Regulation of proto-oncogenic dbl by chaperone-controlled, ubiquitin-mediated degradation

The dbl proto-oncogene product is a prototype of a growing family of guanine nucleotide exchange factors (GEFs) that stimulate the activation of small GTP-binding proteins from the Rho family. Mutations that result in the loss of proto-Dbl's amino terminus produce a variant with constitutive GEF activity and high oncogenic potential. Here, we show that proto-Dbl is a short-lived protein that is kept at low levels in cells by efficient ubiquitination and degradation. The cellular fate of proto-Dbl is regulated by interactions with the chaperones Hsc70 and Hsp90 and the protein-ubiquitin ligase CHIP, and these interactions are mediated by the spectrin domain of proto-Dbl. We show that CHIP is the E3 ligase responsible for ubiquitination and proteasomal degradation of proto-Dbl, while Hsp90 functions to stabilize the protein. Onco-Dbl, lacking the spectrin homology domain, cannot bind these regulators and therefore accumulates in cells at high levels, leading to persistent stimulation of its downstream signaling pathways.     

RAKE and LNA-ISH reveal microRNA expression and localization in archival human brain

microRNAs (miRNAs) are small (approximately 22 nucleotide) regulatory RNAs which play fundamental roles in many biological processes. Recent studies have shown that the expression of many miRNAs is altered in various human tumors and some miRNAs may function as oncogenes or tumor suppressor genes. However, with the exception of glioblastoma multiforme, the expression of miRNAs in brain tumors is unknown. Furthermore, methods to profile miRNAs from formalin-fixed, paraffin-embedded (FFPE) archival tissues or to study their cellular and subcellular localization in FFPE tissues have been lacking. Here we report the coordinated miRNA expression analysis from the tissue level to the subcellular level, using the RAKE (RNA-primed, array-based, Klenow Enzyme) miRNA microarray platform in conjunction with Locked Nucleic Acid (LNA)-based in situ hybridization (LNA-ISH) on archival FFPE human brains and oligodendroglial tumors. The ability to profile miRNAs from archival tissues at the tissue level, by RAKE microarrays, and at the cellular level by LNA-ISH, will accelerate studies of miRNAs in human diseases.     

SIRT1 longevity factor suppresses NF‐κB ‐driven immune responses: regulation of aging via NF‐κB acetylation?

The aging process involves changes in immune regulation, i.e. adaptive immunity declines whereas innate immunity becomes activated. NF-kappaB signaling is the master regulator of the both immune systems. Two recent articles highlight the role of the NF-kappaB system in aging and immune responses. Adler et al showed that the NF-kappaB binding domain is the genetic regulatory motif which is most strongly associated with the aging process. Kwon et al studying HIV-1 infection and subsequent immune deficiency process demonstrated that HIV-1 Tat protein binds to SIRT1 protein, a well-known longevity factor, and inhibits the SIRT1-mediated deacetylation of the p65 component of the NF-kappaB complex. As a consequence, the transactivation efficiency of the NF-kappaB factor was greatly potentiated, leading to the activation of immune system and later to the decline of adaptive immunity. These observations support the scenario where immune responses and aging process can be enforced by the potentiation of NF-kappaB transactivation efficiency. Longevity factors, such as SIRT1 and its activators, might regulate the efficiency of the NF-kappaB signaling, the major outcome of which is inflamm-aging via proinflammatory responses.     

Carbon and Nitrogen Mineralization of Lead Treated Soils in the Eastern Mediterranean Region,Turkey

The aim of this study was to determine the first effect of lead on microbial activity in soil. The study was carried out in the soil samples from four different radish (Raphanus sativus L. var. radicula, Brassicaceae) fields along the highway in a district (Kadirli, Osmaniye) of the Eastern Mediterranean Region, Turkey. After the calculation of Pb contents, the Pb amounts of the soil samples were brought up to 50 and 100 mg Pb kg^?1 by treatment with Pb(NO ₃ ) ₂ , and the samples for the carbon and the nitrogen mineralization were incubated under controlled conditions (28°C, constant moist). The carbon mineralization was determined by a CO ₂ respiration method for 30 days. The nitrogen mineralization was observed in vitro for 6 weeks. The untreated group was statistically different from the 50 and 100 mg Pb kg^?1 treatments in the aspect of the C(CO ₂ ) outlet during mineralization (P ≤ 0.05), but difference between the 50 and 100 mg Pb kg^?1 treatments was not significant. NH ₄ -N and NO ₃ -N contents of each soil were shown differences between across treatments. Based on these results, it is possible to conclude that the addition of 50 and 100 mg Pb kg^?1 provided a toxic effect threshold for the microbial activity into 30 days.     

Incidence of mycoflora and mycotoxins in some edible fruits and seeds of forest origin

Twelve fungi namelyAlternaria alternata, Aspergillus flavus, A niger, A ochraceus, Actinomucor repens, Capnodoium spp., Curvularia lunata, Fusarium pallidoroseum, F solani, F verticillioides, Penicillium citrinum and Rhizopus stolonifer were recorded from samples ofAegle marmelos, Aesculus indica, Buchanania lanzan andPinus gerardiana. In case ofPrunus amygdalus only Rstolonifer was recorded. A significant variation in pattern of mycoflora incidence was observed in terms of source and season. Fungal infestation in most of the substrates was found to be highest during monsoon. Aflatoxins were the most common mycotoxins elaborated by different isolates ofA flavus obtained fromA marmelos, B lanzan andP gerardiana. The amount of aflatoxins produced by the toxigenic isolates ofA flavus was in the range of traces to 0.9–26.0 μg/ml inA marmelos, 0.8–17.5 μg/ml inP gerardiana and 0.65–13.2 μg/ml inB lanzan. The percentage toxigenicity was comparatively lower in the isolates of other mycotoxigenic fungi. Aflatoxins were detected almost in all the samples analyzed for mycotoxin contamination. However, traces of zearalenone were detected inA marmelos. The concentration of aflatoxin B₁ was in the range of 0.13–0.75 μg/g inA marmelos, 0.09–0.60 μg/g inP gerardiana and 0.01–0.20 ug/g inB lanzan. Mycotoxins were not detected inAesculus indica andPrunus amygdalus.     

Detection of thymosin beta 4 in situ in a guinea pig cerebral cortex preparation using 1H NMR spectroscopy.

In the present work we have investigated the macromolecules that contribute to the brain 1H NMR spectrum. The cerebral cortex showed distinct resonances at the uncrowded methyl- and methylene chemical shift scale of the spin-echo 1H NMR spectrum. The peaks at 1.22 and 1.40 ppm (relative to the methyl protons of N-acetyl aspartate at 2.02 ppm) arise from cerebral macromolecules without evidence for co-resonances from low molecular weight metabolites as shown by the spin-spin relaxation decays of these resonances. In addition to these NMR signals, peaks at 0.9 and 1.7 ppm from macromolecules were detected. These resonances are from proteins, and we have identified the polypeptides that contributed to the 1H NMR peaks. Two proteins that were present at concentrations of 250 and 350 micrograms/g of dryed tissue showed 1H NMR spectra that resembled the macromolecular pattern in the cerebral 1H NMR spectrum. They were identified as thymosin beta 4 and histone H1, respectively. Thymosin beta 4 was present in soluble high speed cytoplasmic fraction and in P2 pellet, whereas histone H1 was detected in nuclear enriched fraction. A chemical shift-correlated two-dimensional 1H NMR spectrum of thymosin beta 4 in vitro revealed a coupling pattern that matched the macromolecule in the cerebral cortex which we have previously noted (Kauppinen R. A., Kokko, H., and Williams, S. R. (1992) J. Neurochem. 58, 967-974). On the basis of both one- and two-dimensional NMR evidence, subcellular distribution and high concentration, we assign the 1H NMR signals at 0.9, 1.22, 1.40, and 1.7 ppm in the cerebral cortex to thymosin beta 4.     

RFLP analysis of three different types of acute intermittent porphyria

Summary Restriction fragment length polymorphism (RFLP) analysis was performed in three Finnish families with different subtypes of acute intermittent porphyria (AIP): 1) cross-reacting immunological material (CRIM)-negative with low erythrocyte porphobilinogen (PBG)-deaminase activity, 2) CRIM-positive with low PBG-deaminase activity and 3) CRIM-negative with normal PBG-deaminase activity. The disease-associated RFLP haplotype (A2B1C2) of the PBG-deaminase gene was the same in each family. In all three families, RFLP linkage analysis resulted in highly positive lod scores. The maximal lod score (4.3) was obtained at the recombinant fraction of zero, thus confirming a tight linkage of AIP to the PBG-deaminase locus. Of the 62 family members tested, 30 had the disease-associated haplotype; in 5 of them, conventional tests for AIP were normal and in one, uncertain. RFLP analysis can thus reveal new gene carriers and help in the diagnosis of individuals with uncertain results in other laboratory tests.