Nuclear GIT2 Is an ATM Substrate and Promotes DNA Repair

Insults to nuclear DNA induce multiple response pathways to mitigate the deleterious effects of damage and mediate effective DNA repair. G-protein-coupled receptor kinase-interacting protein 2 (GIT2) regulates receptor internalization, focal adhesion dynamics, cell migration, and responses to oxidative stress. Here we demonstrate that GIT2 coordinates the levels of proteins in the DNA damage response (DDR). Cellular sensitivity to irradiation-induced DNA damage was highly associated with GIT2 expression levels. GIT2 is phosphorylated by ATM kinase and forms complexes with multiple DDR-associated factors in response to DNA damage. The targeting of GIT2 to DNA double-strand breaks was rapid and, in part, dependent upon the presence of H2AX, ATM, and MRE11 but was independent of MDC1 and RNF8. GIT2 likely promotes DNA repair through multiple mechanisms, including stabilization of BRCA1 in repair complexes; upregulation of repair proteins, including HMGN1 and RFC1; and regulation of poly(ADP-ribose) polymerase activity. Furthermore, GIT2-knockout mice demonstrated a greater susceptibility to DNA damage than their wild-type littermates. These results suggest that GIT2 plays an important role in MRE11/ATM/H2AX-mediated DNA damage responses.     

Animals as an indicator of carbon sequestration and valuable landscapes

Possibilities of the assessment of a landscape with the use of succession development stages, monitored with the value of the Mean Individual Biomass (MIB) of carabid beetles and the occurrence of bird species are discussed on the basis of an example from Poland. Higher variability of the MIB value in space signifies a greater biodiversity. Apart from the variability of MIB, it is suggested to adopt the occurrence of the following animals as indicators, (in the order of importance), representing underlying valuable landscapes: black stork, lesser spotted eagle, white-tailed eagle, wolf, crane and white stork. The higher number of these species and their greater density indicate a higher value of the landscape for biodiversity and ecosystem services, especially carbon sequestration. All these indicators may be useful to assess measures for sustainable land use.     

Endogenous ADP-Ribosylation in Brain: Initial Characterization of Substrate Proteins

Cholera and pertussis toxin-mediated ADP-ribosylation has been used extensively to study regulation of guanine nucleotide binding proteins (G proteins) in the nervous system, but much less is known about possible endogenous ADP-ribosylation of G proteins in brain. The present study demonstrates endogenous ADP-ribosylation, in the absence of cholera and pertussis toxins, of four predominate proteins in homogenates of rat cerebral cortex. These proteins showed apparent molecular masses of 20, 42, 45, and 50 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 42- and 45-kDa proteins comigrated precisely with the major cholera toxin-labeled bands. Furthermore, the endogenous ADP-ribosylated and cholera toxin-ADP-ribosylated bands yielded identical 32P-labeled peptide fragments by one-dimensional peptide mapping, indicating that they are probably the same proteins, presumably the alpha-subunits of Gs. In contrast, peptide maps of the 50-kDa protein, which migrated close to a 48-kDa cholera toxin-labeled band, demonstrated that this protein is distinct from the toxin-labeled band and from Gs alpha. Levels of endogenous ADP-ribosylation activity showed regional heterogeneity in brain, with a nearly threefold variation observed among the brain regions examined. Chronic administration (7 days) of corticosterone significantly increased overall levels of endogenous ADP-ribosylation, indicating that components of this system may be under hormonal control in vivo. Attempts to identify neurotransmitters or second messenger systems that regulate endogenous ADP-ribosylation activity in brain have so far been unsuccessful with one exception.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of Aging on Nuclear DNA in Corn Leaves

Leaves of Zea mays L. were selected at a young stage when theyhad accumulated about two-thirds of their maximum chlorophyll,at a mature stage when approximately full dimensions and maximumchlorophyll levels were attained, and following pollen shedwhen lower leaves were dying from the tip. Data show a lossof one-fourth of the nuclear DNA, no effect of aging on eu-and heterochromatin levels and a marked decline in the guanine(G) and cytosine (C) percentages. Although DNA levels mightbe under hormonal control, it is not clear that the loss detectedwould be physiologically influential in aging sequences. Yetthis decline preceded those of leaf nitrogen and chlorophyll. (Received July 7, 1983; Accepted November 23, 1983)     

Frontiers of Model Animals for Neuroscience:Two Prosperous Aging Model Animals forPromoting Neuroscience Research

A model animal showing spontaneous onset is a useful tool for investigating the mechanism of disease. Here, I would like to introduce two aging model animals expected to be useful for neuroscience research: the senescence-accelerated mouse (SAM) and the klotho mouse. The SAM was developed as a mouse showing a senescence-related phenotype such as a short lifespan or rapid advancement of senescence. In particular, SAMP8 and SAMP10 show age-related impairment of learning and memory. SAMP8 has spontaneous spongy degeneration in the brain stem and spinal cord with aging, and immunohistochemical studies reveal excess protein expression of amyloid precursor protein and amyloid β in the brain, indicating that SAMP8 is a model for Alzheimer’s disease. SAMP10 also shows age-related impairment of learning and memory, but it does not seem to correspond to Alzheimer’s disease because senile plaques primarily composed of amyloid β or neurofibrillary tangles primarily composed of phosphorylated tau were not observed. However, severe atrophy in the frontal cortex, entorhinal cortex, amygdala, and nucleus accumbens can be seen in this strain in an age-dependent manner, indicating that SAMP10 is a model for normal aging. The klotho mouse shows a phenotype, regulated by only one gene named α-klotho, similar to human progeria. The α-klotho gene is mainly expressed in the kidney and brain, and oxidative stress is involved in the deterioration of cognitive function of the klotho mouse. These animal models are potentially useful for neuroscience research now and in the near future.     

Identification of She3 as an SCFGrr1 Substrate in Budding Yeast

The highly orchestrated progression of the cell cycle depends on the degradation of many regulatory proteins at different cell cycle stages. One of the key cell cycle ubiquitin ligases is the Skp1-cullin-F-box (SCF) complex. Acting in concert with the substrate-binding F-box protein Grr1, SCF^Grr1 promotes the degradation of cell cycle regulators as well as various metabolic enzymes. Using a yeast two-hybrid assay with a Grr1 derivative as the bait, we identified She3, which is an adaptor protein in the asymmetric mRNA transport system, as a novel Grr1 substrate. We generated stabilized She3 mutants, which no longer bound to Grr1, and found that the degradation of She3 is not required for regulating asymmetric mRNA transport. However, She3 stabilization leads to slower growth compared to wild-type cells in a co-culture assay, demonstrating that the degradation of She3 by Grr1 is required for optimal cell growth.     

Initial growth deceleration as an immanent property of plants

A previously published sectional model of the system of tree branches (spruce) was expanded in the range (0,1) of the fractal model parameter μ, which links the value of green biomass of a “tree” to its size. The presence of branches in this range indicates the realization of green biomass in the form of photosynthe-sizing “points”, and is interpreted as endosymbiosis of cyanobacteria with protists. Using the method of box dimensionality, the parameter μ was estimated for the sets of points within an interval. The properties of the uniform and group distributions are shown to be different. For the group distribution, the trajectories of parameter μ are fundamentally different, depending on the method of point grouping, i.e., they decrease with an increase in the total number of points for a fixed number of points n _g in the groups and increase for a fixed number N _g groups. The initial endosymbiosis of cyanobacteria and “protists” is characterized by a lack of the necessary infrastructure in protists for feeding cyanobacteria and distributing their products and, thus, by a fixed number of “points” in the group. As the infrastructure of protists developed in the course of evolution, endosymbiosis tended to move towards an increased number of points in the group and, accordingly, increased μ. A special form of the dependence of parameter μ(N _g *n _g ) provides the inherent nature of the initial growth deceleration and modularity of architecture, which are occasionally observed in extant plants.     

Mortality as an Indicator of Aging: Possibilities and Limitations

Biophysics - Abstract—Using data gathered from 40 countries throughout a historical period covering two and a half centuries, we studied the peculiarities of age-related changes in the aging...     

Animals are dependent on preformed alpha-amino nitrogen as an essential nutrient

It has traditionally been thought that animals can utilize ammonia for amino acid biosynthesis, and that for them some amino acids are nutritionally nonessential. Presumably this idea originates from the notions of Schoenheimer (G. L. Foster et al. [1939] J. Biol. Chem. 127, 319-327) and of Rose (W. C. Rose et al. [1948] J. Biol. Chem. 176, 753-762), which we question for the following reasons. First, Schoenheimer's experiments only showed the incorporation of ammonia into amino acids. This may occur simply as an exchange between ammonia and the alpha-amino group of endogenous amino acids and reflects the enzymatic properties of glutamate dehydrogenase, which is a reversible enzyme. Second, Rose's nutritional experiments were concerned with whether carbon skeletons of particular amino acids can (nonessential) or cannot (essential) be synthesized from common intermediates of carbohydrate metabolism. We propose that mammals, living as they do at the top of the food web, are absolutely dependent directly or indirectly on higher plants and microorganisms for preformed alpha-amino nitrogen per se and that the first joining of C- and N-atoms to make glutamate constitutes a basic anabolic system in nature after the fixation of CO2 and N2.     

Nuclear transport as an ultimate step of multidrug resistance

Adriamycin (ADM) incorporation into nuclei of whole multidrug resistant (MDR) CEM cells is lower than into sensitive ones (S), that is mostly thought to be the consequence of a decrease of drug related to the activity of the multidrug resistance plasma membrane protein P 170. Isolated nuclei of the lymphoblastic tumor cell line CEM, which structures were controlled by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and confocal microscopy, where incubated with 10(-6) mole/l of ADM. Incorporation into DNA was quantified by spectrofluorimetry. It was lower and slower into MDR nuclei than into S ones. Different modulators of active transport influence drug transfer into S nuclei and had no effect in MDR nuclei. The nuclear transfer into S nuclei appeared divided into two components: one was decreased by WGA, increased by cytosolic factors and an other part was purely passive in an identical intensity to MDR nuclei. Resistance of MDR nuclei seemed indebt to a defect, in these cells, of factors that mediate and/or activate nuclear transport of drug.     

Ratios of Carbon Isotopes in Microbial Lipids as an Indicator of Substrate Usage

The occurrence and abundance of microbial fatty acids have been used for the identification of microorganisms in microbial communities. However, these fatty acids can also be used as indicators of substrate usage. For this, a systematic investigation of the discrimination of the stable carbon isotopes by different microorganisms is necessary. We grew 11 strains representing major bacterial and fungal species with four different isotopically defined carbon sources and determined the isotope ratios of fatty acids of different lipid fractions. A comparison of the differences of δ¹³C values of palmitic acid (C_16:0) with the δ¹³C values of the substrates revealed that the isotope ratio is independent of the growth stage and that most microorganisms showed enrichment of C_16:0 with ¹³C when growing on glycerol. With the exception of Burkholderia gladioli, all microorganism showed depletion of ¹³C in C_16:0 while incorporating the carbons of glucose, and most of them were enriched with ¹³C from mannose, with the exception of Pseudomonas fluorescens and the Zygomycotina. Usually, the glycolipid fractions are depleted in ¹³C compared to the phospholipid fractions. The δ¹³C pattern was not uniform within the different fatty acids of a given microbial species. Generally, tetradecanoic acid (C_14:0) was depleted of ¹³C compared to palmitic acid (C_16:0) while octadecanoic acid (C_18:0) was enriched. These results are important for the calibration of a new method in which δ¹³C values of fatty acids from the environment delineate the use of bacterial substrates in an ecosystem.     

Use of a Nylon Manufacturing Waste as an Industrial Fermentation Substrate

Nonvolatile residue (NVR), a waste stream from the manufacture of nylon 6′6′, contains mainly small carboxylic acids and alcohols, making it a potential fermentation substrate. Above a concentration of 1.3% (wt/vol), NVR inhibited the growth of all microorganisms tested. The most inhibitory of the major NVR components were the monocarboxylic acids (C₄ to C₆) and ε-caprolactone. The inhibitory effects of NVR could be avoided by using a carbon-limited chemostat. Microorganisms were found that could use all of the major NVR components as carbon and energy sources. One such organism, Pseudomonas cepacia, was grown in a carbon-limited chemostat with a medium feed concentration of 20.5 g of NVR liter⁻¹. At a dilution rate of 0.14 h⁻¹ the yield of biomass (Y_x/s, where x is biomass produced and s is substrate used) from NVR was 18% (neglecting the water content of NVR). It was concluded that NVR would be a suitable carbon source for certain industrial fermentation processes such as the production of poly-β-hydroxybutyric acid.     

Initial Genetic Analysis of Xylella fastidiosa in Texas

Xylella fastidiosa is the causative agent of Pierce’s Disease of grape. No published record of X. fastidiosa genetics in Texas exists despite growing financial risk to the U.S. grape industry, a Texas population of the glassy-winged sharpshooter insect vector (Homalodisca vitripennis) now spreading in California, and evidence that the bacterium is ubiquitous to southern states. Using sequences of conserved gyrB and mopB genes, we have established at least two strains in Texas, grape strain and ragweed strain, corresponding genetically with subsp. piercei and multiplex, respectively. The grape strain in Texas is found in Vitis vinifera varieties, hybrid vines, and wild Vitis near vineyards, whereas the ragweed strain in Texas is found in annuals, shrubs, and trees near vineyards or other areas. RFLP and QRT PCR techniques were used to differentiate grape and ragweed strains with greater efficiency than sequencing and are practical for screening numerous X. fastidiosa isolates for clade identity.     

The Use of Hybrid Somatic Cells as an Approach to Mitochondrial Genetics in Animals

We have studied the fate of parental mitochondrial DNA (mtDNA) in hybrid somatic cells derived by Sendai virus-induced fusion of human cells and mouse or rat cells. Many hybrid cell strains were obtained which contained sequences from both human and rodent mtDNA after 40 to 60 population doublings. Some strains were subcloned and cultured further for up to 150 doublings; a large fraction of these strains contained both parental mtDNA sequences at that time.The relation between human and rodent mtDNA sequences was tested in some of the hybrid cell strains. In a high fraction of strains tested the human and rodent mtDNA sequences were linked to each other by what are most likely covalent bonds. This linkage may be described as recombination of mtDNA sequences from two different animals.