A new pathway for mitogen-dependent cdk2 regulation uncovered in p27(Kip1)-deficient cells

BACKGROUND: The ability of cyclin-dependent kinases (CDKs) to promote cell proliferation is opposed by cyclin-dependent kinase inhibitors (CKIs), proteins that bind tightly to cyclin-CDK complexes and block the phosphorylation of exogenous substrates. Mice with targeted CKI gene deletions have only subtle proliferative abnormalities, however, and cells prepared from these mice seem remarkably normal when grown in vitro. One explanation may be the operation of compensatory pathways that control CDK activity and cell proliferation when normal pathways are inactivated. We have used mice lacking the CKIs p21(Cip1) and p27(Kip1) to investigate this issue, specifically with respect to CDK regulation by mitogens. RESULTS: We show that p27 is the major inhibitor of Cdk2 activity in mitogen-starved wild-type murine embryonic fibroblasts (MEFs). Nevertheless, inactivation of the cyclin E-Cdk2 complex in response to mitogen starvation occurs normally in MEFs that have a homozygous deletion of the p27 gene. Moreover, CDK regulation by mitogens is also not affected by the absence of both p27 and p21. A titratable Cdk2 inhibitor compensates for the absence of both CKIs, and we identify this inhibitor as p130, a protein related to the retinoblastoma gene product Rb. Thus, cyclin E-Cdk2 kinase activity cannot be inhibited by mitogen starvation of MEFs that lack both p27 and p130. In addition, cell types that naturally express low amounts of p130, such as T lymphocytes, are completely dependent on p27 for regulation of the cyclin E-Cdk2 complex by mitogens. CONCLUSIONS: Inhibition of Cdk2 activity in mitogen-starved fibroblasts is usually performed by the CKI p27, and to a minor extent by p21. Remarkably p130, a protein in the Rb family that is not related to either p21 or p27, will directly substitute for the CKIs and restore normal CDK regulation by mitogens in cells lacking both p27 and p21. This compensatory pathway may be important in settings in which CKIs are not expressed at standard levels, as is the case in many human tumors.     

Reproduction rates and secondary production of three species of the rotifer genus Synchaeta in the estuarine Potomac River

Rotifers are a relatively well-studied component of lacustrinesystems but their role is only poorly understood in estuaries.Three species of the genus Synchaeta—S. baltica, S. triophthalmaand S.cecilia—dominate the cold-water assemblage of rotifersin Chesapeake Bay. Laboratory experiments were conducted todetermine the temperature dependence of egg development time(EDT) for each species; EDT varied over an approximate rangeof 90–9 h as temperature (T) varied from 2 to 22°C.The EDT/temperature relationships could be closely fitted bya simple polynomial equation of the form log(EDT) = a + b(logT) + c(log T)² for each species. Natural populations of thesethree rotifers were sampled during a cruise in the Potomac River(7–11 March 1983). Estimates of specific reproductiverates (b) were calculated based on the previously defined EDT/temperaturerelationship and the observed ratio of eggs/rotifers for eachspecies. The two most abundant species, S.triophthalma and S.cecilia,showed a clear dependence of b on the observed chlorophyll aconcentrations. Maximum reproductive rates ({small tilde}0.015h^–1) were attained only at relatively high phytoplanktondensities within a bloom of Heterocapsa triquetra where thechlorophyll a concentrations exceeded 10 µg l^–1.Estimates of secondary production suggest that Synchaeta spp.may contribute to the trophic flow of carbon in this systemwith a significance at least similar to that of the planktoniccopepods.     

Marine metagenomics: strategies for the discovery of novel enzymes with biotechnological applications from marine environments

Metagenomic based strategies have previously been successfully employed as powerful tools to isolate and identify enzymes with novel biocatalytic activities from the unculturable component of microbial communities from various terrestrial environmental niches. Both sequence based and function based screening approaches have been employed to identify genes encoding novel biocatalytic activities and metabolic pathways from metagenomic libraries. While much of the focus to date has centred on terrestrial based microbial ecosystems, it is clear that the marine environment has enormous microbial biodiversity that remains largely unstudied. Marine microbes are both extremely abundant and diverse; the environments they occupy likewise consist of very diverse niches. As culture-dependent methods have thus far resulted in the isolation of only a tiny percentage of the marine microbiota the application of metagenomic strategies holds great potential to study and exploit the enormous microbial biodiversity which is present within these marine environments.     

Hierarchical gene expression profiles of HUVEC stimulated by different lipid A structures obtained from Porphyromonas gingivalis and Escherichia coli

The ability of lipid A structural variants to elicit unique endothelial cell gene expression was examined by measuring global gene expression profiles in human umbilical cord vein endothelial cells (HUVEC) using Affymetrix full genome chips. Two lipid A structural variants obtained from Porphyromonas gingivalis designated PgLPS(1435/1449) and PgLPS(1690) as well as LPS obtained from Escherichia coli wild type and an E. coli msbB mutant (missing myristic acid in the lipid A) were examined. Each of these lipid A structures has been shown to interact with TLR4; however, PgLPS(1435/1449) and E. coli msbB LPS have been shown to be TLR4 antagonists while PgLPS(1690) and wild-type E. coli LPS are TLR4 agonists. It was found that PgLPS(1435/1449) and PgLPS(1690) as well as E. coli msbB LPS activated a subset of those genes significantly transcribed in response to E. coli wild-type LPS. Furthermore, the subset of genes expressed in response to the different lipid A structural forms were those most significantly activated by wild-type E. coli LPS demonstrating a hierarchy in TLR4-dependent endothelial cell gene activation. A unique gene expression profile for the weak TLR4 agonist PgLPS(1690) was observed and represents a TLR4 hierarchy in endothelial cell gene activation.     

Pan trapping soybean aphids (Hemiptera: Aphididae) using attractants

Since its introduction into the United States in the past 10 yr, soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), has been a damaging pest to soybean, Glycine max (L.) Merrill. During 2008 and 2009, fields in central and north central Iowa experienced pockets of high soybean aphid populations. Electroantennograms have shown that soybean aphid alatae are capable of detecting host plant volatiles and sex pheromones. Here, we evaluated baited pan traps as a potential soybean aphid attractant. Yellow pan traps were placed in soybean fields after planting along with lures that contained plant volatiles and sex pheromones in 2008 or sex pheromones only in 2009. Pan trap contents were collected weekly, and plant counts also were conducted. Aphids were identified, and soybean aphids were counted to determine whether one chemical lure was more attractive to spring migrants than other lures. In both years, soybean aphids collected in pan traps with lures were not significantly different from the other products tested.     

The lens organizes the anterior segment: specification of neural crest cell differentiation in the avian eye

During the development of the anterior segment of the eye, neural crest mesenchyme cells migrate between the lens and the corneal epithelium. These cells contribute to the structures lining the anterior chamber: the corneal endothelium and stroma, iris stroma, and trabecular meshwork. In the present study, removal of the lens or replacement of the lens with a cellulose bead led to the formation a disorganized aggregate of mesenchymal cells beneath the corneal epithelium. No recognizable corneal endothelium, corneal stroma, iris stroma, or anterior chamber was found in these eyes. When the lens was replaced immediately after removal, a disorganized mass of mesenchymal cells again formed beneath the corneal epithelium. However, 2 days after surgery, the corneal endothelium and the anterior chamber formed adjacent to the lens. When the lens was removed and replaced such that only a portion of its anterior epithelial cells faced the cornea, mesenchyme cells adjacent to the lens epithelium differentiated into corneal endothelium. Mesenchyme cells adjacent to lens fibers did not form an endothelial layer. The cell adhesion molecule, N-cadherin, is expressed by corneal endothelial cells. When the lens was removed the mesenchyme cells that accumulated beneath the corneal epithelium did not express N-cadherin. Replacement of the lens immediately after removal led to the formation of an endothelial layer that expressed N-cadherin. Implantation of lens epithelia from older embryos showed that the lens epithelium maintained the ability to support the expression of N-cadherin and the formation of the corneal endothelium until E15. This ability was lost by E18. These studies provide evidence that N-cadherin expression and the formation of the corneal endothelium are regulated by signals from the lens. N-cadherin may be important for the mesenchymal-to-epithelial transformation that accompanies the formation of the corneal endothelium.     

The BOS™ as a species-specific method to deliver baits to wild boar in a Mediterranean area

The impact of wild boar Sus scrofa and feral pigs on ecosystems and human activities is of interest worldwide. Bait-delivered pharmaceuticals such as contraceptives or disease vaccines are increasingly advocated to assist the management of such impacts. We evaluated the Boar-Operated-System (BOS?) to deliver baits to wild boar in a Mediterranean area with a large community of potential nontarget species. In a pre-trial phase (BOS? open), both wild boar and 12 nontarget species (wildlife and livestock) visited the BOS? and eight species consumed the baits. In the trial phase, when the BOS? were closed, only wild boar consumed baits. From pre-trial to trial, the rate of visits by nontarget species to the BOS? decreased significantly, but that of wild boar did not change. We observed that crested porcupines Hystrix cristata prevented the wild boar from using BOS?. We confirmed the effectiveness of BOS? to deliver baits selectively to wild boar in a Mediterranean area.     

DINOFLAGELLATE HOST‐PARASITE STEROL PROFILES DICTATE KARLOTOXIN SENSITIVITY1

We examined the sterol profile of Karlodinium veneficum (D. Ballant.) J. Larsen, Akashiwo sanguinea (Hiraska) Ge. Hansen et Moestrup, Alexandrium tamarense (M. Lebour) Balech, Alexandrium affine (H. Inoue et Fukuyo) Balech, Gonyaulax polygramma F. Stein, and Gymnodinium instriatum (Freud. et J. J. Lee) Coats, along with their Amoebophyra parasites. There were no consistent sterol profiles that characterized the genus Amoebophyra. Instead, in five out of six comparisons, the host and parasite sterol profiles where highly correlated. The one exception, Amoebophyra sp. ex Alex. tamarense, was least like its host in sterol profile and also possessed the widest host range for infection. There was little correlation between host and parasite in fatty acid profiles, with the parasite being deficient in fatty acids characteristic of the plastid [e.g., 18:5(n‐3) associated with galactolipids of the thylakoids, as previously published by Adolf et al. (2007)]. Those hosts and parasites with sterol profiles dominated by desmethyl sterols were most sensitive to karlotoxin toxicity. In the host‐parasite pairs most sensitive to karlotoxin addition, recovery of the intact karlotoxin molecule was poorest. Given the sensitivity to karlotoxin, some species of Amoebophyra may avoid infection of K. veneficum.     

The Effects of CFTR and Mucoid Phenotype on Susceptibility and Innate Immune Responses in a Mouse Model of Pneumococcal Lung Disease

Recent studies have reported the isolation of highly mucoid serotype 3 Streptococcus pneumoniae (Sp) from the respiratory tracts of children with cystic fibrosis (CF). Whether these highly mucoid Sp contribute to, or are associated with, respiratory failure among patients with CF remains unknown. Other mucoid bacteria, predominately Pseudomonas aeruginosa, are associated with CF respiratory decline. We used a mouse model of CF to study pneumococcal pneumonia with highly mucoid serotype 3 and non-mucoid serotype 19A Sp isolates. We investigated susceptibility to infection, survival, and bacterial counts from bronchoaviolar lavage samples and lung homogenates, as well as associated inflammatory cytokines at the site of infection, and lung pathology. Congenic CFTR^–/– mice and wild-type (WT)-mice were infected intranasally with CHB756, CHB1126, and WU2 (highly mucoid capsular serotype 3, intermediately mucoid serotype 3, and less mucoid serotype 3, respectively), or CHB1058 (non-mucoid serotype 19A). BAL, lung homogenates, and blood were collected from mice 5 days post-infection. Higher CFU recovery and shorter survival were observed following infection of CFTR^–/– mice with CHB756 compared to infection with CHB1126, WU2, or CHB1058 (P≤0.001). Additionally, CFTR^–/– mice infected with CHB756 and CHB1126 were more susceptible to infection than WT-mice (P≤0.05). Between CFTR^–/– mice and WT-mice, no significant differences in TNF-α, CXCL1/KC concentrations, or lung histopathology were observed. Our results indicate that highly mucoid type 3 Sp causes more severe lung disease than non-mucoid Sp, and does so more readily in the lungs of CFTR^–/– than WT-mice.