cAMP-mediated increase in the critical cell size required for the G1 to S transition in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, cyclic AMP is required for cellular growth. In this study we show that cAMP also specifically inhibits the G1-S transition of the S. cerevisiae cell cycle by increasing the critical cell size required at start, the major yeast cell cycle control step. In fact: (a) addition of cAMP delays the time of entering into the S budded phase of small G1 cells, while it is ineffective on large fast-growing cells. (b) If cell growth is strongly depressed, cAMP permanently inhibits cell cycle commitment of cells arrested at the alpha-factor-sensitive step. The cell fraction inhibited by cAMP is inversely correlated with the average cell size of treated populations. (c) The critical protein content (Ps) and the critical cell volume (VB) required for budding in unperturbed exponentially growing yeast populations are largely increased by cAMP. On these bases, we propose a new cAMP role at start.     

Distribution of artemisinin and bioactive flavonoids from Artemisia annua L. during plant growth

Artemisia annua L. (Qinghao, Asteraceae) is a promising and potent antimalarial herbal drug. Its activity has been ascribed to the content of artemisinin, a sesquiterpene lactone that is very effective against drug-resistant Plasmodium. Many studies have pointed out that the presence of polymethoxyflavonoids in the phytocomplex can enhance the bioavailability or the activity of artemisinin. In this study the production of both artemisinin and flavonoids by plants of an aromatic ecotype of A. annua L. was characterized in different aerial parts of the plants at different developmental stages. The qualitative profile of the investigated plant parts was similar; in addition to artemisinin, four flavonoids were identified: chrysoplenetin, casticin, eupatin and artemetin. The highest contents of both flavonoids and artemisinin were found at the full blooming stage. At this developmental stage, artemisinin was higher in leaves than in inflorescences, while the total flavonoid levels were similar in both plant organs.     

Raft-like microdomains play a key role in mitochondrial impairment in lymphoid cells from patients with Huntington's disease

Huntington's disease (HD) is a genetic neurodegenerative disease characterized by an exceedingly high number of contiguous glutamine residues in the translated protein, huntingtin (Htt). The primary site of cell toxicity is the nucleus, but mitochondria have been identified as key components of cell damage. The present work has been carried out in immortalized lymphocytes from patients with HD. These cells, in comparison with lymphoid cells from healthy subjects, displayed: i) a redistribution of mitochondria, forming large aggregates; ii) a constitutive hyperpolarization of mitochondrial membrane; and iii) a constitutive alteration of mitochondrial fission machinery, with high apoptotic susceptibility. Moreover, mitochondrial fission molecules, e.g., protein dynamin-related protein 1, as well as Htt, associated with mitochondrial raft-like microdomains, glycosphingolipid-enriched structures detectable in mitochondria. These findings, together with the observation that a ceramide synthase inhibitor and a raft disruptor are capable of impairing the peculiar mitochondrial remodeling in HD cells, suggest that mitochondrial alterations occurring in these cells could be due to raft-mediated defects of mitochondrial fission/fusion machinery.     

Clostridium difficile toxin B induces senescence in enteric glial cells: A potential new mechanism of Clostridium difficile pathogenesis

Clostridium difficile infection (CDI) causes nosocomial/antibiotic-associated diarrhea and pseudomembranous colitis, with dramatic incidence/mortality worldwide. C. difficile virulence factors are toxin A and toxin B (TcdB) which cause cytopathic/cytotoxic effects and inflammation. Until now studies were focused on molecular effects of C. difficile toxins (Tcds) on different cells while unexplored aspect is the status/fate of cells that survived their cytotoxicity. Recently we demonstrated that enteric glial cells (EGCs) are susceptible to TcdB cytotoxicity, but several EGCs survived and were irreversibly cell-cycle arrested and metabolically active, suggesting that EGCs could became senescent. This is important because allowed us to evaluate the not explored status/fate of cells surviving Tcds cytotoxicity, and particularly if TcdB induces senescence in EGCs.Rat-transformed EGCs were treated with 10?ng/ml TcdB for 6?h–48?h, or for 48?h, followed by incubation for additional 4 or 11?days in absence of TcdB (6 or 13 total days). Senescence markers/effectors were examined by specific assays.TcdB induces senescence in EGCs, as demonstrated by the senescence markers: irreversible cell-cycle arrest, senescence-associated-β?galactosidase positivity, flat morphology, early and persistent DNA damage (ATM and H2AX phosphorylation), p27 overexpression, pRB hypophosphorylation, c?Myc, cyclin B1, cdc2 and phosphorylated-cdc2 downregulation, Sirtuin?2 and Sirtuin?3 overexpression. TcdB-induced EGC senescence is dependent by JNK and AKT activation but independent by ROS, p16 and p53/p21 pathways.In conclusion, TcdB induces senescence in EGCs. The extrapolation of these results to CDI leads to hypothesize that EGCs that survived TcdB, once they have acquired a senescence state, could cause irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), and tumors due to persistent inflammation, transfer of senescence status and stimulation of pre-neoplastic cells.     

Fertility in postpartum dairy cows in winter or summer following estrus synchronization and fixed time AI after the induction of an LH surge with GnRH or hCG

In this study, the fertility of postpartum dairy cows after a sequence of treatments with GnRH (Day 0), PGF2alpha (Day 7) and GnRH (Day 9) (GnRH group; n = 164) or hCG (Day 0), PGF2alpha (Day 7) and hCG (Day 9) (group hCG; n = 166) was investigated in summer and winter seasons. All cows were artificially inseminated without estrus detection, 16-18 h after the end of treatment. Control cows (CONT; n = 226) were not treated and were inseminated at natural estrus. The pregnancy rates at Day 90 (46% versus 33%; P < 0.05) and at Day 135 (76% versus 62%; P < 0.05) postpartum were significantly lower in CONT cows in summer compared to winter months but this effect was not observed in the two treated groups. The number of days from calving to conception was significantly lower in GnRH and hCG treatment groups compared to CONT cows in cold months (102 +/- 3.2, 106 +/- 4.2, 126 +/- 3.1, respectively; P < 0.001) and in hot months (112 +/- 3.2, 114 +/- 4.2, 139 +/- 3.1, respectively; P < 0.001). The concentration of insulin was significantly higher in winter (P < 0.001). There were no differences in average plasma concentration of glucose (P = 0.474), GH (P = 0.441) or IGF-I (P = 0.190). In conclusion, we have shown that veterinary supervision combined with a program of estrous synchronization and fixed time insemination can improve fertility of cows suffering heat stress.     

Effectiveness of a microbial formula,as a bioaugmentation agent,tailored for bioremediation of diesel oil and heavy metal co-contaminated soil

The activation of natural bioremediation potentials is the challenge that research is currently addressing for overcoming bottlenecks still affecting bioremediation applications. Bioaugmentation is one possible way to activate such natural potentials, provided that the biodiversity introduced to increase catabolically relevant capacity is identified also considering the ecological context. The present work deals with bioaugmentation aimed at the remediation of a soil co-contaminated (spiked) with both diesel oil (1%, v/w), and heavy metals (Pb and Zn), using intact soil core microcosms in different experimental conditions. We supposed that both heavy metal resistance and active metabolism towards organic pollutants are essential metabolic traits to trap the energetic flux, which drives the microbial community towards biodegradation under the given experimental conditions. Consequently, the bioaugmentation was performed by introducing a tailor made microbial formula composed of 12 allochthonous strains. They belong to a stable population previously isolated from a chronic polluted site and are both hydrocarbon degraders and heavy metal resistant and, also, compatible with the autochthonous microbial community. The active role of the microbial formula in pushing the entire community towards an effective bioremediation of diesel oil close to 75%, in the presence of bioavailable metals, has been proven through hydrocarbons analysis, metabolic and molecular profiling at community level (Biolog system, DGGE).     

Constitutive localization of DR4 in lipid rafts is mandatory for TRAIL-induced apoptosis in B-cell hematologic malignancies

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) acts as an apoptosis inducer for cancer cells sparing non-tumor cell targets. However, several phase I/II clinical trials have shown limited benefits of this molecule. In the present work, we investigated whether cell susceptibility to TRAIL ligation could be due to the presence of TRAIL death receptors (DRs) 4 and 5 in membrane microdomains called lipid rafts. We performed a series of analyses, either by biochemical methods or fluorescence resonance energy transfer (FRET) technique, on normal cells (i.e. lymphocytes, fibroblasts, endothelial cells), on a panel of human cancer B-cell lines as well as on CD19⁺ lymphocytes from patients with B-chronic lymphocytic leukemia, treated with different TRAIL ligands, that is, recombinant soluble TRAIL, specific agonistic antibodies to DR4 and DR5, or CD34⁺ TRAIL-armed cells. Irrespective to the expression levels of DRs, a molecular interaction between ganglioside GM3, abundant in lymphoid cells, and DR4 was detected. This association was negligible in all non-transformed cells and was strictly related to TRAIL susceptibility of cancer cells. Interestingly, lipid raft disruptor methyl-beta-cyclodextrin abrogated this susceptibility, whereas the chemotherapic drug perifosine, which induced the recruitment of TRAIL into lipid microdomains, improved TRAIL-induced apoptosis. Accordingly, in ex vivo samples from patients with B-chronic lymphocytic leukemia, the constitutive embedding of DR4 in lipid microdomains was associated per se with cell death susceptibility, whereas its exclusion was associated with TRAIL resistance. These results provide a key mechanism for TRAIL sensitivity in B-cell malignances: the association, within lipid microdomains, of DR4 but not DR5, with a specific ganglioside, that is the monosialoganglioside GM3. On these bases we suggest that lipid microdomains could exert a catalytic role for DR4-mediated cell death and that an ex vivo quantitative FRET analysis could be predictive of cancer cell sensitivity to TRAIL.     

Rrp1, a cyclic-di-GMP-producing response regulator, is an important regulator of Borrelia burgdorferi core cellular functions

Two-component systems (TCS) are universal among bacteria and play critical roles in gene regulation. Our understanding of the contributions of TCS in the biology of the Borrelia is just now beginning to develop. Borrelia burgdorferi , a causative agent of Lyme disease, harbours a TCS comprised of open reading frames (ORFs) BB0419 and BB0420. BB0419 encodes a response regulator designated Rrp1, and BB0420 encodes a hybrid histidine kinase–response regulator designated Hpk1. Rrp1, which contains a conserved GGDEF domain, undergoes phosphorylation and produces the secondary messenger, cyclic diguanylate (c-di-GMP), a critical signaling molecule in numerous organisms. However, the regulatory role of the Rrp1–Hpk1 TCS and c-di-GMP signaling in Borrelia biology are unexplored. In this study, the distribution, conservation, expression and potential global regulatory capability of Rrp1 were assessed. rrp1 was found to be universal and highly conserved among isolates, co-transcribed with hpk1 , constitutively expressed during in vitro cultivation, and significantly upregulated upon tick feeding. Allelic exchange replacement and microarray analyses revealed that the Rrp1 regulon consists of a large number of genes encoded by the core Borrelia genome (linear chromosome, linear plasmid 54 and circular plasmid 26) that encode for proteins involved in central metabolic processes and virulence mechanisms including immune evasion.     

Upward squatting in individuals with and without patellofemoral pain syndrome: a biomechanical study

The purpose of the study was to test the hypothesis on whether individuals with patellofemoral pain syndrome (PFPS) try to avoid knee position during upward squatting so as not to aggravate this syndrome. Also, we tested whether PFPS would generate changes in the kinetic and electromyographic (EMG) strategies used to perform this task. Eight healthy subjects and 8 subjects with PFPS, but without a history of pain for at least 30 days, took part in the experiment. They were asked to perform upward squatting with knees initially flexed at 60° (very flexed) until reaching an upright position. Angle, velocity, and acceleration (kinematic) were reconstructed for knee and ankle joints. The torques at these joints were calculated using inverse dynamics, taking into account anthropometric and inertial characteristics of each subject, including records from force data. Only activities of major muscles were recorded. The kinetic and EMG profiles were quantified during acceleration and deceleration phases of the upward squatting. Both healthy and PFPS subjects used the same kinetic and EMG strategies to perform the upward squatting, even though the magnitude of the muscle activities were decreased for the latter group. Compared to the control group, the PFPS subjects presented larger joint ankle torques and smaller knee joint torques. However, the subjects avoided keeping their knees very flexed at the initial position. Group differences in the kinetic and EMG strategies can be explained by differences in the initial position, suggesting a protective strategy used by subjects with PFPS. Therefore, for these subjects, coaches and therapists should avoid using this exercise when the knee is required to move above 40° flexion.