Regulation of mitochondrial fission by GIPC-mediated Drp1 retrograde transport

Dynamin-related protein 1 (Drp1) is a key regulator of mitochondrial fission, a large cytoplasmic GTPase recruited to the mitochondrial surface via transmembrane adaptors to initiate scission. While Brownian motion likely accounts for the local interactions between Drp1 and the mitochondrial adaptors, how this essential enzyme is targeted from more distal regions like the cell periphery remains unknown. Based on proteomic interactome screening and cell-based studies, we report that GAIP/RGS19-interacting protein (GIPC) mediates the actin-based retrograde transport of Drp1 toward the perinuclear mitochondria to enhance fission. Drp1 interacts with GIPC through its atypical C-terminal PDZ-binding motif. Loss of this interaction abrogates Drp1 retrograde transport resulting in cytoplasmic mislocalization and reduced fission despite retaining normal intrinsic GTPase activity. Functionally, we demonstrate that GIPC potentiates the Drp1-driven proliferative and migratory capacity in cancer cells. Together, these findings establish a direct molecular link between altered GIPC expression and Drp1 function in cancer progression and metabolic disorders.     

Increase in Alfalfa Nodulation, Nitrogen Fixation, and Plant Growth by Specific DNA Amplification in Sinorhizobium meliloti

To improve symbiotic nitrogen fixation on alfalfa plants, Sinorhizobium meliloti strains containing different average copy numbers of a symbiotic DNA region were constructed by specific DNA amplification (SDA). A DNA fragment containing a regulatory gene (nodD1), the common nodulation genes (nodABC), and an operon essential for nitrogen fixation (nifN) from the nod regulon region of the symbiotic plasmid pSyma of S. meliloti was cloned into a plasmid unable to replicate in this organism. The plasmid then was integrated into the homologous DNA region of S. meliloti strains 41 and 1021, which resulted in a duplication of the symbiotic region. Sinorhizobium derivatives carrying further amplification were selected by growing the bacteria in increased concentrations of an antibiotic marker present in the integrated vector. Derivatives of strain 41 containing averages of 3 and 6 copies and a derivative of strain 1021 containing an average of 2.5 copies of the symbiotic region were obtained. In addition, the same region was introduced into both strains as a multicopy plasmid, yielding derivatives with an average of seven copies per cell. Nodulation, nitrogenase activity, plant nitrogen content, and plant growth were analyzed in alfalfa plants inoculated with the different strains. The copy number of the symbiotic region was critical in determining the plant phenotype. In the case of the strains with a moderate increase in copy number, symbiotic properties were improved significantly. The inoculation of alfalfa with these strains resulted in an enhancement of plant growth.     

Diacylglycerol kinase inhibition prevents IL-2-induced G1 to S transition through a phosphatidylinositol-3 kinase-independent mechanism.

Stimulation via IL-2R ligation causes T lymphocytes to transit through the cell cycle. Previous experiments by our group have demonstrated that, in human T cells, IL-2 binding induces phosphatidic acid production through activation of the alpha isoform of diacylglycerol kinase. In this study, using the IL-2-dependent mouse T cell line CTLL-2, we demonstrate that pharmacological inhibition of IL-2-induced diacylglycerol kinase activation is found to block IL-2-induced late G1 to S transition without affecting cell viability. Herein, we demonstrate that diacylglycerol kinase inhibition has a profound effect on the induction of the protooncogenes c-myc, c-fos, and c-raf by IL-2, whereas expression of bcl-2 and bcl-xL are not affected. When the IL-2-regulated cell cycle control checkpoints are examined in detail, we demonstrate that inhibition of diacylglycerol kinase activation prevents IL-2 induction of cyclin D3 without affecting p27 down-regulation. The strict control of cell proliferation exerted by phosphatidic acid through activation of diacylglycerol kinase is independent of other well-characterized IL-2R-derived signals, such as the phosphatidylinositol-3 kinase/Akt pathway, indicating the existence of a different and important mechanism to control cell division.     

Kinetic characterization in batch and continuous culture of Escherichia coli mutants affected in phosphoenolpyruvate metabolism: differences in acetic acid production

The growth kinetics of an Escherichia coli wild type strain and two derivative mutants were examined in batch cultures and in glucose-limited chemostats. One mutant (PB12) had an inactive phosphotranferase transport system and the other (PB25) had interrupted pykA and pykF genes that code for the two pyruvate kinase isoenzymes. In both batch and continuous culture, important differences in acetic acid accumulation and other metabolic activities were found. Compared to the wild type strain, we observed a reduction in acetic acid accumulation of 25 and 80% in PB25 and PB12 strains respectively, in batch culture. Continuous culture experiments revealed that compared to the other two strains, PB25 accumulated less acetic acid as a function of dilution rate. In continuous cultures, oxidoreductase metabolic activities were substantially affected in the two mutant strains. These changes in turn were reflected in different levels of biomass and CO₂ production, and in oxygen consumption.     

By-product formation during exposure of respiring Saccharomyces cerevisiae cultures to excess glucose is not caused by a limited capacity of pyruvate carboxylase

Upon exposure to excess glucose, respiring cultures of Saccharomyces cerevisiae produce substantial amounts of ethanol and acetate. A possible role of a limited anaplerotic capacity in this process was investigated by overexpressing pyruvate carboxylase and by replacing it with a heterologous enzyme (Escherichia coli phosphoenolpyruvate carboxylase). Compared to the wild-type, neither the pyruvate carboxylase (Pyc)-overexpressing nor the transgenic strain exhibited reduced by-product formation after glucose pulses to aerobic glucose-limited chemostat cultures. An increased intracellular malate concentration was observed in the two engineered strains. It is concluded that by-product formation in S. cerevisiae is not caused by a limited anaplerotic capacity.     

Viroids with hammerhead ribozymes: some unique structural and functional aspects with respect to other members of the group.

Viroids, subviral pathogens of plants, are composed of a single-stranded circular RNA of 246-399 nucleotides. Within the 27 viroids sequenced, avocado sunblotch, peach latent mosaic and chrysanthemum chlorotic mottle viroids (ASBVd, PLMVd and CChMVd, respectively) can form hammerhead structures in both of their polarity strands. These ribozymes mediate self-cleavage of the oligomeric RNAs generated in the replication through a rolling circle mechanism, whose two other steps are catalyzed by an RNA polymerase and an RNA ligase. ASBVd, and presumably PLMVd and CChMVd, replicate and accumulate in the chloroplast, whereas typical viroids replicate and accumulate in the nucleus. PLMVd and CChMVd do not adopt a rod-like or quasi rod-like secondary structure as typical viroids do but have a highly branched conformation. A pathogenicity determinant has been mapped in a defined region of the CChMVd molecule.     

Subtropical Front fluctuations south of Australia (45°09′S, 146°17′E) for the last 130 ka years based on calcareous nannoplankton

Calcareous nannoplankton assemblages from a Late Quaternary deep-sea core (GC07; 46°09′S, 146°17′E) south of Australia provide information on regional palaeoceanography and palaeoclimate changes in the Southern Ocean, in particular the movement of the Subtropical Front for the past 130 ka years. Marine Isotope Stages 1–5 are identified through changes in calcareous nannoplankton assemblages, supported by ¹⁴C dates, and oxygen isotope and %CaCO data.Two distinct assemblages are recognised: a warm water assemblage with higher abundances of Calcidiscus leptoporus, Emiliania huxleyi, Helicosphaera.carteri, Syracosphaera pulchra, Gephyrocapsa caribbeanica and Gephyrocapsa oceanica; and, a cold water assemblage with higher abundances of Gephyrocapsa muellerae and Coccolithus pelagicus. Alternation between these two assemblages downcore in GC07 reflect movement of the Subtropical Front across the location and can be correlated to Marine Isotope Stages (MIS) 1–5. Sediments with a cold water assemblage indicate the position of the Subtropical Front equatorward of the site when transitional to sub-antarctic waters were overlying the site. Conversely sediments with a warm water assemblage indicate the Subtropical Front was poleward of GC07 when warmer, subtropical waters were over the site. MIS 1 and 5 are interpreted as warmer than MIS 3 (based on species composition) with the Subtropical Front more poleward than for MIS 3. During MIS 3 the Subtropical Front is interpreted as adjacent to or immediately poleward of GC07. Some species including C. leptoporus and C. pelagicus show negative covariance and are considered to be reliable species in identifying glacial and interglacial intervals in this region.Comparison with established biostratigraphy based on calcareous nannoplankton showed the datum event for the reversal between E. huxleyi and G. muellerae of 73 ka in transitional waters is not applicable in this region. The reversal between these two species occurs between 48 and 30 cm downcore in GC07 with a ¹⁴C date of 11 020 year BP at 49–48 cm, i.e. the reversal event is younger than this date.     

A field based statistical approach for validating a remotely sensed mangrove forest classification scheme

Amongst the most threatened ecosystems on Earth, mangrove forests are also one of the more difficult to work in due to their growth in mud and open water coastal zones and their dense tangled stems, branches and prop roots. Consequently, there has been an impetus to employ remotely sensed imagery as a means for rapid inventory of these coastal wetlands. To date, the majority of mangrove maps derived from satellite imagery utilize a simple mangrove classification scheme which does not distinguish mangrove species and may not be useful for conservation and management purposes. Although more elaborate satellite based mangrove classification schemes are being developed, given their enhanced complexity they deserve additional justification for end users. The purpose of this study was to statistically examine the appropriateness of one such classification scheme based on an inventory of field data. In January of 2007 and May of 2008, 61 field sample plots were selected in a stratified random fashion based on a previous classification of a degraded mangrove forest of the Isla La Palma (Sinaloa, Mexico) using Landsat TM5 data. Unlike other previous Landsat TM based classifications of this region, which simply identified the mangrove forests as one class, the mangroves were classified (i.e. mapped) according to four conditions; healthy tall, healthy dwarf, poor condition, and dead mangroves. Within each sample plot, all mangroves of diameter of breast height (dbh) greater than 2.5 cm were identified and their height, condition and dbh recorded. An estimated Leaf Area Index (LAI) value also was obtained for each sample and the shortest distance from the center of each sample plot to open flowing water was determined using a geographic information system (GIS) overlay procedure. These data were then used to calculate mean values for the four classes as well as to determine stem densities, basal areas, and the Shannon–Wiener diversity index. In order to assess the appropriateness of this mangrove classification scheme a discriminant analysis approach was then applied to these field data. The results indicate this forest has undergone severe degradation, with decreasing mean tree heights, mean dbh and species diversity. In regards to the discriminant analysis procedure, further classification of these field plots and cross-validation based on these significant variables provided high classification accuracy thus validating the appropriateness of the satellite based image classification scheme. Moreover, the discriminant analysis indicated that the estimated LAI, mean height, and mean dbh are significant in the separation of the classification of mangrove forest condition along these field sample plots.     

Disease states associated with telomerase deficiency appear earlier in mice with short telomeres.

Mice deficient for the mouse telomerase RNA (mTR-/-) and lacking telomerase activity can only be bred for approximately six generations due to decreased male and female fertility and to an increased embryonic lethality associated with a neural tube closure defect. Although late generation mTR-/- mice show defects in the hematopoietic system, they are viable to adulthood, only showing a decrease in viability in old age. To assess the contribution of genetic background to the effect of telomerase deficiency on viability, we generated mTR-/- mutants on a C57BL6 background, which showed shorter telomeres than the original mixed genetic background C57BL6/129Sv. Interestingly, these mice could be bred for only four generations and the survival of late generation mTR-/- mice decreased dramatically with age as compared with their wild-type counterparts. Fifty percent of the generation 4 mice die at only 5 months of age. This decreased viability with age in the late generation mice is coincident with telomere shortening, sterility, splenic atrophy, reduced proliferative capacity of B and T cells, abnormal hematology and atrophy of the small intestine. These results indicate that telomere shortening in mTR-/- mice leads to progressive loss of organismal viability.