AAK1-mediated micro2 phosphorylation is stimulated by assembled clathrin

AAK1, the adaptor-associated kinase 1, phosphorylates the μ2 subunit of AP2 and regulates the recruitment of AP2 to tyrosine-based internalization motifs found on membrane-bound receptors. AAK1 overexpression specifically inhibits the AP2-dependent internalization of transferrin receptor and LDL-receptor related protein by functionally sequestering AP2 (Conner and Schmid. J Cell Biol 2003; 162: 773). However, while AAK1 stably associates with AP2 and specifically targets the μ2 subunit in vitro , μ2 phosphorylation in vivo was not altered by overexpression of either wild-type or kinase-inactive AAK1. These results suggested that AAK1 might be tightly regulated in the cell. Here, we report that AAK1 is an atypical kinase that is rate limited by its stable association with AP2 and that clathrin stimulates μ2 phosphorylation by AAK1. Efficient stimulation of AAK1 by clathrin involves multiple interactions between several domains on AAK1 and both heavy and light chains on clathrin. Importantly, incubation of AAK1 with clathrin cages resulted in even greater stimulation when compared to that of unassembled clathrin triskelia. Collectively, our observations indicate that clathrin function is not limited to structural and/or mechanical roles in endocytic vesicle formation: the stimulatory effects of clathrin on AAK1 activity argue that it also plays a regulatory role by modulating the activity of AP2 complexes through activation of AAK1. We suggest a model in which AAK1 is specifically activated in coated pits to enhance cargo recruitment and efficient internalization.     

Phosphate release and heavy metal accumulation by biofilm-immobilized and chemically-coupled cells of a Citrobacter sp. pre-grown in continuous culture

A heavy metal-accumulating Citrobacter sp. was grown in carbon-limiting continuous culture in an air-lift fermentor containing raschig rings as support for biofilm development. Planktonic cells from the culture outflow were immobilized in parallel on raschig rings by chemical coupling (silanization), for quantitative comparison of phosphatase activity and uranyl uptake by both types of immobilized cell. The flow rate giving 50% conversion of substrate to product (phosphate) in flow-through reactors was higher, by 35-40%, for the biofilm-immobilized cells, possibly exploiting a pH-buffering effect of inorganic phosphate species within the extracellular polymeric material. Upon incorporation of uranyl ions (0.2 mM UO22+), both types of cell removed more than 90% of the input UO22+ at slow flow rates, but the chemically-coupled cells performed better at higher flow rates. The deposited material (HUO2PO4) subsequently removed Ni2+ from a second flow via intercalative ion exchange of Ni2+ into the crystalline HUO2PO4.4H2O lattice. This occurred irrespective of the method of coupling of the biomass to the support and suggested that uranyl phosphate accumulated by both types of cell has potential as a bio-inorganic ion exchanger-a potential use for the uranium recoved from primary waste treatment processes.     

S-protein mutants indicate a functional role for SBP in the self-incompatibility reaction of Papaver rhoeas

The self-incompatibility response involves S-allele specific recognition between stigmatic S proteins and incompatible pollen, resulting in S-specific pollen inhibition. In Papaver rhoeas, the pollen S gene product is predicted to be a receptor that interacts with the stigmatic S protein in an S specific manner. We recently identified an S protein binding protein (SBP) in pollen that binds stigmatic S proteins, although apparently not in an S-allele-specific manner. In order to investigate the functional significance of the interaction between S proteins and SBP, we constructed mutant derivatives of the S1 protein and tested their SBP-binding activity and their biological activity. Here we present an evaluation of nine mutant derivatives of the S1 protein. Western ligand blotting was used to show that mutations to amino acid residues in predicted loops 2 and 6 of the S1 protein cause significant reductions in their SBP-binding activity. These same mutants show a concomitant reduction in their ability to inhibit incompatible pollen. This establishes a direct link between SBP binding and inhibition of incompatible pollen and implicates SBP as a pollen component playing a key role in the self-incompatibility reaction. We discuss the possible nature of the contribution of SBP in the S-specific rejection of incompatible pollen.     

Role of hyaluronan and reactive oxygen species in tissue kallikrein-mediated epidermal growth factor receptor activation in human airways

In human airways, oxidative stress-induced submucosal gland cell hypertrophy and hyperplasia, histological features of chronic bronchitis, have been linked to epidermal growth factor receptor (EGFR) activation. To explore mechanisms of oxidative stress-induced EGFR activation and signaling, primary cultures of human tracheal submucosal gland (SMG) cells were used to assess EGFR ligand release, EGFR phosphorylation, p44/42 MAPK phosphorylation, and mucin 5AC synthesis in response to reactive oxygen species generated by xanthine/xanthine oxidase (X/XO). Exposure to X/XO increased release of epidermal growth factor (EGF) from these cells, thereby activating EGFR, phosphorylating MAPK, and increasing mucin 5AC production. The importance of EGF was confirmed by transfection of small interfering RNA inhibiting pro-EGF production, which resulted in inhibition of EGFR and MAPK phosphorylation despite X/XO exposure. Blocking signaling by using specific protease inhibitors showed that tissue kallikrein (TK) processed pro-EGF in response to X/XO. Airway TK is bound and inactivated by luminal hyaluronan (HA), and treatment of submucosal gland cells with X/XO induced HA depolymerization and TK activation. These events were blocked by reactive oxygen species scavengers and addition of exogenous excess HA and TK inhibitors. Thus, HA plays a crucial role in regulating airway TK activity and thereby TK-mediated release of active EGF from human SMG cells. Sustained HA depolymerization is expected to cause TK activation, EGF release, and EGFR signaling and to lead to SMG cell hypertrophy and hyperplasia as well as mucus hypersecretion with subsequent airflow obstruction.     

Synthesis of quinolinyl/isoquinolinyl[a]pyrrolo [3,4-c] carbazoles as cyclin D1/CDK4 inhibitors

A novel series of pyrrolo[3,4-c] carbazoles fused with a quinolinyl/isoquinolinyl moiety were synthesized and their D1/CDK4 inhibitory and antiproliferative activity were evaluated. Compound 8H, 14H-isoquinolinyl[6,5-a]-pyrrolo[3,4-c]carbazole-7,9-dione (1d) was found to be a highly potent D1/CDK4 inhibitor with an IC(50) of 69 nM. Compound 1d also inhibited tumor cell growth, arrested tumor cells in G1 phase and inhibited pRb phosphorylation.     

Characterisation of Triticum vavilovii-derived stripe rust resistance using genetic,cytogenetic and molecular analyses and its marker-assisted selection

Stripe rust resistance was identified in Triticum vavilovii (T. vavilovii Aus22498)-derived Russian wheat aphid (RWA)-resistant germplasm. Inheritance studies indicated monogenic control of resistance. The resistance gene was tentatively designated as Yrvav and was located on chromosome 1B by monosomic analysis. A close association (1.5±0.9% recombination) of Yrvav with a T. vavilovii-derived gliadin allele (Gli-B1vav) placed it in chromosome arm 1BS. Yrvav was allelic with Yr10. Tests with Yr10 avirulent and virulent pathotypes showed that Yrvav and Yr10 possess identical pathogenic specificity. Yrvav and Yr10 showed close genetic associations with alternate alleles at the Xpsp3000 (microsatellite marker), Gli-B1 and Rg1 loci. Based on these observations Yrvav was named as Yr10vav. The close association between Xpsp3000 and Gli-B1 was also confirmed. The Yr10vav-linked Xpsp3000 allele (285 bp) was not present in 65 Australian cultivars, whereas seven Australian wheats lacking Yr10 carried the same Xpsp3000 allele (260 bp) as Yr10 carrying wheat cultivar Moro. Xpsp3000 and/or Gli-B1 could be used in marker-assisted selection for pyramiding Yr10vav or Yr10 with other stripe rust resistance genes. Yr10vav was inherited independently of the T. vavilovii-derived RWA resistance. Received: 5 December 2000 / Accepted: 3 April 2001     

Analysis of Host Range Restriction Determinants in the Rabbit Model: Comparison of Homologous and Heterologous Rotavirus Infections

The main limitation of both the rabbit and mouse models of rotavirus infection is that human rotavirus (HRV) strains do not replicate efficiently in either animal. The identification of individual genes necessary for conferring replication competence in a heterologous host is important to an understanding of the host range restriction of rotavirus infections. We recently reported the identification of the P type of the spike protein VP4 of four lapine rotavirus strains as being P[14]. To determine whether VP4 is involved in host range restriction in rabbits, we evaluated infection in rotavirus antibody-free rabbits inoculated orally with two P[14] HRVs, PA169 (G6) and HAL1166 (G8), and with several other HRV strains and animal rotavirus strains of different P and G types. We also evaluated whether the parental rhesus rotavirus (RRV) (P5B[3], G3) and the derived RRV-HRV reassortant candidate vaccine strains RRV × D (G1), RRV × DS-1 (G2), and RRV × ST3 (G4) would productively infect rabbits. Based on virus shedding, limited replication was observed with the P[14] HRV strains and with the SA11 Cl3 (P[2], G3) and SA11 4F (P6[1], G3) animal rotavirus strains, compared to the homologous ALA strain (P[14], G3). However, even limited infection provided complete protection from rotavirus infection when rabbits were challenged orally 28 days postinoculation (DPI) with 10³ 50% infective doses of ALA rabbit rotavirus. Other HRVs did not productively infect rabbits and provided no significant protection from challenge, in spite of occasional seroconversion. Simian RRV replicated as efficiently as lapine ALA rotavirus in rabbits and provided complete protection from ALA challenge. Live attenuated RRV reassortant vaccine strains resulted in no, limited, or productive infection of rabbits, but all rabbits were completely protected from heterotypic ALA challenge. The altered replication efficiency of the reassortants in rabbits suggests a role for VP7 in host range restriction. Also, our results suggest that VP4 may be involved in, but is not exclusively responsible for, host range restriction in the rabbit model. The replication efficiency of rotavirus in rabbits also is not controlled by the product of gene 5 (NSP1) alone, since a reassortant rotavirus with ALA gene 5 and all other genes from SA11 was more severely replication restricted than either parental rotavirus strain.Rotaviruses are the leading cause of acute viral gastroenteritis in humans and animals throughout the world. Rotaviruses belong to the Reoviridae family and are characterized by a genome consisting of 11 segments of double-stranded RNA (dsRNA), enclosed in a triple-layered protein capsid (28). Serotype designations are based on independent neutralization determinants on the two outer capsid proteins VP4 (P serotypes, for protease-sensitive protein) and VP7 (G serotypes, for glycoprotein) (28). Serotype specificity determined by cross-neutralization assays using hyperimmune sera against the whole virus is mainly defined by VP7, and 14 G serotypes have been identified (28). Recently, antisera or monoclonal antibodies raised to VP4 and sequence analysis of VP4 identified 12 P serotypes and 20 P genotypes, respectively (28, 39). Rotavirus VP4 protein is responsible for a number of important biological functions, such as the enhancement of infectivity by proteolytic cleavage of VP4 into VP8* and VP5*, hemagglutination, restricted growth in cell culture, virulence, initial virus attachment to cells, and protease sensitivity associated with plaque formation (1, 4, 25, 34, 40, 51).The use of animal models, including the rabbit and mouse models, has been essential to the understanding of rotavirus infection, pathology, disease, immunity, and testing of prospective vaccines in children (21). The limitations of the rabbit and adult mouse models of rotavirus infection for vaccine testing are as follows: (i) human rotavirus (HRV) strains do not efficiently replicate in either animal, (ii) clinical disease is not observed, and (iii) only homologous virus strains (isolated from the same species) replicate efficiently and spread horizontally to uninoculated control animals, whereas heterologous virus strains (isolated from a different species) do not (6, 15, 16, 29, 31, 35, 37, 44, 50, 55). We and others developed a rabbit model of rotavirus infection that is useful for defining basic parameters of active immunity, immunogenicity, and protective efficacy of vaccines (12, 15–21, 36, 61). Rabbits are productively infected with homologous lapine rotavirus strains up to at least the age of 5 years, which allows examination of active and long-term immunity for vaccine studies (13, 15–17, 36, 61). Group A lapine rotavirus strains have been isolated in Canada, Japan, Italy, and the United States, and those that have been characterized are serotype G3 (8, 11, 15, 53, 56, 61). Recently, the P type of four different strains was identified as genotype P[14] (11). Previously, limited infection of rabbits with a heterologous strain had been obtained only with SA11 Cl3 (P[2], G3) (15).Attempts to identify host range and virulence determinants for rotavirus have implicated different constellations of genes, including genes 2, 3, 4, 5, 8, 9, 10, and 11 (5, 23, 30, 33, 37, 38, 41, 43, 44, 60, 62, 65). Although host range restriction and virulence may be multigenic, two genes, 4 and 5, are of interest because they cluster according to species of origin, suggesting a role in host range restriction. The finding that genome segment 5 (NSP1) sequences cluster according to species of origin (24, 39, 65) and that, in the mouse model, gene 5 segregates with transmission of virus among littermates (5), led to the hypothesis that NSP1 is involved in host range restriction. VP4 sequence analyses of rotavirus strains isolated from different species revealed that specific VP4 types also generally correlate with the species of origin of each rotavirus strain (43, 60). Therefore, once we identified the P type of four lapine rotaviruses as P[14], we tested two P[14] HRV strains, PA169 (G6) and HAL1166 (G8) (32) to determine if VP4 is involved in host range restriction. We also tested several other HRV strains, live attenuated reassortant candidate vaccine strains [rhesus rotavirus (RRV) × D (G1), RRV × DS-1 (G2), and RRV × ST3 (G4)], and animal rotavirus strains of different P and G types to determine if they could productively infect rabbits. In addition, to evaluate whether the single rotavirus gene 5 is responsible for replication efficiency in rabbits, rabbits were inoculated with a reassortant rotavirus with the lapine ALA gene 5 and all the other genes from the simian rotavirus SA11 Cl3 strain.     

Histodifferentiation and cytodifferentiation in rabbit endometrium. A comparison of the effects of progesterone and various progesterone metabolites

The effects of progesterone, 20α-hydroxy-pregn-4-en-3-one, (20α-OH-P) 17 α hydroxyprogesterone (17α-OH-P) and 5α-pregnan-3, 20-dione (5α-P) on endometrium of ovariectomized rabbits have been examined. Progesterone, at 5 mg/kg/day and at 1.7 mg/kg/day increased the number of mitotic figures observed in luminal and glandular epithelium after 5 days of treatment and induced considerable arborization. None of the other compounds induced significant arborization and only 10 mg/kg/day of 20α-OH-P increased the mitotic index in luminal and glandular epithelium. Administrations of 20α-OH-P, 17α-OH-P or 5α-P resulted in the appearance of very large, pale cells in the luminal epithelium which were not present in progesterone treated rabbits. Cytochemical techniques revealed no specific staining in these cells for polysaccharides (PAS, Alcian blue), lipid (Oil Red O) or for blastokinin, a progesterone-inducible, uterine protein, examined by the fluoroscein isothiocyanate labeled antibody technique. The possibility of an apparent dissociation between histodifferentiation and cytodifferentiation is discussed.     

Population structure and skeletal variation in the Late Woodland of west-central Illinois

This paper analyzes nonmetric trait variation in 11 late Late Woodland (ca. AD 700-1000) and one Mississippian (AD 1000-1300) skeletal samples from west-central Illinois from a population-structure perspective. Most of the sites are of the Bluff phase of Late Woodland in the lower Illinois River valley; others are from a nearby, contemporary archaeological phase. Late Woodland as a whole era (ca. AD 250-1000) was a period of marked population growth and expansion into new regional environments, trends that accompanied horticultural intensification in the area. Overall variation between sites was low, but males, females, and the total sample exhibited a significant geographic component to variation due to interregional morphological differences. The Bluff sites tended to group together relative to the non-Bluff sites. However, there was no significant geographic component to variation among the Bluff sites. The results are only partially consistent with archaeological data suggesting population growth and expansion through fissioning. Previous studies have demonstrated significant heterogeneity for nonmetric trait frequencies among Middle Woodland (ca. 100 BC to AD 250) sites, suggesting a Middle to Late Woodland change in population structure that lowered levels of morphological variation. This supports a model of increased intra- and interregional interaction from Middle to Late Woodland times developed from ceramic data by Braun and by Braun and Plog.     

Photosynthesis and water relations of four oak species: impact of flooding and salinity

 As global climate changes, sea level rise and increased frequency of hurricanes will expose coastal forests to increased flooding and salinity. Quercus species are frequently dominant in these forest, yet little is known about their salinity tolerance, especially in combination with flooding. In this study, 1-year-old seedlings of Quercus lyrata Walt. (overcup oak), Q. michauxii Nutt. (swamp chestnut oak), Q. nigra L. (water oak), and Q. nuttallii Palmer (Nuttall oak) were chronically (simulating sea level rise) and acutely (simulating hurricane storm surge) exposed to increased flooding and salinity, individually and in combination. The four species demonstrated two response patterns of photosynthesis (A), conductance, and leaf water potential, apparently related to their relative flood tolerance. In Q. lyrata, Q. nuttallii, and Q. nigra (moderately flood-tolerant), A was not immediately reduced after the initiation of the freshwater flooding, but was reduced as the duration of flooding increased. In the second pattern, demonstrated by the weakly flood-tolerant Q. michauxii, A was immediately reduced by freshwater flooding with an increasing impact over time. Watering with 2 parts per thousand (ppt) saline water did not consistently reduce A, but flooding with 2 ppt reduced A of all species, similar to the response with freshwater flooding. Photosynthesis of all species was reduced by 6 ppt watering or flooding, with the latter treatment killing all species within 8 weeks. When acutely exposed to 30 ppt salinity, A was quickly and severely reduced regardless of whether the seedlings were watered or flooded. Acutely flooded seedlings exposed to high salinity died within 2 weeks, but seedlings watered with 30 ppt saline water recovered and A was not reduced the following spring. As saline flooding of coastal areas increases due to sea level rise, photosynthesis of these species will be differentially affected based primarily on their flood tolerance. This suggests that increased flooding associated with sea level rise will impact these tree species to a greater extent than small increases in soil salinity. High salinity accompanying storm surges will be very harmful to all of these species. Received: 20 October 1997 / Accepted: 2 December 1998