A gustatory mutant of Drosophila defective in pyranose receptors

Summary Mutations in an X-linked gene, gust-A, block the responses of Drosophila melanogaster to a group of pyranose sugars. It is shown that the behavioural effects of this mutation are correlated with a loss of electrical responses in taste receptors. The mutation affects the chemoacceptors for pyranose sugars leaving the furanose acceptors intact.     

alpha2-Adrenoceptors control the release of noradrenaline but not neuropeptide Y from perivascular nerve terminals

Neuropeptide Y (NPY) and noradrenaline (NA) are co-transmitters at many sympathetic synapses, but it is not yet clear if their release is independently regulated. To address this question, we quantified the electrically evoked release of these co-transmitters from perivascular nerve terminals to the mesenteric circulation in control and drug-treated rats. 6-Hydroxydopamine reduced the tissue content and the electrically evoked release of ir-NPY and NA as well as the rise in perfusion pressure. A 0.001 mg/kg reserpine reduced the content of ir-NPY and NA, but did not modify their release nor altered the rise in perfusion pressure elicited by the electrical stimuli. However, 0.1mg/kg reserpine reduced both the content and release of NA but decreased only the content but not the release of ir-NPY; the rise in perfusion pressure was halved. Clonidine did not affect the release of ir-NPY while it lowered the outflow of NA, not altering the rise in perfusion pressure elicited by the electrical stimuli. Yohimbine, did not modify the release of ir-NPY but increased the NA outflow, it antagonized the clonidine effect. Therefore, presynaptic alpha2-adrenoceptors modulate the release of NA but not NPY, implying separate regulatory mechanisms.     

Fused in Sarcoma (FUS) Protein Lacking Nuclear Localization Signal (NLS) and Major RNA Binding Motifs Triggers Proteinopathy and Severe Motor Phenotype in Transgenic Mice

Dysfunction of two structurally and functionally related proteins, FUS and TAR DNA-binding protein of 43 kDa (TDP-43), implicated in crucial steps of cellular RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. The proteins are intrinsically aggregate-prone and form non-amyloid inclusions in the affected nervous tissues, but the role of these proteinaceous aggregates in disease onset and progression is still uncertain. To address this question, we designed a variant of FUS, FUS 1–359, which is predominantly cytoplasmic, highly aggregate-prone, and lacks a region responsible for RNA recognition and binding. Expression of FUS 1–359 in neurons of transgenic mice, at a level lower than that of endogenous FUS, triggers FUSopathy associated with severe damage of motor neurons and their axons, neuroinflammatory reaction, and eventual loss of selective motor neuron populations. These pathological changes cause abrupt development of a severe motor phenotype at the age of 2.5–4.5 months and death of affected animals within several days of onset. The pattern of pathology in transgenic FUS 1–359 mice recapitulates several key features of human ALS with the dynamics of the disease progression compressed in line with shorter mouse lifespan. Our data indicate that neuronal FUS aggregation is sufficient to cause ALS-like phenotype in transgenic mice.     

Hindlimb Suspension and SPE-Like Radiation Impairs Clearance of Bacterial Infections

A major risk of extended space travel is the combined effects of weightlessness and radiation exposure on the immune system. In this study, we used the hindlimb suspension model of microgravity that includes the other space stressors, situational and confinement stress and alterations in food intake, and solar particle event (SPE)-like radiation to measure the combined effects on the ability to control bacterial infections. A massive increase in morbidity and decrease in the ability to control bacterial growth was observed using 2 different types of bacteria delivered by systemic and pulmonary routes in 3 different strains of mice. These data suggest that an astronaut exposed to a strong SPE during extended space travel is at increased risk for the development of infections that could potentially be severe and interfere with mission success and astronaut health.     

The nuclear experience of CPEB: Implications for RNA processing and translational control

CPEB is a sequence-specific RNA binding protein that promotes polyadenylation-induced translation in early development, during cell cycle progression and cellular senescence, and following neuronal synapse stimulation. It controls polyadenylation and translation through other interacting molecules, most notably the poly(A) polymerase Gld2, the deadenylating enzyme PARN, and the eIF4E-binding protein Maskin. Here, we report that CPEB shuttles between the nucleus and cytoplasm and that its export occurs via the CRM1-dependent pathway. In the nucleus of Xenopus oocytes, CPEB associates with lampbrush chromosomes and several proteins involved in nuclear RNA processing. CPEB also interacts with Maskin in the nucleus as well as with CPE-containing mRNAs. Although the CPE does not regulate mRNA export, it influences the degree to which mRNAs are translationally repressed in the cytoplasm. Moreover, CPEB directly or indirectly mediates the alternative splicing of at least one pre-mRNA in mouse embryo fibroblasts as well as certain mouse tissues. We propose that CPEB, together with Maskin, binds mRNA in the nucleus to ensure tight translational repression upon export to the cytoplasm. In addition, we propose that nuclear CPEB regulates specific pre-mRNA alternative splicing.     

Cytokine profiles of HeLa and human diploid cells induced by different fractions of Vibrio parahaemolyticus cultures exposed to stress conditions

Vibrio (V.) parahaemolyticus is an aquatic halophilic bacteria which produces gastroenteritis and in rare cases septicaemia after the consumption of raw or under-cooked contaminated seafood.The severity of diarrheal illness caused by this bacterium is closely related to the presence of two types of hemolysins (the thermostable direct hemolysin-TDH and TDH related hemolysin-TRH) and also of type III secretion system (TTSS) proteins. The TTSS type 1 induces a wide array of effects on infected HeLa cells such as autophagy, oncosis, cell rounding and lysis. Previous studies have shown that heat shock proteins have the ability to stimulate the production of interleukins in different cellular cultures. In our studies we have stimulated two cellular lines (HeLa and human diploid cells) with different V. parahaemolyticus culture fractions in order to observe the effect on cytokines production. Thus, the purpose of this study was to analyze the expression of IL-1, IL-2, IL-4, IL-6, IL-10 and TNF-alpha induced by the cell treatment with total cellular lysate, periplasmic fractions and culture supernatants extracted from V. parahaemolyticus exposed to normal and also to stress conditions. The ELISA assay of the cytokine profile of the HeLa and HDC cell lines stimulated with different bacterial fractions revealed that in the V. parahemolyticus cultures submitted to osmotic and heat shock stress are accumulating factors (probably heat shock proteins) which are exhibiting immunomodulatory activity, responsible for the induction of a pro-inflammatory response associated with increased levels of IL-6 and TNF-alpha expression, however balanced by the stimulation of the anti-inflammatory cytokine IL-4 synthesis.     

Anhydrobiosis-associated nuclear DNA damage and repair in the sleeping chironomid: linkage with radioresistance

Anhydrobiotic chironomid larvae can withstand prolonged complete desiccation as well as other external stresses including ionizing radiation. To understand the cross-tolerance mechanism, we have analyzed the structural changes in the nuclear DNA using transmission electron microscopy and DNA comet assays in relation to anhydrobiosis and radiation. We found that dehydration causes alterations in chromatin structure and a severe fragmentation of nuclear DNA in the cells of the larvae despite successful anhydrobiosis. Furthermore, while the larvae had restored physiological activity within an hour following rehydration, nuclear DNA restoration typically took 72 to 96 h. The DNA fragmentation level and the recovery of DNA integrity in the rehydrated larvae after anhydrobiosis were similar to those of hydrated larvae irradiated with 70 Gy of high-linear energy transfer (LET) ions ((4)He). In contrast, low-LET radiation (gamma-rays) of the same dose caused less initial damage to the larvae, and DNA was completely repaired within within 24 h. The expression of genes encoding the DNA repair enzymes occurred upon entering anhydrobiosis and exposure to high- and low-LET radiations, indicative of DNA damage that includes double-strand breaks and their subsequent repair. The expression of antioxidant enzymes-coding genes was also elevated in the anhydrobiotic and the gamma-ray-irradiated larvae that probably functions to reduce the negative effect of reactive oxygen species upon exposure to these stresses. Indeed the mature antioxidant proteins accumulated in the dry larvae and the total activity of antioxidants increased by a 3-4 fold in association with anhydrobiosis. We conclude that one of the factors explaining the relationship between radioresistance and the ability to undergo anhydrobiosis in the sleeping chironomid could be an adaptation to desiccation-inflicted nuclear DNA damage. There were also similarities in the molecular response of the larvae to damage caused by desiccation and ionizing radiation.     

Polyamine depletion with two different polyamine analogues causes DNA damage in human breast cancer cell lines

It is well known that the positively charged polyamines have a DNA-stabilizing function and that polyamine depletion alters chromatin function. We have previously shown that polyamine depletion causes an S phase prolongation, and others have shown that there is an accumulation of Okazaki-like fragments in polyamine-depleted cells. In the present study, we have used the comet assay to investigate polyamine depletion-induced DNA strand breaks. Three breast cancer cell lines and one normal-like breast cell line were treated with the polyamine analogue N(1),N(11)-diethylnorspermine or with the polyamine biosynthesis inhibitor 4-amidinoindan-1-one 2'-amidinohydrazone (CGP 48664). The comet assay showed that polyamine depletion resulted in DNA strand breaks. We also show that these DNA strand breaks occurred in cells where there was no expression of gamma-H2AX, which is a marker of DNA double-strand breaks. Thus, our conclusion is that polyamine depletion causes DNA single-strand breaks, which may be the cause for the observed delay in S phase progression.