A mouse model for monitoring calpain activity under physiological and pathological conditions

Calpains are Ca(2+)-dependent cysteine proteases known to be important for the regulation of cell functions and which aberrant activation causes cell death in a number of degenerative disorders. To provide a tool for monitoring the status of calpain activity in vivo under physiological and pathological conditions, we created a mouse model that expresses ubiquitously a fluorescent reporter consisting of eCFP and eYFP separated by a linker cleavable by the ubiquitous calpains. We named this mouse CAFI for calpain activity monitored by FRET imaging. Our validation studies demonstrated that the level of calpain activity correlates with a decrease in FRET (fluorescence resonance energy transfer) between the two fluorescent proteins. Using this model, we observed a small level of activity after denervation and fasting, a high level of activity during muscle regeneration and ischemia, and local activity in damaged myofibers after exercise. Finally, we crossed the CAFI mouse with the alpha-sarcoglycan-deficient model, demonstrating an increase of calpain activity at the steady state. Altogether, our results present evidence that CAFI mice could be a valuable tool in which to follow calpain activity at physiological levels and in disease states.     

Activation and regulation of platelet-activating factor receptor: role of G(i) and G(q) in receptor-mediated chemotactic, cytotoxic, and cross-regulatory signals

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycerolphosphocholine; PAF) induces leukocyte accumulation and activation at sites of inflammation via the activation of a specific cell surface receptor (PAFR). PAFR couples to both pertussis toxin-sensitive and pertussis toxin-insensitive G proteins to activate leukocytes. To define the role(s) of G(i) and G(q) in PAF-induced leukocyte responses, two G-protein-linked receptors were generated by fusing G alpha(i3) (PAFR-G alpha(i3)) or G alpha(q) (PAFR-G alpha(q)) at the C terminus of PAFR. Rat basophilic leukemia cell line (RBL-2H3) stably expressing wild-type PAFR, PAFR-G alpha(i3), or PAFR-G alpha(q) was generated and characterized. All receptor variants bound PAF with similar affinities to mediate G-protein activation, intracellular Ca2+ mobilization, phosphoinositide (PI) hydrolysis, and secretion of beta-hexosaminidase. PAFR-G alpha(i3) and PAFR-G alpha(q) mediated greater GTPase activity in isolated membranes than PAFR but lower PI hydrolysis and secretion in whole cells. PAFR and PAFR-G alpha(i3), but not PAFR-G alpha(q), mediated chemotaxis to PAF. All three receptors underwent phosphorylation and desensitization upon exposure to PAF but only PAFR translocated beta arrestin to the cell membrane and internalized. In RBL-2H3 cells coexpressing the PAFRs along with CXCR1, IL-8 (CXCL8) cross-desensitized Ca2+ mobilization to PAF by all the receptors but only PAFR-G alpha(i3) activation cross-inhibited the response of CXCR1 to CXCL8. Altogether, the data indicate that G(i) exclusively mediates chemotactic and cross-regulatory signals of the PAFR, but both G(i) and G(q) activate PI hydrolysis and exocytosis by this receptor. Because chemotaxis and cross-desensitization are exclusively mediated by G(i), the data suggest that differential activation of both G(i) and G(q) by PAFR likely mediate specific as well as redundant signaling pathways.     

Effect of recombinant bovine somatotropin (bST) on follicular population and on in vitro buffalo embryo production

The objective of this study was to evaluate the effect of bovine somatotropin (bST) on ovarian follicular population in buffalo heifers and its influence on oocyte quality, recovery rates and in vitro embryo production. We tested the hypothesis that bST treatment in buffalo females submitted to an ovum pick-up (OPU) program would improve the number of follicles recruited, oocyte quality and in vitro embryo production. A total of 10 heifers were assigned into two treatment groups: group bST (n=5; receiving 500 mg of bST in regular intervals) and control group (n=5; without additional treatment). Both groups were subjected to OPU sessions twice a week (every 3 or 4 days), for a total of 10 sessions per female, although due to procedural problems, only the first five OPU sessions produced embryos. The number of follicles and the diameters were recorded at all OPU sessions. The harvested oocytes were counted and classified according to their quality as either A, B, C, D or E, with A and B considered good quality. Cleavage and blastocyst production rates were evaluated 2 and 7 days after in vitro fertilization, respectively. The bST treatment increased the total number of antral follicles (>3mm in diameter; 12.2 compared with 8.7; p<0.05) and of small antral follicles (<5mm; 9.1 compared with 6.5; p<0.05) per OPU session. The bST also tended to increase the number of oocytes recovered per session (5.2 compared with 4.1; p=0.07), and enhanced the percentage of good quality oocytes (48.8% compared with 40.6%; p=0.07). bST showed no effect on cleavage and blastocyst production rates (p>0.05). The significant effects of performing repeated OPU sessions were decreasing the follicular population (p<0.001) as well as the number of follicles aspirated (p<0.001), and oocytes recovered (p<0.02). In conclusion, bST treatment improves the follicular population, demonstrating its possible application in buffalo donors submitted to OPU programs.     

Molecular Mechanism of the Bifunctional Role of Lipopolysaccharide in Osteoclastogenesis

Lipopolysaccharide (LPS), a common bacteria-derived product, has long been recognized as a key factor implicated in periodontal bone loss. However, the precise cellular and molecular mechanisms by which LPS induces bone loss still remains controversial. Here, we show that LPS inhibited osteoclastogenesis from freshly isolated osteoclast precursors but stimulated osteoclast formation from those pretreated with RANKL in vitro in tissue culture dishes, bone slices, and a co-culture system containing osteoblasts, indicating that RANKL-mediated lineage commitment is a prerequisite for LPS-induced osteoclastogenesis. Moreover, the RANKL-mediated lineage commitment is long term, irreversible, and TLR4-dependent. LPS exerts the dual function primarily by modulating the expression of NFATc1, a master regulator of osteoclastogenesis, in that it abolished RANKL-induced NFATc1 expression in freshly isolated osteoclast precursors but stimulated its expression in RANKL-pretreated cells. In addition, LPS prolonged osteoclast survival by activating the Akt, NF-κB, and ERK pathways. Our current work has not only unambiguously defined the role of LPS in osteoclastogenesis but also has elucidated the molecular mechanism underlying its complex functions in osteoclast formation and survival, thus laying a foundation for future delineation of the precise mechanism of periodontal bone loss.LPS,² a common bacteria-derived product, has long been recognized as a key factor implicated in the development of chronic periodontitis. LPS plays an important role in periodontitis by initiating a local host response in gingival tissues that involves recruitment of inflammatory cells, production of prostanoids and cytokines, elaboration of lytic enzymes and activation of osteoclast formation and function to induce bone loss (1-3).Osteoclasts, the body''s sole bone-resorbing cells, are multinucleated giant cells that differentiate from cells of hematopoietic lineage upon stimulation by two critical factors: the macrophage/monocyte colony-forming factor (M-CSF) and the receptor activator of NF-κB ligand (RANKL) (4-6). RANKL exerts its effects on osteoclast formation and function by binding to its receptor, RANK (receptor activator of NF-κB) expressed on osteoclast precursors and mature osteoclasts (7-9). RANKL also has a decoy receptor, osteoprotegerin, which inhibits RANKL action by competing with RANK for binding RANKL (10, 11).RANK is a member of the tumor necrosis factor receptor (TNFR) family (12). Members of the TNFR family lack intrinsic enzymatic activity, and hence they transduce intracellular signals by recruiting various adaptor proteins including TNF receptor-associated factors (TRAFs) through specific motifs in the cytoplasmic domain (13, 14). It has been established that RANK contains three functional TRAF-binding sites (³⁶⁹PFQEP³⁷³, ⁵⁵⁹PVQEET⁵⁶⁴, and ⁶⁰⁴PVQEQG⁶⁰⁹) that, redundantly, play a role in osteoclast formation and function (15, 16). Collectively, through these functional TRAF-binding motifs, RANK activates six major signaling pathways, NF-κB, JNK, ERK, p38, NFATc1, and Akt, which play important roles in osteoclast formation, function, and/or survival (15, 17-19). In particular, NFATc1 has been established as a master regulator of osteoclast differentiation (20-22).The involvement of osteoclasts in the pathogenesis of periodontal bone loss is supported by observations that osteoclasts are physically present and functionally involved in bone resorption in periodontal tissues (23-27). RANKL and RANK knockout mice develop osteopetrosis and show failure in tooth eruption due to a lack of osteoclasts (24, 25, 28). Moreover, op/op mice, in which a mutation in the coding region of the M-CSF gene generates a stop codon that leads to premature termination of translation of M-CSF mRNA, also show osteopetrosis and failure in tooth eruption due to a defect in osteoclast development (26, 27).Whereas the role of osteoclasts in periodontal disease associated alveolar bone destruction has been well established, the precise role of LPS in osteoclastogenesis still remains controversial. The vast majority of the previous studies demonstrated that LPS stimulates osteoclastogenesis. This is consistent with the role that LPS, a well recognized pathogenic factor in periodontitis, presumably plays in periodontal bone loss (29-33). However, two previous studies demonstrated, surprisingly, that LPS plays bifunctional roles in osteoclastogenesis in that although it inhibits osteoclast formation from normal osteoclast precursors, it reverses to promote osteoclastogenesis from osteoclast precursors pretreated with RANKL (34, 35). Given that this finding is inconsistent with the presumed role of LPS as a pathogenic factor in periodontal bone loss and lacks careful and further validation, the prevalent view is still that LPS stimulates osteoclastogenesis (1-3). Importantly, if LPS indeed has a dual function in osteoclastogenesis, the molecular mechanism by which LPS exerts a dual effect on osteoclastogenesis need to be further elucidated.In the present work, using various in vitro assays, we have demonstrated independently that LPS inhibits osteoclastogenesis from normal osteoclast precursors but promotes the development of osteoclasts from RANKL-pretreated cells in tissue culture dishes and bone slices in single-cell and co-culture settings, confirming the two previous observations that LPS play a bifunctional role in osteoclastogenesis (34, 35). Moreover, we have further shown that the RANKL-mediated lineage commitment is long term and irreversible in LPS-mediated osteoclastogenesis. More importantly, we have revealed that LPS inhibits osteoclastogenesis by suppressing NFATc1 expression and JNK activation while it prolongs osteoclast survival by activating the Akt, NF-κB, and ERK pathways. These studies have not only unambiguously and precisely defined the role of LPS in osteoclastogenesis but, more importantly, may also lead to a paradigm shift in future investigation of the molecular mechanism of periodontal bone loss.     

Epigenetics,oxidative stress,and Alzheimer disease

Alzheimer disease (AD) is a progressive neurodegenerative disorder whose clinical manifestations appear in old age. The sporadic nature of 90% of AD cases, the differential susceptibility to and course of the illness, as well as the late age onset of the disease suggest that epigenetic and environmental components play a role in the etiology of late-onset AD. Animal exposure studies demonstrated that AD may begin early in life and may involve an interplay between the environment, epigenetics, and oxidative stress. Early life exposure of rodents and primates to the xenobiotic metal lead (Pb) enhanced the expression of genes associated with AD, repressed the expression of others, and increased the burden of oxidative DNA damage in the aged brain. Epigenetic mechanisms that control gene expression and promote the accumulation of oxidative DNA damage are mediated through alterations in the methylation or oxidation of CpG dinucleotides. We found that environmental influences occurring during brain development inhibit DNA-methyltransferases, thus hypomethylating promoters of genes associated with AD such as the β-amyloid precursor protein (APP). This early life imprint was sustained and triggered later in life to increase the levels of APP and amyloid-β (Aβ). Increased Aβ levels promoted the production of reactive oxygen species, which damage DNA and accelerate neurodegenerative events. Whereas AD-associated genes were overexpressed late in life, others were repressed, suggesting that these early life perturbations result in hypomethylation as well as hypermethylation of genes. The hypermethylated genes are rendered susceptible to Aβ-enhanced oxidative DNA damage because methylcytosines restrict repair of adjacent hydroxyguanosines. Although the conditions leading to early life hypo- or hypermethylation of specific genes are not known, these changes can have an impact on gene expression and imprint susceptibility to oxidative DNA damage in the aged brain.     

Effect of host plant on susceptibility of whitefly Bemisia tabaci (Homoptera: Aleyrodidae) to the entomopathogenic fungus Beauveria bassiana (Ascomycota: Hypocreales)

We determined host plant effect on susceptibility of whitefly Bemisia tabaci to the entomopathogenic fungus Beauveria bassiana under controlled conditions. Insects were reared on cucumber, eggplant, tomato or cabbage. Fungal suspensions of 1×10⁴, 10⁵, 10⁶, 10⁷ and 10⁸ conidia/mL were applied on second-instar nymphs. Nymphal survival significantly differed among different host plant species on which the nymphs were reared. Ten days after inoculation with 1×10⁸ conidia/mL, percent survival was 4.2±0.7, 9.6±0.4, 13.4±0.8, and 24.3±0.9% on cucumber, eggplant, tomato and cabbage, respectively. Average survival times of nymphs were also significantly influenced by host plant species. After inoculation with 1×10⁸ conidia/mL, survival times were 4.8±0.15, 6.0±0.11, 5.7±0.13, and 6.2±0.08 days for nymphs reared on cucumber, eggplant, tomato, and cabbage, respectively. Virulence also differed depending on host plant species; 10 days after inoculation, LC₅₀ values were 4.6×10⁴, 1.6×10⁵, 4.2×10⁵ and 2.1×10⁶ conidia/mL on cucumber, eggplant, tomato and cabbage, respectively. Nymphs on cucumber showed highest susceptibility.     

A facile synthesis, structure, and antimicrobial evaluation of novel 4-arylhydrazono-5-trifluoromethyl-2,4-dihydropyrazol-3-ones, their N- and N,O-bis-β-d-glucosides

Synthesis of some novel 4-arylhydrazono-5-trifluoromethyl-2,4-dihydropyrazol-3-ones, their N- and N,O-bis- β-d-glucosides is described. Antimicrobial evaluation of eight selected compounds against Aspergillus fumigatus RCMB 002008 (1), Penicillium italicum RCMB 001018 (1), Syncephalastrum racemosum RCMB 016001, Candida albicans RCMB 005003, Staphylococcus aureus RCMB 106-001 (1), Pseudomonas aeruginosa RCMB 102-002, Bacillus subtilis RCMB 101-001, and Escherichia coli RCMB 103-001 has been achieved. The screening results indicated that all the tested compounds exhibited different inhibitory effects against five to seven different organisms of the eight test organisms.     

MicroRNA-122 Inhibits Tumorigenic Properties of Hepatocellular Carcinoma Cells and Sensitizes These Cells to Sorafenib

MicroRNAs are negative regulators of protein coding genes. The liver-specific microRNA-122 (miR-122) is frequently suppressed in primary hepatocellular carcinomas (HCCs). In situ hybridization demonstrated that miR-122 is abundantly expressed in hepatocytes but barely detectable in primary human HCCs. Ectopic expression of miR-122 in nonexpressing HepG2, Hep3B, and SK-Hep-1 cells reversed their tumorigenic properties such as growth, replication potential, clonogenic survival, anchorage-independent growth, migration, invasion, and tumor formation in nude mice. Further, miR-122-expressing HCC cells retained an epithelial phenotype that correlated with reduced Vimentin expression. ADAM10 (a distintegrin and metalloprotease family 10), serum response factor (SRF), and insulin-like growth factor 1 receptor (Igf1R) that promote tumorigenesis were validated as targets of miR-122 and were repressed by the microRNA. Conversely, depletion of the endogenous miR-122 in Huh-7 cells facilitated their tumorigenic properties with concomitant up-regulation of these targets. Expression of SRF or Igf1R partially reversed tumor suppressor function of miR-122. Further, miR-122 impeded angiogenic properties of endothelial cells in vitro. Notably, ADAM10, SRF, and Igf1R were up-regulated in primary human HCCs compared with the matching liver tissue. Co-labeling studies demonstrated exclusive localization of miR-122 in the benign livers, whereas SRF predominantly expressed in HCC. More importantly, growth and clonogenic survival of miR-122-expressing HCC cells were significantly reduced upon treatment with sorafenib, a multi-kinase inhibitor clinically effective against HCC. Collectively, these results suggest that the loss of multifunctional miR-122 contributes to the malignant phenotype of HCC cells, and miR-122 mimetic alone or in combination with anticancer drugs can be a promising therapeutic regimen against liver cancer.     

Forms of rarity of tree species in the southern Brazilian Atlantic rainforest

The assessment of species rarity considers local abundance (scarce or abundant population), habitat affinity (stenoecious or euryecious species), and geographic distribution (stenotopic or eurytopic species). When analyzed together these variables classify species into eight categories, from common species to those having small populations, unique habitats, and restricted geographic distribution (form 7), as proposed by Rabinowitz in 1981. Based on these categories, it is possible to calculate the frequency of the different forms of rarity of the species present in a given site. The Brazilian Atlantic rainforest is considered a hotspot of the world biodiversity harboring many endemic species, which have restricted geographic distribution. Our objective was to identify the forms of rarity of tree species and their proportions in the southern portion of the Brazilian Atlantic rainforest using Rabinowitz’s forms of rarity. All the seven forms of rarity are present in the 846 tree species we analyzed: 46% eurytopic and 54% stenotopic, 73% euryecious and 27% stenoecious, 76% locally abundant and 24% locally scarce species. Eurytopic, euryecious locally abundant species accounted for 41.1%, whereas 58.9% were somehow rare: 4.5% eurytopic, euryecious locally scarce, 0.2% eurytopic, stenoecious locally abundant, 0.1% eurytopic, stenoecious locally scarce, 19.5% stenotopic, euryecious locally abundant, 8.0% stenotopic, euryecious locally scarce, 15.6% stenotopic, stenoecious locally abundant, and 11.0% stenotopic, stenoecious locally scarce. Considering that the most restrictive forms of rarity precedes extinction, the application of Rabinowitz’s system demonstrated that most tree species of the southern Brazilian Atlantic rainforest are threatened due to their restricted geographic distribution, restriction to a single habitat, reduced local abundance, or even to a combination of these variables.