Feasibility and limits of an orthotopic human colon cancer model in nude mice

We sought to develop an accurate colorectal cancer model in nude mice with stable local growth, tumor cell dissemination, and reproducible metastatic capacity. To this end, we orthotopically transplanted histologically intact human colorectal cancer tissue from 10 human patients into nude mice. After successful local tumor growth, tumor tissues were retransplanted as many as 9 times in serial passage. All specimens were transplanted using microsurgical techniques. Histologic, immunohistochemical, and polymerase chain reaction techniques were used to determine tumor growth rates and kinetics, development of regional lymph node and distant hepatic metastases, and the induction of minimal residual disease (MRD). Stable local tumor growth rates with variable growth kinetics were detected in 73.4% of all mice. The lymph node and hepatic metastasis rates were low, at 18.4% and 4.9%, respectively. MRD, as reflected by CK20 positivity of the bone marrow in animals with lymph node and hepatic metastases, was present in 22.2%. The orthotopic colorectal cancer model described here is feasible for the induction of reproducible local tumor growth but is limited by variable growth kinetics and the low rate of lymph node and hepatic metastases. Cytokeratin-positive cells indicative of MRD could be detected in the bone marrow of approximately 25% of the nude mice with metastases. The observed induction of MRD after orthotopic implantation of intact human colon cancer in animals with lymph node and hepatic metastases might be improved if established colon cancer cell lines were used.     

An archaeal b-type cytochrome containing a nonfunctional carbonic anhydrase-like domain

A new type of cytochrome b was isolated from the cytoplasmatic fraction of the archaeon Acidianus ambivalens, which is the first soluble cytochrome found in this member of the thermoacidophilic order of the Sulfolobales. The protein is a monomeric and monohemic cytochrome b with a molecular mass of 22 kDa. Visible spectroscopy of the as-purified protein shows a Soret peak at 405 nm and a broad band at 625 nm, indicating the presence of a high-spin ferric heme. Upon reduction, the Soret band shifts to 422 nm and a broad band at 560 nm develops, again characteristic of high-spin ferrous heme. The reduced form can bind carbon monoxide, with visible absorption bands arising at 411 and 566 nm. EPR spectroscopy of the oxidized protein shows a spectrum typical of a high-spin heme, with major g values at 6.56 and 5.85. The reduction potential of the heme cofactor was determined to be -16+/-10 mV, at pH 6.5. Analysis of the protein amino acid sequence shows that it consists of a novel arrangement of domains. The first domain, at the N-terminus, has a remarkable similarity towards beta class carbonic anhydrases, whereas the second region comprises a putative cytochrome domain. The latter presumably consists of a novel fold, as it bears no sequence similarities towards other known cytochromes, or towards known domains. Strikingly, the first module contains the C-X (n)-H-X(2)-C motif that accounts for the binding of the catalytic zinc in carbonic anhydrases, but lacks several other critical residues required for substrate binding and proper active site geometry. In agreement with this finding, the isolated cytochrome contains one bound zinc atom, but has no carbonic anhydrase activity. Inspection of the sequences available from the genomic sequencing project of the close relative archaeon Sulfolobus solfataricus shows the presence of an identical protein, suggesting its dissemination among the Sulfolobales. The role of zinc as a key element for the intrinsic thermal stability of these proteins is discussed.     

First insight into the genome of an uncultivated crenarchaeote from soil

Molecular phylogenetic surveys based on the characterization of 16S rRNA genes have revealed that soil is an environment particularly rich in microbial diversity. A clade of crenarchaeota (archaea) has frequently been detected among many other novel lineages of uncultivated bacteria. In this study we have initiated a genomic approach for the characterization of uncultivated microorganisms from soil. We have developed a procedure based on a two-phase electrophoresis technique that allows the fast and reliable purification of concentrated and clonable, high molecular weight DNA. From this DNA we have constructed complex large-insert genomic libraries. Using archaea-specific 16S rRNA probes we have isolated a 34 kbp fragment from a 900 Mbp fosmid library of soil DNA. The clone contained a complete 16S/23S rRNA operon and 17 genes encoding putative proteins. Phylogenetic analyses of the rRNA genes and of several protein encoding genes (e.g. DNA polymerase, FixAB, glycosyl transferase) confirmed the specific affiliation of the genomic fragment with the non-thermophilic clade of the crenarchaeota. Content and structure of the genomic fragment indicated that the archaea from soil differ significantly from their previously studied uncultivated marine relatives. The protein encoding genes gave the first insights into the physiological potential of these organisms and can serve as a basis for future genomic and functional genomic studies.     

Effect of Free Malonate on the Utilization of Glutamate by Rat Brain Mitochondria

Malonate is an effective inhibitor of succinate dehydrogenase in preparations from brain and other organs. This property was reexamined in isolated rat brain mitochondria during incubation with L-glutamate. The biosynthesis of aspartate was determined by a standard spectrofluorometric method and a radiometric technique. The latter was suitable for aspartate assay after very brief incubations of mitochondria with glutamate. At a concentration of 1 mM or higher, malonate totally inhibited aspartate biosynthesis. At 0.2 mM, the inhibitory effect was still present. It is thus possible that the natural concentration of free malonate in adult rat brain of 192 nmol/g wet weight exerts an effect on citric acid cycle reactions in vivo. The inhibition of glutamate utilization by malonate was readily overcome by the addition of malate which provided oxaloacetate for the transamination of glutamate. The reaction was accompanied by the accumulation of 2-oxoglutarate. The metabolism of glutamate was also blocked by inclusion of arsenite and gamma-vinyl-gamma-aminobutyric acid but again added malate allowed transamination to resume. When arsenite and gamma-vinyl-gamma-aminobutyric acid were present, the role of malonate as an inhibitor of malate entry into the mitochondrial interior could be determined without considering the inhibition of succinate dehydrogenase. The apparent Km and Vmax values for uninhibited malate entry were 0.01 mM and 100 nmol/mg protein/min, respectively. Malonate was a competitive inhibitor of malate transport (Ki = 0.75 mM).     

Probing lymphoma infiltration in spleen of AKR/J mice chronically exposed to electromagnetic fields for risk assessment – toward noninvasive modeling

Using the AKR/J mouse model, the potential of Raman spectroscopy for monitoring lymphoma in predisposed subjects is demonstrated by discriminating lymphoma infiltration in spleens; the relevance of different excitation profiles is shown. Under green excitation with optimal fluorescence bleaching, stronger DNA bands, intensity variations at amide‐III and phenylalanine bands, and the behavior of the 1606/1639 cm^–1 doublet correlate with tumorigenesis. Under red excitation, Raman fingerprints with multivariate models help to discriminate AKR/J‐mouse histological subtypes: Lymphoblastic lymphoma (LB) is found significantly distant from both separated lymphocytic lymphoma (LL) and healthy spleen; this agrees with histology since LB has well differentiated large lymphoma cells, while LL, with smaller cells similar to normal lymphocytes, usually cannot be discriminated from normal tissue without histoimmunoassays. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Does ammonium affect accumulation of starch in rhizomes ofPhragmites australis (Cav.) Trin. ex Steud.?

Reed rhizomes from four habitats differing in soil nitrogen availability were collected and analysed for carbohydrate content during one year. Rhizomes collected from sediments where nitrate was the prevailing form of available nitrogen accumulated predominantly starch. On the other hand, rhizomes buried in sediments rich in ammonium contained predominantly sucrose. These observations together with the fact that ammonium stimulates the activity of enzymes involved in degradation of starch suggest that ammonium affects starch storage by inducing conversion of starch into sucrose. During spring when sprouting takes place, the rates of sucrose or starch mobilization differed remarkably, the sucrose in rhizomes buried in ammonium-rich sediments being depleted much faster than the starch in rhizomes at ammonium-poor habitats. The consequent reduction of total carbohydrate content in rhizomes at ammonium-rich habitats places the plants in a vulnerable situation. Eutrophication and reed decline are postulated to be events causally related via toxicity of ammonium, of which considerable amounts are microbiologically released from decaying organic matter in reed beds under excessive nutrient supply.     

The conversion of wheat RFLP probes into STS markers via the single-stranded conformation polymorphism technique.

We describe a flexible and general strategy for converting a wheat RFLP-based assay into a PCR-based sequence-tagged site (STS), and have applied it to derive markers for a powdery mildew resistance gene present in a wheat-rye translocation. The concept is based on deriving PCR primers that amplify all of the homoeoloci defined by a single-copy cDNA sequence, and separating the resulting mixture of homoeoamplicons via single-stranded conformation polymorphism (SSCP) gels, which are able to detect minor differences between related DNA sequences. After their separation, the individual homoeoamplicons were sequenced and these were used to define nucleotide polymorphisms that could be exploited to design locus-specific PCR primers. In one case, we were able to demonstrate that the assay was allele specific.