Calibrating estimates of phage-induced mortality in marine bacteria: Ultrastructural studies of marine bacteriophage development from one-step growth experiments

The timing of lytic phage development and the relationship between host generation times and latent periods were investigated by electron microscopy of one-step growth experiments in two strains of marine Vibrio species. Results were used in a correction factor developed to interpret field studies of phage-infected marine bacteria. Both the number of mature phage per average cell section and the percentage of cells with mature phage increased exponentially by 73–86% into the latent periods. Assuming that bacterial infection and lysis take place continually in the ocean, conversion factors for relating the percentage of visibly infected bacteria to the total percentage of the bacterial community that are phage-infected were calculated as 3.70–7.14. When this range of factors was applied to previously-collected field data [Proctor LM, Fuhrman JA (1990) Nature (Lond) 343:60–62; Proctor LM, Fuhrman JA (1991) Mar Ecol Prog Ser 69:133–142] from 3 to 31% of the free-living bacteria and 3 to 26% of particulate-associated bacteria appeared to be phage-infected at any given time. Based upon a steady-state model in which half the daughter cells survive to divide again, the percent of total mortality would be twice the total percentage of phage-infected cells. From 6 to 62% and from 6 to 52% of mortality for the free-living and particulate-associated bacterial community, respectively, may be due to viruses. Offprint requests to: L. M. Proctor.     

Effect of pH and temperature on protein kinase release by Leishmania donovani

During their life cycle Leishmania are exposed to environments that differ markedly in pH and temperature. The effect of these factors on protein kinase release into the surrounding environment by Leishmania donovani promastigotes was examined. Promastigotes release protein kinase activity both constitutively and following induction by incubation with an exogenous substrate, phosvitin. The substrate specificity of the constitutive and induced activities was similar, unlike that previously described for Leishmania major promastigotes. The Leishmania donovani enzymes phosphorylate phosvitin, but not casein, mixed histones or protamine sulphate, and both activities are shed over a wide pH range from 6 to 9. Transfer of promastigotes from pH 7.4/30 degrees C to pH 5.0-5.5/37 degrees C, conditions that mimic those encountered by parasites following transmission from sandflies to a mammalian host and uptake by macrophages, inhibited release of the constitutive activity. Identical conditions had only a minor effect on induced protein kinase release. Both types of protein kinase activities released at pH 7.4 were still active when assayed at pH 5.0. Characterisation of the constitutive and induced promastigote protein kinases showed that casein kinase 1- and casein kinase 2-like activities are released by Leishmania donovani. Constitutive enzyme release decreased over time, however, the addition of phosvitin to these "casein kinase-depleted" promastigotes induced elevated casein kinase 1 and casein kinase 2 shedding. These results suggest that shed protein kinase might play a role in parasite survival and adaptation to host environments.     

Comparative genome analysis of potential regulatory elements in the ABCG5-ABCG8 gene cluster

The excretion of sterols from the liver and intestine is regulated by the ABCG5 and ABCG8 transporters. To identify potential regulatory elements, 152 kb of the human ABCG5-ABCG8 gene cluster was sequenced and comparative genome analysis was performed. The two genes are oriented in a head-to-head configuration and are separated by a 374-bp intergenic region, which is highly conserved among several species. Using a reporter construct, the intergenic region was found to act as a bidirectional promoter. A conserved GATA site in the intergenic region was shown by site-directed mutagenesis to act as a repressor for the ABCG5 promoter. The intergenic region was also shown to be partially responsive to treatment by LXR agonists. In summary, several potential regulatory elements were found for the ABCG5 and ABCG8 genes, and the intergenic region was found to act as a bidirectional promoter.     

Identifying novel regulators of shoot meristem development

New organs are initiated throughout the life span of higher plants. This process occurs at the shoot meristem, which is initiated during embryogenesis and is later responsible for generating the above-ground portion of the plant. The shoot meristem can be thought of as having two zones, a central zone containing meristematic cells in an undifferentiated state, and a surrounding peripheral zone where cells enter a specific developmental pathway toward a differentiated state. Recent advances have revealed several genes that specifically regulate meristem development inArabidopsis. However, extensive mutagenesis by several labs have identified only a handful, of loci that appear to specifically regulate shoot meristem development. We have undertaken an enhancer/suppressor mutagenesis of an existing meristem mutant (clv1) and have identified novel regulators of meristem development. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology”     

A Voltage-Gated Calcium Channel Regulates Lysosomal Fusion with Endosomes and Autophagosomes and Is Required for Neuronal Homeostasis

Autophagy helps deliver sequestered intracellular cargo to lysosomes for proteolytic degradation and thereby maintains cellular homeostasis by preventing accumulation of toxic substances in cells. In a forward mosaic screen in Drosophila designed to identify genes required for neuronal function and maintenance, we identified multiple cacophony (cac) mutant alleles. They exhibit an age-dependent accumulation of autophagic vacuoles (AVs) in photoreceptor terminals and eventually a degeneration of the terminals and surrounding glia. cac encodes an α1 subunit of a Drosophila voltage-gated calcium channel (VGCC) that is required for synaptic vesicle fusion with the plasma membrane and neurotransmitter release. Here, we show that cac mutant photoreceptor terminals accumulate AV-lysosomal fusion intermediates, suggesting that Cac is necessary for the fusion of AVs with lysosomes, a poorly defined process. Loss of another subunit of the VGCC, α2δ or straightjacket (stj), causes phenotypes very similar to those caused by the loss of cac, indicating that the VGCC is required for AV-lysosomal fusion. The role of VGCC in AV-lysosomal fusion is evolutionarily conserved, as the loss of the mouse homologues, Cacna1a and Cacna2d2, also leads to autophagic defects in mice. Moreover, we find that CACNA1A is localized to the lysosomes and that loss of lysosomal Cacna1a in cerebellar cultured neurons leads to a failure of lysosomes to fuse with endosomes and autophagosomes. Finally, we show that the lysosomal CACNA1A but not the plasma-membrane resident CACNA1A is required for lysosomal fusion. In summary, we present a model in which the VGCC plays a role in autophagy by regulating the fusion of AVs with lysosomes through its calcium channel activity and hence functions in maintaining neuronal homeostasis.     

The effect of phospholipid composition of reconstituted HDL on its cholesterol efflux and anti-inflammatory properties

The goal of this study was to understand how the reconstituted HDL (rHDL) phospholipid (PL) composition affects its cholesterol efflux and anti-inflammatory properties. An ApoA-I mimetic peptide, 5A, was combined with either SM or POPC. Both lipid formulations exhibited similar in vitro cholesterol efflux by ABCA1, but 5A-SM exhibited higher ABCG1- and SR-BI-mediated efflux relative to 5A-POPC (P < 0.05). Injection of both rHDLs in rats resulted in mobilization of plasma cholesterol, although the relative potency was 3-fold higher for the same doses of 5A-SM than for 5A-POPC. Formation of preβ HDL was observed following incubation of rHDLs with both human and rat plasma in vitro, with 5A-SM inducing a higher extent of preβ formation relative to 5A-POPC. Both rHDLs exhibited anti-inflammatory properties, but 5A-SM showed higher inhibition of TNF-α, IL-6, and IL-1β release than did 5A-POPC (P < 0.05). Both 5A-SM and 5A-POPC showed reduction in total plaque area in ApoE^−/− mice, but only 5A-SM showed a statistically significant reduction over placebo control and baseline (P < 0.01). The type of PL used to reconstitute peptide has significant influence on rHDL’s anti-inflammatory and anti-atherosclerosis properties.     

Histone tail-independent chromatin binding activity of recombinant cohesin holocomplex

Cohesin, an SMC (structural maintenance of chromosomes) protein-containing complex, governs several important aspects of chromatin dynamics, including the essential chromosomal process of sister chromatid cohesion. The exact mechanism by which cohesin achieves the bridging of sister chromatids is not known. To elucidate this mechanism, we reconstituted a recombinant cohesin complex and investigated its binding to DNA fragments corresponding to natural chromosomal sites with high and low cohesin occupancy in vivo. Cohesin displayed uniform but nonspecific binding activity with all DNA fragments tested. Interestingly, DNA fragments with high occupancy by cohesin in vivo showed strong nucleosome positioning in vitro. We therefore utilized a defined model chromatin fragment (purified reconstituted dinucleosome) as a substrate to analyze cohesin interaction with chromatin. The four-subunit cohesin holocomplex showed a distinct chromatin binding activity in vitro, whereas the Smc1p-Smc3p dimer was unable to bind chromatin. Histone tails and ATP are dispensable for cohesin binding to chromatin in this reaction. A model for cohesin association with chromatin is proposed.     

Biomass allocation in herbaceous plants under grazing impact in the high semi-arid Andes

Among the plant traits that affect performance, vitality and herbivore resistance in rangeland vegetation, biomass partitioning ranks top, commonly outweighing processes at single leaves (e.g., photosynthesis, respiration). We explored the allometry of a broad sample of herbaceous species from two high elevation sites in the Andes in order to explain biomass partitioning under harsh climatic conditions and risk of biomass losses under strong camelid grazing pressure. We combined data from NW-Argentina and W-Bolivia from elevations between 4200 and 4250 m in a landscape dominated by tall Festuca orthophylla tussocks and a drought driven seasonality (rainfall only between November and March). Across 10–20 taxa per region we found less investment in leaves with a mean leaf mass fraction of only 11% and instead a massive storage in below-ground compartments (rhizomes, tap roots), particularly at the colder Argentinean site. Though grazing pressure was much greater in Bolivia, the foliage mass fraction was there larger than in Argentina. The inter-tussock space in these open, dry plains (‘pajonal’) was dominated by rosette forming species with a below-ground shoot apex and massive tap roots (70% of all species), rendering these species less sensitive to grazing and trampling. The storage organs of these species represented more than 50% of total biomass. Llamas, which represented the main vertebrate herbivore in these open plains at the Bolivian site, preferred non tap-root herbs and species with low leaf nitrogen concentration. Palatable forbs for llamas (22% of all species at the Bolivian site) only survived when nested (facilitated) in the rigid, tall Festuca orthophylla tussocks or thorny shrubs. In conclusion, these extremely high elevation rangeland herbs invest preferentially in structures for persistence (K-strategy) rather than maximizing carbon gain.     

Towards measuring brain function on groups of people in the real world

In three studies, EEGs from three groups of participants were recorded during progressively more real world situations after drinking alcoholic beverages that brought breath alcohol contents near the limit for driving in California 30 minutes after drinking. A simple equation that measured neurophysiological effects of alcohol in the first group of 15 participants performing repetitive cognitive tasks was applied to a second group of 15 operating an automobile driving simulator, and to a third group of 10 ambulatory people recorded simultaneously during a cocktail party. The equation derived from the first group quantified alcohol's effect by combining measures of higher frequency (beta) and lower frequency (theta) power into a single score. It produced an Area Under the Receiver Operator Characteristic Curve of .73 (p<.05; 67% sensitivity in recognizing alcohol and 87% specificity in recognizing placebo). Applying the same equation to the second group operating the driving simulator, AUC was .95, (p<.0001; 93% sensitivity and 73% specificity), while for the cocktail party group AUC was .87 (p<.01; 80% sensitivity and 80% specificity). EEG scores were significantly related to breath alcohol content in all studies. Some individuals differed markedly from the overall response evident in their respective groups. The feasibility of measuring the neurophysiological effect of a psychoactive substance from an entire group of ambulatory people at a cocktail party suggests that future studies may be able to fruitfully apply brain function measures derived under rigorously controlled laboratory conditions to assess drug effects on groups of people interacting in real world situations.     

Crag Is a GEF for Rab11 Required for Rhodopsin Trafficking and Maintenance of Adult Photoreceptor Cells

Rhodopsins (Rhs) are light sensors, and Rh1 is the major Rh in the Drosophila photoreceptor rhabdomere membrane. Upon photoactivation, a fraction of Rh1 is internalized and degraded, but it remains unclear how the rhabdomeric Rh1 pool is replenished and what molecular players are involved. Here, we show that Crag, a DENN protein, is a guanine nucleotide exchange factor for Rab11 that is required for the homeostasis of Rh1 upon light exposure. The absence of Crag causes a light-induced accumulation of cytoplasmic Rh1, and loss of Crag or Rab11 leads to a similar photoreceptor degeneration in adult flies. Furthermore, the defects associated with loss of Crag can be partially rescued with a constitutive active form of Rab11. We propose that upon light stimulation, Crag is required for trafficking of Rh from the trans-Golgi network to rhabdomere membranes via a Rab11-dependent vesicular transport.