DNA sequence analysis of spontaneous histidine mutations in a polA1 strain of Escherichia coli K12 suggests a specific role of the GTGG sequence

Summary Spontaneously arising histidine mutations in an Escherichia coli K12 strain deficient for DNA polymerase I were analysed at the DNA sequence level. We screened approximately 150000 colonies and isolated 106 histidine auxotrophs. Of these, 98 were unstable hisC mutations; 12 representative mutants analysed were shown to have arisen by the excision of a single quadruplet repeat in the sequence 5-GCTGGCTGGCTGGCTG-3. Of the eight mutations at other sites, three hisA deletions and one hisD deletion occurred as a consequence of misalignment of tandemly repeated pentamers (hisD) or decamers (hisA). A single hisA point mutation was found to be a missense mutation. Two extended deletions, covering the his operon were not analysed. We could not identify the hisC deletion by sequencing. We conclude that polA1 is a strong imitator that induces mutations mostly of the minus frameshift and deletion type by a Streisinger-type of mispairing in repetitive DNA sequences. Finally, the possible role of a 5-GTGG-3 sequence and its inverted or direct complements, which are found in the vicinity of all the deletions and frameshifts, is discussed.     

Rhizobacteria associated with Miscanthus x giganteus improve metal accumulation and plant growth in the flotation tailings

Plant and Soil - Flotation tailings represent an extremely unfriendly substrate for plant colonization due to toxic metal concentrations and marked macronutrient deficiencies. The perennial grass...     

Fragile X‐associated tremor/ataxia syndrome: Regional decrease of mitochondrial DNA copy number relates to clinical manifestations

Fragile X‐associated tremor/ataxia syndrome (FXTAS) is a late‐onset neurodegenerative disorder that appears in at least one‐third of adult carriers of a premutation (55‐200 CGG repeats) in the fragile X mental retardation 1 (FMR1) gene. Several studies have shown that mitochondrial dysfunction may play a central role in aging and also in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease as well as in FXTAS. It has been recently proposed that mtDNA copy number, measured by the number of mitochondrial genomes per nuclear genome (diploid), could be a useful biomarker of mitochondrial dysfunction. In order to elucidate the role of mtDNA variation in the pathogenesis of FXTAS, mtDNA copy number was quantified by digital droplet Polymerase chain reaction. In human brain samples, mtDNA levels were measured in the cerebellar vermis, dentate nucleus, parietal and temporal cortex, thalamus, caudate nucleus and hippocampus from a female FXTAS patient, a FMR1 premutation male carrier without FXTAS and from three male controls. The mtDNA copy number was further analyzed using this technology in dermal fibroblasts primary cultures derived from three FXTAS patients and three controls as well as in cortex and cerebellum of a CGG knock in FXTAS mice model. Finally, qPCR was carried out in human blood samples. Results indicate reduced mtDNA copy number in the specific brain region associated with disease progression in FXTAS patients, providing new insights into the role of mitochondrial dysfunction in the pathogenesis of FXTAS.     

Involvement of an autocrine stromal cell derived factor-1/CXCR4 system on the distant metastasis of human oral squamous cell carcinoma

We have previously shown that a stromal cell-derived factor-1 (SDF-1; CXCL12)/CXCR4 system is involved in the establishment of lymph node metastasis, but not in that of distant metastasis, in oral squamous cell carcinoma (SCC). In this study, we investigated the role of the autocrine SDF-1/CXCR4 system, with a focus on distant metastasis in oral SCC cells. The immunohistochemical staining of SDF-1 and CXCR4 using primary oral SCCs and metastatic lymph nodes showed a significantly higher number of SDF-1-positive cases among the metastatic lymph nodes than among the primary oral SCCs, which was associated with a poor survival rate among those of the former group. The forced expression of SDF-1 in B88 cells, which exhibit functional CXCR4 and lymph node metastatic potential (i.e., the autocrine SDF-1/CXCR4 system), conferred enhanced cell motility and anchorage-independent growth potential onto the cells. Orthotopic inoculation of the transfectant into nude mice was associated with an increase in the number of metastatic lymph nodes and more aggressive metastatic foci in the lymph nodes. Furthermore, the SDF-1 transfectant (i.e., the autocrine SDF-1/CXCR4 system) exhibited dramatic metastasis to the lung after i.v. inoculation, whereas the mock transfectant (i.e., the paracrine SDF-1/CXCR4 system) did not. Under the present conditions, AMD3100, a CXCR4 antagonist, significantly inhibited the lung metastasis of the SDF-1 transfectant, ameliorated body weight loss, and improved the survival rate of tumor-bearing nude mice. These results suggested that, in cases of oral SCC, the paracrine SDF-1/CXCR4 system potentiates lymph node metastasis, but distant metastasis might require the autocrine SDF-1/CXCR4 system.     

NEUROTOXICITY OF A NON-METABOLIZABLE AMINO ACID, 1-AMINOCYCLOPENTANE-1-CARBOXYLIC ACID: ANTAGONISM BY AMINO ACIDS IN CULTURES OF CEREBELLUM

Abstract— The non-metabolizable amino acid 1-aminocyclopentane-1-carboxylic acid (ACPC) induced degeneration of myelinated axons but spared nerve cell bodies in well myelinated organotypic cultures of cerebellum. The ACPC concentrations used were comparable to those which induce axonal degeneration in vivo. Developing unmyelinated cultures were more sensitive to ACPC than mature cultures and newly myelinating axons appeared to be particularly affected. Supplementing the medium with amino acids, but not with vitamins, prevented toxicity at the lower concentrations of ACPC and afforded considerable protection against the highest concentrations. The protective effect of amino acids could not be accounted for by inhibition of intracellular ACPC transport. These results are considered in terms of other evidence indicating defective protein metabolism in ACPC-treated mice.     

Effects of small-sided games and high-intensity interval training on physical performance in young female handball players

This study was designed to compare the effectiveness of small-sided handball games in combination with handball training (SSG group) versus high-intensity interval training in combination with handball training (HIIT group) on physical performance of young female handball players during pre-competitive period. Twenty-four young female handball players, who have a 6.17 ± 1.54 years training experience and competition in the national league participated in this study. SSG group (n = 12; age 16.06 ± 0.80 years, body mass 61.27 ± 3.68 kg, body height 1.64 ± 4.7 m, body mass index 22.7 kg/m²) while HIIT group (n = 12; 16.20 ± 1.28 years, body mass 62.46 ± 7.86 kg, body height 1.68 ± 6.8 m, body mass index 22 kg/m²). Both groups applied training programs twice-a-week for 8 weeks. Before and after the training programs physical performances were assessed: Countermovement jump (CMJ), Squat jump (SJ), Sprint on 0–10 m; Sprint on 0–20 m; Sprint on 0–30 m, Throwing medicine ball and total distance covered during the Yo-YoIRT1. After 8 weeks SSG and HIIT groups significantly improved CMJ, SJ, 0–20 m sprint, 0–30 m sprint, throwing medicine ball and Yo-YoIRT1 (p ≤ 0.05). However, significantly greater improvement was achieved in Yo-YoIRT1 (m) in HIIT group (28.40%) than SSG group (17.63%). These results indicate that SSG group and HIIT group equally improve of physical performances (jump, sprint and upper explosive strength) among young female handball players in pre-competitive period.     

Two novel mouse genes--Nubp2, mapped to the t-complex on chromosome 17, and Nubp1, mapped to chromosome 16--establish a new gene family of nucleotide-binding proteins in eukaryotes.

Two novel mouse genes and one novel human gene that define distinctive eukaryotic nucleotide-binding proteins (NUBP) and are related to the mrp gene of prokaryotes are characterized. Phylogenetic analyses of the genes, encoding a short form (Nubp2) and a long form (Nubp1) of NUBP, clearly establish them as a new NUBP/MRP gene family that is well conserved throughout phylogeny. In addition to conserved ATP/GTP-binding motifs A (P-loop) and A', members of this family share at least two highly conserved sequence motifs, NUBP/MRP motifs alpha and beta. Only one type of NUBP/MRP gene has been observed thus far in prokaryotes, but there are two types in eukaryotes. One group includes mouse Nubp1, human NBP, yeast NBP35, and Caenorhabditis elegans F10G8.6 and is characterized by a unique N-terminal sequence with four cysteine residues that is lacking in the other group, which includes mouse Nubp2, human NUBP2, and yeast YIA3w. Northern blot analyses of the two mouse genes show distinctive patterns consistent with this classification. Mouse Nubp2 is mapped to the t-complex region of mouse Chromosome 17, whereas Nubp1 is mapped to the proximal region of mouse Chromosome 16. Interestingly, both regions are syntenic with human chromosome 16p13.1-p13.3, suggesting that a chromosomal breakage between Nubp2 and Nubp1 probably occurred during the evolution of mouse chromosomes.     

The extent of whole‐genome copy number alterations predicts aggressive features in primary melanomas

Recent evidence indicates that melanoma comprises distinct types of tumors and suggests that specific morphological features may help predict its clinical behavior. Using a SNP‐array approach, we quantified chromosomal copy number alterations (CNA) across the whole genome in 41 primary melanomas and found a high degree of heterogeneity in their genomic asset. Association analysis correlating the number and relative length of CNA with clinical, morphological, and dermoscopic attributes of melanoma revealed that features of aggressiveness were strongly linked to the overall amount of genomic damage. Furthermore, we observed that melanoma progression and survival were mainly affected by a low number of large chromosome losses and a high number of small gains. We identified the alterations most frequently associated with aggressive melanoma, and by integrating our data with publicly available gene expression profiles, we identified five genes which expression was found to be necessary for melanoma cells proliferation. In conclusion, this work provides new evidence that the phenotypic heterogeneity of melanoma reflects a parallel genetic diversity and lays the basis to define novel strategies for a more precise prognostic stratification of patients.