indicating that the DNA in the cob cells had not degraded during senescence.     

Further evidence for stroma lamellae as a source of Photosystem 1 fractions from spinach chloroplasts

The mechanism of digitonin action on spinach chloroplasts was investigated by thin sectioning. Evidence is presented which shows that digitonin continues to modify membranes for many minutes after the addition of the fixative glutaraldehyde. However, the action of digitonin can be stopped by simultaneous fixation and dilution of the detergent. Such experiments indicate that the initial action of digitonin is to release stroma lamellae which in turn yield a Photosystem 1 fraction. This interpretation is further supported by a significant correlation between the chlorophyll a/chlorophyll b ratio and the ratio of stroma to grana lamellae in spinach chloroplasts.     

Buccal cells as a source of DNA for comparative animal genomic analysis

The source of DNA of adequate quality and quantity is an important consideration in genome analysis. In many animal and livestock species, easy access to DNA will facilitate the rapid and reliable genotyping of a large number of individual individuals. Here, we describe the use, for the first time, of buccal cells from non-human mammalian species as a source of DNA template for PCR and restriction analysis. The buccal cells from the pig, cow and human, were used to amplify PCR fragments that were scanned SNPs and for comparative genome analysis. The work indicates that buccal cells are also adequate sources of DNA for genome analysis of animals that have been identified as priorities in comparative genomics.     

Experimental evidence for allosteric modifier saturation as predicted by the bi-substrate Hill equation

The cooperative enzyme reaction rates predicted by the bi-substrate Hill equation and the bi-substrate Monod-Wyman-Changeux (MWC) equation when allosterically inhibited are compared in silico. Theoretically, the Hill equation predicts that when the maximum inhibitory effect at a certain substrate condition has been reached, an increase in allosteric inhibitor concentration will have no effect on reaction rate, that is the Hill equation shows allosteric inhibitor saturation. This saturating inhibitory effect is not present in the MWC equation. Experimental in vitro data for pyruvate kinase, a bi-substrate cooperative enzyme that is allosterically inhibited, are presented. This enzyme also shows inhibitor saturation, and therefore serves as experimental evidence that the bi-substrate Hill equation predicts more realistic allosteric inhibitor behaviour than the bi-substrate MWC equation.     

Biophoton emission. New evidence for coherence and DNA as source

The phenomenon of ultraweak photon emission from living systems was further investigated in order to elucidate the physical properties of this radiation and its possible source. We obtained evidence that the light has a high degree of coherence because of (1) its photon count statistics, (2) its spectral distribution, (3) its decay behavior after exposure to light illumination, and (4) its transparency through optically thick materials. Moreover, DNA is apparently at least an important source, since conformational changes induced with ethidium bromide in vivo are clearly reflected by changes of the photon emission of cells. The physical properties of the radiation are described, taking DNA as an exciplex laser system, where a stable state can be reached far from thermal equilibrium at threshold.     

Competition as a source of errors in RAPD analysis

We have used artificial 11 DNA mixtures of all pairwise combinations of four doubled haploid Brassica napus lines to test the ability of RAPDs to function as reliable dominant genetic markers. In situations where a specific RAPD band is present in one homozygous line but absent in the other, the band is expected in the artificial heterozygote, i.e. in the 11 DNA mixture. In 84 of all 613 heterozygous situations analysed, the expected band failed to amplify in the RAPD reaction. Thus, RAPD markers will lead to an erroneous genetic interpretation in 14% of all cases. In contrast, the formation of non-parental heteroduplex bands was found at a frequency of only 0.2%. Analysis of 1 1 mixtures using (1) a different set of optimized reaction conditions and (2) a material with low genomic complexity (Bacillus cereus) gave identical results. Serial dilutions of one genome into another, in steps of 10%, showed that all of the polymorphic bands decreased in intensity as a linear function of their respective proportion in the mixture. In dilutions with water no differences in band intensity were detected. Thus, competition occurs in the amplification of all RAPD fragments and is a major source of genotyping errors in RAPD analysis.     

Semen as a rich source of diagnostic biomarkers for prostate cancer: latest evidence and implications

Molecular and Cellular Biochemistry - Prostate cancer (PCa) is one of the most common cancers in men and the cause of numerous cancer deaths in the world. Nowadays, based on diagnostic criteria,...     

Experimental evidence for a semi-flexible conformation for arabinoxylans

Purified water-soluble arabinoxylans from wheat flour were deferuloylated and fractionated into six fractions by graded ethanol precipitation. Further fractionation by HPSEC on Sephacryl S500 resulted in 48 subfractions with low polydispersity index. Conformational characteristics (persistence length q, hydrodynamic parameter v and Mark-Houwink exponent a) were similar among all subfractions and fitted with a semi-flexible conformation, whatever their structural characteristics. Substitution degree of the xylan backbone by arabinose residues has no influence on the conformation of arabinoxylans.     

Canalization as a non-genetic source of adaptiveness during morphogenesis: experimental evidence from analysis of reproductive development in Sorghum bicolor

In Sorghum bicolor, perturbations in reproductive development observed following salt-treatment also influence progeny grown in the absence of NaCl. However, a developmental reversion of these modifications may be observed throughout two successive generations. This response, termed canalization, does not spontaneously occur following growth in the absence of NaCl, but is triggered by the level of perturbation in parental expression of reproductive characters. Moreover, canalization is not specific to the perturbed character, but it includes modifications in reproductive development as a whole. A decrease in developmental variability coincides with amplitude of the developmental reversion. This phenomenon is interpreted as an evidence for orientation of the developmental process towards the lowest free-energy state of the 'epigenetic landscape'. Involvement of this phenomenon of canalization in developmental stability, adaptiveness, and evolution is discussed. Moreover, these results point to the need for a posteriori methods of investigations in order to analyze self-organized transformations in biological systems.     

Acetate as a carbon source for hydrogen production by photosynthetic bacteria

Hydrogen is a clean energy alternative to fossil fuels. Photosynthetic bacteria produce hydrogen from organic compounds by an anaerobic light-dependent electron transfer process. In the present study hydrogen production by three photosynthetic bacterial strains (Rhodopseudomonas sp., Rhodopseudomonas palustris and a non-identified strain), from four different short-chain organic acids (lactate, malate, acetate and butyrate) was investigated. The effect of light intensity on hydrogen production was also studied by supplying two different light intensities, using acetate as the electron donor. Hydrogen production rates and light efficiencies were compared. Rhodopseudomonas sp. produced the highest volume of H2. This strain reached a maximum H2 production rate of 25 ml H2 l(-1) h(-1), under a light intensity of 680 micromol photons m(-2) s(-1), and a maximum light efficiency of 6.2% under a light intensity of 43 micromol photons m(-2) s(-1). Furthermore, a decrease in acetate concentration from 22 to 11 mM resulted in a decrease in the hydrogen evolved from 214 to 27 ml H2 per vessel.     

Archived Guthrie blood spots as a novel source for quantitative DNA methylation analysis

Sodium bisulfite treatment followed by PCR and DNA sequencing is widely considered the gold standard for the analysis of DNA methylation patterns. However, this technique generally requires substantial quantities of genomic DNA as starting material and is often associated with degradation of DNA. Here, we assess the feasibility of performing bisulfite sequencing on DNA isolated from 3-mm diameter punches of dried blood Guthrie spots. We demonstrate that it is possible to perform bisulfite sequencing from both freshly prepared and archived dried blood spots, using a combination of high purity DNA extraction and efficient bisulfite conversion. With the number of new technologies available for DNA methylation studies, we have extended this analysis and have successfully used a high-throughput mass spectrometry method for DNA methylation analysis on these samples. This provides a new source of material for epigenetic analysis of birth samples and provides an invaluable reference point to track temporal change in epigenetic profiles possibly linked with health and disease.     

Experimental evidence for multistability in a photobiochemical system

The kinetic behavior of a typical Hill reaction catalyzed by thylakoids and using the oxidized form of 2,6-dichloroindophenol (DCPIPox) as the artificial electron acceptor, is considered. Here, the light absorption process and the reduction of DCPIPox are autocatalytically coupled, leading to the occurrence of multiple steady states with respect to either the acceptor concentration or the incident light intensity. Experimental evidence is presented for both cases and the emergence of autocatalysis is discussed. The effect of the spatial arrangement on the global behavior of the system described is emphasized.