Mutations in PIP5K3 are associated with François-Neetens mouchetée fleck corneal dystrophy

Fran?ois-Neetens fleck corneal dystrophy (CFD) is a rare, autosomal dominant corneal dystrophy characterized by numerous small white flecks scattered in all layers of the stroma. Linkage analysis localized CFD to a 24-cM (18-Mb) interval of chromosome 2q35 flanked by D2S2289 and D2S126 and containing PIP5K3. PIP5K3 is a member of the phosphoinositide 3-kinase family and regulates the sorting and traffic of peripheral endosomes that contain lysosomally directed fluid phase cargo, by controlling the morphogenesis and function of multivesicular bodies. Sequencing analysis disclosed missense, frameshift, and/or protein-truncating mutations in 8 of 10 families with CFD that were studied, including 2256delA, 2274delCT, 2709C-->T (R851X), 3120C-->T (Q988X), IVS19-1G-->C, 3246G-->T (E1030X), 3270C-->T (R1038X), and 3466A-->G (K1103R). The histological and clinical characteristics of patients with CFD are consistent with biochemical studies of PIP5K3 that indicate a role in endosomal sorting.     

Function of the cytochrome bc1-aa3 branch of the respiratory network in mycobacteria and network adaptation occurring in response to its disruption

The aerobic electron transport chain in Mycobacterium smegmatis can terminate in one of three possible terminal oxidase complexes. The structure and function of the electron transport pathway leading from the menaquinol-menaquinone pool to the cytochrome bc1 complex and terminating in the aa3-type cytochrome c oxidase was characterized. M. smegmatis strains with mutations in the bc1 complex and in subunit II of cyctochome c oxidase were found to be profoundly growth impaired, confirming the importance of this respiratory pathway for mycobacterial growth under aerobic conditions. Disruption of this pathway resulted in an adaptation of the respiratory network that is characterized by a marked up-regulation of cydAB, which encodes the bioenergetically less efficient and microaerobically induced cytochrome bd-type menaquinol oxidase that is required for the growth of M. smegmatis under O2-limiting conditions. Further insights into the adaptation of this organism to rerouting of the electron flux through the branch terminating in the bd-type oxidase were revealed by expression profiling of the bc1-deficient mutant strain using a partial-genome microarray of M. smegmatis that is enriched in essential genes. Although the expression profile was indicative of an increase in the reduced state of the respiratory chain, blockage of the bc1-aa3 pathway did not induce the sentinel genes of M. smegmatis that are induced by oxygen starvation and are regulated by the DosR two-component regulator.     

Effect of Oxygen on Temporary Stabilization of Photoreduced Quinone Acceptors in Rhodobacter sphaeroides Reaction Centers

The effect of molecular oxygen on the photochemical activity of the Rhodobacter sphaeroides reaction centers frozen to 160 K under actinic illumination was investigated by the ESR method. About 90% of initially photochemically active bacteriochlorophyll (P) were fixed at 160 K for a long time in aerobic samples in an inactive form. In anaerobic samples, not more than 65% were fixed in an inactive form under the same conditions. In aerobic preparations, a small portion of photochemically active bacteriochlorophyll (about 10%) that retains its photochemical activity at 160 K after freezing under illumination has dark reduction kinetics similar to that of samples at room temperature after several seconds of actinic illumination. In anaerobic samples frozen under illumination, the remaining photochemically active reaction centers (35%) have the same dark reduction kinetics as samples illuminated at 295 K for 1-2 min. The conclusion is that the irreversible stabilization of bacteriochlorophyll P in the oxidized inactive state formed in the reaction centers frozen under illumination is brought about by light-induced conformational changes fixed under low temperatures.     

Effect of mercuric chloride and methylmercury on photosynthetic activity of the diatom Thalassiosira weissflogii by a fluorescent method

It was shown by the pulse-amplitude modulation fluorescent method that, at a weak illumination (6 microE m-2.s-1), methylmercury at a concentration of 10(-6)-10(-7) M decreases the photochemical activity of the reaction centers of photosystem II in cells of microalgae Thalassiosira weissflogii after a prolonged lag phase. Cells resistant to methylmercury at these low concentrations were detected by the microfluorimetric method. Chloride mercury decreased the activity of photosystem II of the algae only when at higher concentrations. Both toxicants at a concentration of 10(-6) M decreased the rate of recovery of photoinduced damage of centers of photosystem II and led to an increase in the energization component of nonphotochemical fluorescence quenching. These results indicate that the complex of fluorescent methods can be used to monitor early changes in the photosynthetic apparatus of algae in response to the toxic action of heavy metals.     

The photochemical activity of photosystem II in sulfur-deprived Chlamydomonas reinhardtii cells depends on the redox state of the quinone pool during the transition to anaerobiosis

Measurements with a PAM fluorometer showed that the photochemical activity of photosystem II (PS II) in sulfur-deprived Chlamydomonas reinhardtii cells (media TAP-S) decreases slowly under aerobic conditions. In a closed cultivator, when the rate of O2 photosynthetic evolution declines below the rate of respiration, the cell culture is under anaerobic conditions in which the activation of hydrogenase and the production of hydrogen take place. We found that the slow decrease in PS II activity is followed by an abrupt inactivation of PS II centers just after the onset of anaerobiosis. This fast PS II inactivation is reversed by aeration of the media and is accompanied by an increase in the fluorescence parameter Ft. Moreover, the rate of the abrupt PS II inactivation diminished after the addition into the medium of electron acceptors such as CO2 (carbonate-bicarbonate buffer), NO3- and SO4(2-) , the assimilation of which in chloroplasts requires a lot of reductants. We suggest that the PS II inactivation is due to the overreduction of the plastoquinone pool after the onset of anaerobiosis.     

Terminal osteoblast differentiation, mediated by runx2 and p27KIP1, is disrupted in osteosarcoma

The molecular basis for the inverse relationship between differentiation and tumorigenesis is unknown. The function of runx2, a master regulator of osteoblast differentiation belonging to the runt family of tumor suppressor genes, is consistently disrupted in osteosarcoma cell lines. Ectopic expression of runx2 induces p27KIP1, thereby inhibiting the activity of S-phase cyclin complexes and leading to the dephosphorylation of the retinoblastoma tumor suppressor protein (pRb) and a G1 cell cycle arrest. Runx2 physically interacts with the hypophosphorylated form of pRb, a known coactivator of runx2, thereby completing a feed-forward loop in which progressive cell cycle exit promotes increased expression of the osteoblast phenotype. Loss of p27KIP1 perturbs transient and terminal cell cycle exit in osteoblasts. Consistent with the incompatibility of malignant transformation and permanent cell cycle exit, loss of p27KIP1 expression correlates with dedifferentiation in high-grade human osteosarcomas. Physiologic coupling of osteoblast differentiation to cell cycle withdrawal is mediated through runx2 and p27KIP1, and these processes are disrupted in osteosarcoma.     

Effect of a Single Excitation Stimulus on Photosynthetic Activity and Light-dependent pH Banding in <Emphasis Type="Italic">Chara</Emphasis> Cells

Using pH microelectrodes and a Micro-scopy PAM (pulse-amplitude modulated) chlorophyll fluorometer, it is shown that a propagation of an action potential in Chara corallina leads to transient suppression of spatially periodic pH profiles along the illuminated cell. The suppression was manifested as a large pH decrease in the alkaline zones and a slight pH increase in the acid zones. The propagating action potential diminished the maximum yield of chlorophyll fluorescence (F_m′) in the alkaline cell regions, as well as the quantum yield of photosystem II photochemistry, without affecting F_m′ in the acid cell regions. The results indicate an interference of membrane excitation in the mechanisms responsible for pH banding patterns in Characean algae. Apparently, the electrical excitation of the plasma membrane in the alkaline cell regions initiates a pathway that can modulate membrane events at the thylakoid membrane.     

The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes

The Berkeley Drosophila Genome Project (BDGP) strives to disrupt each Drosophila gene by the insertion of a single transposable element. As part of this effort, transposons in >30,000 fly strains were localized and analyzed relative to predicted Drosophila gene structures. Approximately 6300 lines that maximize genomic coverage were selected to be sent to the Bloomington Stock Center for public distribution, bringing the size of the BDGP gene disruption collection to 7140 lines. It now includes individual lines predicted to disrupt 5362 of the 13,666 currently annotated Drosophila genes (39%). Other lines contain an insertion at least 2 kb from others in the collection and likely mutate additional incompletely annotated or uncharacterized genes and chromosomal regulatory elements. The remaining strains contain insertions likely to disrupt alternative gene promoters or to allow gene misexpression. The expanded BDGP gene disruption collection provides a public resource that will facilitate the application of Drosophila genetics to diverse biological problems. Finally, the project reveals new insight into how transposons interact with a eukaryotic genome and helps define optimal strategies for using insertional mutagenesis as a genomic tool.     

Identification of a peptide binding motif for secreted frizzled-related protein-1

Secreted Frizzled-related proteins (sFRPs) bind Wnts and modulate their activity. To identify putative sFRP-1 binding motifs, we screened an M13 phage displayed combinatorial peptide library. A predominant motif, L/V-VDGRW-L/V, was present in approximately 70% of the phage that bound sFRP-1. Use of peptide/alkaline phosphatase chimeras and alanine scanning confirmed that the conserved motif was important for sFRP-1 recognition. The dissociation constant for a peptide/sFRP-1 complex was 3.9 microM. Additional analysis revealed that DGR was the core of the binding motif. Although Wnt proteins lack this sequence, other proteins possessing the DGR motif may function as novel binding partners for sFRP-1.