Early Archean planktonic mode of life: Implications from fluid dynamics of lenticular microfossils

Lenticular, and commonly flanged, microfossils in 3.0–3.4 Ga sedimentary deposits in Western Australia and South Africa are unusually large (20–80 μm across), robust, and widespread in space and time. To gain insight into the ecology of these organisms, we performed simulations of fluid dynamics of virtual cells mimicking lenticular forms of variable sizes, oblateness, flange presence, and flange thickness. Results demonstrate that (a) the flange reduces sedimentation velocity, (b) this flange function works more effectively in larger cells, and (c) modest oblateness lowers sedimentation rate. These observations support interpretations that the lenticular microbes were planktonic—a lifestyle that could have been advantageous in an early Earth harsh environment including violent volcanic activities, repeated asteroid impacts, and relatively high UV‐radiation. Although the robustness of these organisms could have provided additional protection on the early Earth, this architecture may have impeded a planktonic lifestyle by increasing cell density. However, our data suggest that this disadvantage could have been compensated by enlargement of cell volume, which could have enhanced the ability of the flange to slow sedimentation rate, especially if coupled with vacuolation. The results of this simulation study may help to explain the unique morphology and unusually large size of these Archean microfossils.     

Glycation end-product cross-link breaker reduces collagen and improves cardiac function in aging diabetic heart

Aging and diabetes mellitus (DM) both affect the structure and function of the myocardium, resulting in increased collagen in the heart and reduced cardiac function. As part of this process, hyperglycemia is a stimulus for the production of advanced glycation end products (AGEs), which covalently modify proteins and impair cell function. The goals of this study were first to examine the combined effects of aging and DM on hemodynamics and collagen types in the myocardium in 12 dogs, 9-12 yr old, and second to examine the effects of the AGE cross-link breaker phenyl-4,5-dimethylthazolium chloride (ALT-711) on myocardial collagen protein content, aortic stiffness, and left ventricular (LV) function in the aged diabetic heart. The alloxan model of DM was utilized to study the effects of DM on the aging heart. DM induced in the aging heart decreased LV systolic function (LV ejection fraction fell by 25%), increased aortic stiffness, and increased collagen type I and type III protein content. ALT-711 restored LV ejection fraction, reduced aortic stiffness and LV mass with no reduction in blood glucose level (199 +/- 17 mg/dl), and reversed the upregulation of collagen type I and type III. Myocardial LV collagen solubility (%) increased significantly after treatment with ALT-711. These data suggest that an AGE cross-link breaker may have a therapeutic role in aged patients with DM.     

Functions of adaptor protein (AP)-3 and AP-1 in tyrosinase sorting from endosomes to melanosomes

Specialized cells exploit adaptor protein complexes for unique post-Golgi sorting events, providing a unique model system to specify adaptor function. Here, we show that AP-3 and AP-1 function independently in sorting of the melanocyte-specific protein tyrosinase from endosomes to the melanosome, a specialized lysosome-related organelle distinguishable from lysosomes. AP-3 and AP-1 localize in melanocytes primarily to clathrin-coated buds on tubular early endosomes near melanosomes. Both adaptors recognize the tyrosinase dileucine-based melanosome sorting signal, and tyrosinase largely colocalizes with each adaptor on endosomes. In AP-3-deficient melanocytes, tyrosinase accumulates inappropriately in vacuolar and multivesicular endosomes. Nevertheless, a substantial fraction still accumulates on melanosomes, concomitant with increased association with endosomal AP-1. Our data indicate that AP-3 and AP-1 function in partially redundant pathways to transfer tyrosinase from distinct endosomal subdomains to melanosomes and that the AP-3 pathway ensures that tyrosinase averts entrapment on internal membranes of forming multivesicular bodies.     

The discovery and structure-activity relationships of pyrano[3,4-b]indole-based inhibitors of hepatitis C virus NS5B polymerase

We describe the structure-activity relationship of the C7-position of pyrano[3,4-b]indole-based inhibitors of HCV NS5B polymerase. Further exploration of the allosteric binding site led to the discovery of the significantly more potent compounds 13 and 14.     

High-Resolution Physical Map of the X-linked Retinoschisis Interval in Xp22

X-linked retinoschisis (RS) is the leading cause of macular degeneration in young males and has been mapped to Xp22 between DXS418 and DXS999. To facilitate identification of the RS gene, we have constructed a yeast artificial chromosome (YAC) contig across this region comprising 28 YACs and 32 sequence-tagged sites including seven novel end clone markers. To establish the definitive marker order, a PAC contig containing 50 clones was also constructed, and all clones were fingerprinted. The marker order is: Xpter–DXS1317–(AFM205yd12–DXS7175–DXS7992)–60N8T7–DXS1195–DXS7993–DXS7174–60N8-SP6–DXS418–DXS7994–DXS7995–DXS7996–HYAT2–25HA10R–HYAT1–DXS7997–DXS7998–DXS257–434E8R–3542R–DXS6762–DXS7999–DXS6763–434E8L–DXS8000–DXS6760–DXS7176–DXS8001–DXS999–3176R–PHKA2–Xcen. A long-range restriction map was constructed, and the RS region is estimated to be 1300 kb, containing three putative CpG islands. An unstable region was identified between DXS6763 and 434E8L. These data will facilitate positional cloning of RS and other disease genes in Xp22.     

IL-2 secretion by CD4+ T cells in vivo is rapid, transient, and influenced by TCR-specific competition

The secretion of IL-2 is a critical and early landmark in the activation program of CD4(+) T cells in vitro, but the lack of sensitive assays has limited its application for studying T cell activation in vivo. Using a mouse cytokine capture assay we were able to detect the rapid secretion of IL-2 after an in vivo stimulus by 1-2 h in naive T cells and as early as 30 min in memory T cells. Maximal secretion was achieved within 1-2 h for memory cells or 6-8 h for naive T cells. Surprisingly IL-2 production terminated quickly in vivo and secretion was undetectable by 20-24 h in either cell type. We further demonstrated that this short duration of secretion can be influenced by cellular competition between Ag-specific CD4(+) T cells. The consequences of competition were mimicked by reducing the strength of the antigenic stimulus. These data argue that early competition between T cells influences both the eventual frequency of IL-2 producers in the population and also the duration of their secretion, potentially by altering the strength or duration of the stimulus available to each T cell.     

Role of mass spectrometry in the purification of peptides and proteins

Experiments were carried out to evaluate the fractionation of proteins and peptides according to mass. Model mixtures were separated by either reversed-phase or ion-exchange chromatography with mass spectrometry-compatible mobile phase additives. Fraction collection was triggered by the mass/charge ratio of each one of the components of the mixture. Chromatography was additionally monitored with a UV-Vis detector in order to compare the new technique with generally accepted in separations. The results indicated that adequate purification is achieved by this new technique. Fraction collection triggered by changes in the mass/charge ratio reduces sample handling and analysis time. This study demonstrates the utility of mass-directed fractionation of peptides and proteins when mass spectrometry-compatible mobile phase additives are used.     

Use of Bioluminescence for Detection of Genetically Engineered Microorganisms Released into the Environment

The persistence and movement of strain JS414 of Xanthomonas campestris pv. campestris, which was genetically engineered to bioluminesce, were monitored during a limited field introduction. Bioluminescence and traditional dilution plate counts were determined. Strain JS414 was applied to cabbage plants and surrounding soil by mist inoculation, by wound inoculation, by scattering infested debris among plants, and by incorporating bacteria into the soil. Bioluminescent X. campestris pv. campestris was detected in plant samples and in the rhizosphere up to 6 weeks after inoculation. Movement to uninoculated plants was detected on one occasion, but movement from the immediate release area was not detected. Strain JS414 was detected in soil samples beneath mist- and wound-inoculated plants only at intentionally infested locations and in aerial samples only on the day of inoculation. Our bioluminescence methods proved to be as sensitive as plating methods for detecting the genetically engineered microorganisms in environmental samples. Our results demonstrate that transgenic incorporation of the luxCDABE operon provides a non-labor-intensive, sensitive detection method for monitoring genetically engineered microorganisms in nature.