Selective inhibition of Escherichia coli recBC activities by plasmid-encoded GamS function of phage lambda

The gam locus of bacteriophage lambda encompasses two coding sequences with the same reading frame and translational stop, one corresponding to an Mr 11646 polypeptide (gamS gene), the other to an Mr 16349 polypeptide (gamL gene). A DNA segment encoding gamS but not gamL was placed under lambda pR promoter control (regulated by the cIts857-coded repressor) on a multicopy plasmid, and an insertion mutation (gamS201) was constructed. Expression of gamS+, but not gamS201, inhibited Escherichia coli RecBC nuclease in vivo; the criteria were inhibition of chromosomal DNA degradation after UV irradiation and plating of T4 gene 2- phages. The recB+ C+ bacteria expressing gamS+ were completely or partially similar to recC- mutants with respect to certain phenotypes: defective plating of phages P1 and P2, ability to plate (in a recA- background) lambda red- gam- phages, reduced resistance to UV irradiation, defective SOS induction, decreased colony-forming ability.     

Combinatorial G-CSF/AMD3100 Treatment in Cardiac Repair after Myocardial Infarction

Aims

Several studies suggest that circulating bone marrow derived stem cells promote the regeneration of ischemic tissues. For hematopoietic stem cell transplantation combinatorial granulocyte-colony stimulating factor (G-CSF)/Plerixafor (AMD3100) administration was shown to enhance mobilization of bone marrow derived stem cells compared to G-CSF monotherapy. Here we tested the hypothesis whether combinatorial G-CSF/AMD3100 therapy has beneficial effects in cardiac recovery in a mouse model of myocardial infarction.

Methods

We analyzed the effect of single G-CSF (250 µg/kg/day) and combinatorial G-CSF/AMD3100 (100 µg/kg/day) treatment on cardiac morphology, vascularization, and hemodynamics 28 days after permanent ligation of the left anterior descending artery (LAD). G-CSF treatment started directly after induction of myocardial infarction (MI) for 3 consecutive days followed by a single AMD3100 application on day three after MI in the G-CSF/AMD3100 group. Cell mobilization was assessed by flow cytometry of blood samples drawn from tail vein on day 0, 7, and 14.

Results

Peripheral blood analysis 7 days after MI showed enhanced mobilization of white blood cells (WBC) and endothelial progenitor cells (EPC) upon G-CSF and combinatorial G-CSF/AMD3100 treatment. However, single or combinatorial treatment showed no improvement in survival, left ventricular function, and infarction size compared to the saline treated control group 28 days after MI. Furthermore, no differences in histology and vascularization of infarcted hearts could be observed.

Conclusion

Although the implemented treatment regimen caused no adverse effects, our data show that combinatorial G-CSF/AMD therapy does not promote myocardial regeneration after permanent LAD occlusion.     

Glyphosate reduces shoot concentrations of mineral nutrients in glyphosate-resistant soybeans

Although glyphosate-resistant (GR) technology is used in most countries producing soybeans (Glycine max L.), there are no particular fertilize recommendations for use of this technology, and not much has been reported on the influence of glyphosate on GR soybean nutrient status. An evaluation of different cultivar maturity groups on different soil types, revealed a significant decrease in macro and micronutrients in leaf tissues, and in photosynthetic parameters (chlorophyll, photosynthetic rate, transpiration and stomatal conductance) with glyphosate use (single or sequential application). Irrespective of glyphosate applications, concentrations of shoot macro- and micronutrients were found lower in the near-isogenic GR-cultivars compared to their respective non-GR parental lines Shoot and root dry biomass were reduced by glyphosate with all GR cultivars evaluated in both soils. The lower biomass in GR soybeans compared to their isogenic normal lines probably represents additive effects from the decreased photosynthetic parameters as well as lower availability of nutrients in tissues of the glyphosate treated plants.     

Accelerated HER-2 degradation enhanced ovarian tumor recognition by CTL. Implications for tumor immunogenicity

We investigated the ubiquitination and degradation of a tumor antigen, the HER-2/neu (HER-2) protooncogene product which is overexpressed in epithelial cancers. HER-2 degradation was investigated in the ovarian tumor line, SKOV3.A2, that constitutively overexpressed long-life HER-2. We used as agonist geldanamycin (GA), which initiated downmodulation of HER-2 from the cell surface. HER-2 was polyubiquitinated and degraded faster in the presence than in the absence of GA. GA did not decrease HLA-A2 expression. Presentation of the immunodominant cytotoxic T lymphocyte (CTL) epitope, E75 (369–377) from SKOV.A2 was inhibited by proteasome inhibitors, such as LLnL but was enhanced by cysteine protease inhibitors such as E64, indicating that both the proteasome and cysteine proteases are involved in epitope formation but have different effects. Enhanced tumor recognition was not an immediate or early effect of GA treatment, but was evident after 20 h of GA treatment. In contrast, 20 h GA treatment did not increase tumor sensitivity to LAK cell lysis. Twenty hour GA-treated SKOV3.A2 cells expressed an unstable HER-2 protein synthesized in the presence of GA, of faster electrophoretic mobility than control HER-2. This suggested that the newly synthesized HER-2 in the presence of GA was the main source of epitopes recognized by CTL. Twenty hour GA-treated SKOV3.A2 cells were better inducers of CTL activity directed to a number of HER-2 CTL epitopes, in peripheral blood mononuclear cells compared with control untreated SKOV3.A2 cells. Thus, induction of HER-2 protein instability enhanced the sensitivity of tumor for CTL lysis. Increased HER-2 CTL epitopes presentation may have implications for overcoming the poor immuno-genicity of human tumors, and design of epitope precursors for cancer vaccination.     

Stimuli inducing gregarious colouration and behaviour in nymphs of Schistocerca gregaria

Solitarious nymphs of Schistocerca gregaria were reared under various conditions in both Jerusalem and Oxford to tease apart cues involved in behavioural and colour phase change. Treatments included rearing nymphs from the IInd or IIIrd until the final nymphal stadium in physical contact with similarly aged conspecific groups or with another locust species, Locusta migratoria migratorioides, as well as confining single nymphs in mesh cages, which were kept within crowds of S. gregaria or L. migratoria migratorioides, providing visual and olfactory but no physical contact with other locusts. In the Oxford experiments, an extra treatment was included which provided olfactory cues without visual or contact stimulation. Our results confirm that transformation from the solitarious to the gregarious phase of locusts is complex, and that different phase characteristics not only follow different time courses, but are also controlled by different suites of cues. As predicted from earlier studies, behavioural phase change was evoked by non-species-specific cues. Rearing in contact with either species was fully effective in inducing gregarious behaviour, as was the combination of the sight and smell of other locusts, but odour alone was ineffective. Colour phase change was shown to comprise two distinct elements that could be dissociated: black patterning and yellow background. The former of these could be induced as effectively by rearing S. gregaria nymphs in a crowd of L. migratoria migratorioides as by rearing with conspecifics. Sight and smell of other locusts also triggered black patterning and, unlike behavioural change, some black patterning was induced by odour cues alone. Hence, physical contact was not needed to induce gregarious black patterning. Yellow colouration, however, was only fully induced when locusts were reared in contact with conspecifics, implying the presence of a species-specific contact chemical cue.     

Arabidopsis thaliana,a versatile model system for study of eukaryotic genome-maintenance functions

The genome of the model plant Arabidopsis thaliana encodes many orthologs of human genome-maintenance proteins, and in several important cases plant DNA repair and mutation-antagonism functions resemble their mammalian counterparts more closely than do those of established microbial models. These orthologs, in conjunction with the powerful tools now available for work with Arabidopsis and the practical advantages of its small size and rapid life cycle, now make it an attractive model system for study of eukaryotic DNA repair and mutagenesis. Already, null mutations that inactivate proteins involved in repair of DNA double-strand breaks or in DNA translesion synthesis and are lethal in mice have proved to be tolerated by plants. This review compares in some detail the genome-maintenance activities encoded by plants, mammals and microbes, and describes important Arabidopsis tools and life cycle characteristics. It concludes with selected examples that illustrate Arabidopsis advantages and/or reveal new insights into genome-maintenance functions of general interest.     

High-Frequency Stimulation of the Subthalamic Nucleus Counteracts Cortical Expression of Major Histocompatibility Complex Genes in a Rat Model of Parkinson’s Disease

High-frequency stimulation of the subthalamic nucleus (STN-HFS) is widely used as therapeutic intervention in patients suffering from advanced Parkinson’s disease. STN-HFS exerts a powerful modulatory effect on cortical motor control by orthodromic modulation of basal ganglia outflow and via antidromic activation of corticofugal fibers. However, STN-HFS-induced changes of the sensorimotor cortex are hitherto unexplored. To address this question at a genomic level, we performed mRNA expression analyses using Affymetrix microarray gene chips and real-time RT-PCR in sensorimotor cortex of parkinsonian and control rats following STN-HFS. Experimental parkinsonism was induced in Brown Norway rats by bilateral nigral injections of 6-hydroxydopamine and was assessed histologically, behaviorally, and electrophysiologically. We applied prolonged (23h) unilateral STN-HFS in awake and freely moving animals, with the non-stimulated hemisphere serving as an internal control for gene expression analyses. Gene enrichment analysis revealed strongest regulation in major histocompatibility complex (MHC) related genes. STN-HFS led to a cortical downregulation of several MHC class II (RT1-Da, Db1, Ba, and Cd74) and MHC class I (RT1CE) encoding genes. The same set of genes showed increased expression levels in a comparison addressing the effect of 6-hydroxydopamine lesioning. Hence, our data suggest the possible association of altered microglial activity and synaptic transmission by STN-HFS within the sensorimotor cortex of 6-hydroxydopamine treated rats.     

Effects of 8 bromo-cyclic GMP on cyclic AMP levels in urine and tissues of hypothyroid rats.

The effects of 8-bromo-guanosine 3', 5' cyclic monophosphate (8Brcyclic GMP) on the levels of cyclic AMP in urine, brain, liver, and muscle were determined in hypothyroid rats. Cyclic AMP urinary levels were significantly lower in untreated hypothyroid rats than in normal rats, and when hypothyroid rats were treated with replacement thyroxine, urinary cyclic AMP returned to normal after six days of treatment. Hypothyroid rats treated with 8Brcyclic GMP, 30 mg/kg body weight subcutaneously every 12 hours exhibited a biphasic response. From days 2 through 4 of treatment, cyclic AMP excretion rose to approximately 2.5 times normal levels. Subsequently, the cyclic AMP excretion returned to hypothyroid levels and remained low throughout the rest of the treatment period. Tissue cyclic AMP levels in hypthyroid rats treated with 8Br-cyclic GMP were increased in comparison to those of untreated hypothyroid rats. Increase in brain was 134%, in liver 55%, and in muscle 42%. We conclude from these studies that 8Br-cyclic GMP can significantly alter cyclic AMP levels in hypothyroid rats.     

Modeling the onset and propagation of trabecular bone microdamage during low-cycle fatigue

Relatively small amounts of microdamage have been suggested to have a major effect on the mechanical properties of bone. A significant reduction in mechanical properties (e.g. modulus) can occur even before the appearance of microcracks. This study uses a novel non-linear microdamaging finite-element (FE) algorithm to simulate the low-cycle fatigue behavior of high-density trabecular bone. We aimed to investigate if diffuse microdamage accumulation and concomitant modulus reduction, without the need for complete trabecular strut fracture, may be an underlining mechanism for low-cycle fatigue failure (defined as a 30% reduction in apparent modulus). A microCT constructed FE model was subjected to a single cycle monotonic compression test, and constant and variable amplitude loading scenarios to study the initiation and accumulation of low-cycle fatigue microdamage. Microcrack initiation was simulated using four damage criteria: 30%, 40%, 50% and 60% reduction in bone element modulus (el-MR). Evaluation of structural (apparent) damage using the four different tissue level damage criteria resulted in specimen fatigue failure at 72, 316, 969 and 1518 cycles for the 30%, 40%, 50% and 60% el-MR models, respectively. Simulations based on the 50% el-MR model were consistent with previously published experimental findings. A strong, significant non-linear, power law relationship was found between cycles to failure (N) and effective strain (Deltasigma/E(0)): N=1.394x10(-25)(Deltasigma/E(0))(-12.17), r(2)=0.97, p<0.0001. The results suggest that microdamage and microcrack propagation, without the need for complete trabecular strut fracture, are mechanisms for high-density trabecular bone failure. Furthermore, the model is consistent with previous numerical fatigue simulations indicating that microdamage to a small number of trabeculae results in relatively large specimen modulus reductions and rapid failure.