Transport of coenzyme M (2-mercaptoethanesulfonic acid) in Methanobacterium ruminantium.

A system for transport of coenzyme M, 2-mercaptoethanesulfonic acid (HS--CoM), in Methanobacterium ruminatium strain M1 required energy, showed saturation kinetics, and concentrated the coenzyme against a gradient. The process was sensitive to temperature and was maximally active at pH 7.1. Cells took up HS--CoM at a linear rate, with a Vmax of 312 pmol/min per mg (dry weight) and an apparent Km of 73 nM. An intracellular pool of up to 5 mM accumulated which was not exchangeable with the medium. Uptake required both hydrogen and carbon dioxide; it was inhibited by O2. Bromoethanesulfonic acid (BrCH2CH2SO3-), a potent inhibitor of methanogenesis in cell-free extracts, inhibited both uptake and methane production. Results of inhibitor studies with derivatives and analogs of the coenzyme showed that the specificity of the carrier is restricted to a limited range of thioether, thioester, and thiocarbonate derivatives. 2-(Methylthio)ethanesulfonic acid (CH3--S--CoM) showed an apparent Ki for HS--CoM uptake of 15 nM, being taken up itself with a Vmax of 320 pmol/min per mg (dry weight) and an apparent Km of 50 nM. An analysis of intracellular pools after HS--CoM uptake indicated that the predominant forms are a heterodisulfide of unknown composition and CH3--S--CoM.     

Large-scale profiling of Rab GTPase trafficking networks: the membrome

Rab GTPases and SNARE fusion proteins direct cargo trafficking through the exocytic and endocytic pathways of eukaryotic cells. We have used steady state mRNA expression profiling and computational hierarchical clustering methods to generate a global overview of the distribution of Rabs, SNAREs, and coat machinery components, as well as their respective adaptors, effectors, and regulators in 79 human and 61 mouse nonredundant tissues. We now show that this systems biology approach can be used to define building blocks for membrane trafficking based on Rab-centric protein activity hubs. These Rab-regulated hubs provide a framework for an integrated coding system, the membrome network, which regulates the dynamics of the specialized membrane architecture of differentiated cells. The distribution of Rab-regulated hubs illustrates a number of facets that guides the overall organization of subcellular compartments of cells and tissues through the activity of dynamic protein interaction networks. An interactive website for exploring datasets comprising components of the Rab-regulated hubs that define the membrome of different cell and organ systems in both human and mouse is available at http://www.membrome.org/.     

Solution 1H NMR study of the accommodation of the side chain of n-butyl-etiohemin-I incorporated into the active site of cyano-metmyoglobin

In order to identify the most readily deformable portion of the heme pocket in myoglobin, equine myoglobin was reconstituted with a meso-n-butyl substituent on centrosymmetric etiohemin-I. Solution ¹H NMR data for the low-spin iron(III) cyanide complex of oxidized myoglobin that include 2D nuclear Overhauser enhancement spectroscopy contacts, paramagnetic relaxation, and dipolar shifts resulting from magnetic anisotropy show that the heme binds uniquely to the iron in a manner that arranges the methyl and ethyl substituents on a given pyrrole in a clockwise manner when viewed from the proximal side, and with the n-butyl group seated at the canonical -meso position of native protohemin-IX. The butyl group is oriented sharply toward the proximal side and its protein contacts demonstrate that it is oriented largely into the xenon hole in myoglobin. The location of the n-butyl group on the proximal side near the vacancies places it within the region found to be most flexible in molecular dynamics simulation. A small, counterclockwise rotation of the pyrrole N–Fe–N vector of n-butyl-etiohemin-I relative to that for native protohemin, indicated by both the prosthetic group methyl contact shift pattern and the prosthetic group contacts to heme pocket residues, is proposed to allow the xenon hole to accommodate better the n-butyl group. In contrast to previous work, which showed that a bulky polar substituent on etiohemin-I preferentially seats at the canonical -meso position, the nonpolar n-butyl group selects the -meso position.Electronic Supplementary Material Supplementary material is available for this article at .     

The pathophysiology and mechanisms of NP-C disease

The molecular isolation of NPC1 and NPC2, the genes defective in patients with Niemann-Pick disease type C (NP-C), has heralded in an exponential increase in our understanding of this syndrome and thus of human intracellular sterol transport. Despite this, neither the mechanisms of action nor the substrates for these putative transporters have been defined. In this overview, we describe our perspectives on the current awareness of the genetic determination and cellular biology of this syndrome, with emphasis on the underlying events that lead to neurodegeneration and the manner in which they might eventually be treated.     

Sea surface temperature gradients, baroclinicity, and vegetation gradients in the sea

We describe the results of an analysis of the 1984 CalCOFI dataset, in which sea surface temperature (SST) gradients were foundto be well correlated to isopycnal slope (baroclinicity) atthe mesoscale. A link was established between the SST gradientsand gradients of integral biomass [mg chlorophyll (Chl) a m^–2]and integral primary production (mg C m^–2day^–1).The water column photosynthetic efficiency, , was calculatedby combining the CalCOFI data base with satellite-based estimatesof the surface irradiance. It was found that was rather stable,but not constant, for a wide range of isopycnal slopes, in whichintegrated nitrate concentrations varied by up to 500x. Variabilityin was most strongly dependent on calendar date, with the highestvalues in winter months and the lowest values in the summer,which was probably due to an effect of day length or irradiance.The relationship between baroclinicity and SST gradients washighly scale dependent; from 0 to 200 km, the data were randomlydistributed, then as the length scales exceeded 400 km, therelationship between baroclinicity and the SST gradient showeda significantly negative slope, with steadily improving correlationcoefficient. Such relationships for individual cruises generallyshowed improved correlation over the generic annual relationship,which highlights the dynamic hydrography of the region. Baroclinicity/SSTgradient relationships also improved in waters of increasingdepth. A technique is described for calculating net integralbiomass or production gradients from SST imagery. The gradientsin biomass and productivity showed increasingly strong tiesto isopycnal slope at larger size scales. Although this topichas long been described by oceanographers, this paper is thefirst to demonstrate a quantitative coupling between baroclinicityand primary production gradients as a function of length scale.Also significant is the observation that the relationships betweenbaroclinicity versus gradients of integral chlorophyll or integralproductivity are consistent over seasons and over an area exceeding1 million km². We conclude that for satellite algorithms toaccount for >50% of the variance in primary production, thenutrient term must be considered. Information on baroclinicityis relevant to estimates of the along-isopycnal nutrient fluxto the euphotic zone. Merging this with the more traditionallight-based estimates of photosynthesis will improve algorithmperformance since the methods utilize entirely independent sourcesof information.     

p53/58 Binds COPI and Is Required for Selective Transport through the Early Secretory Pathway

p53/58 is a transmembrane protein that continuously recycles between the ER and pre-Golgi intermediates composed of vesicular-tubular clusters (VTCs) found in the cell periphery and at the cis face of the Golgi complex. We have generated an antibody that uniquely recognizes the p53/58 cytoplasmic tail. Here we present evidence that this antibody arrests the anterograde transport of vesicular stomatitis virus glycoprotein and leads to the accumulation of p58 in preGolgi intermediates. Consistent with a role for the KKXX retrieval motif found at the cytoplasmic carboxyl terminus of p53/58 in retrograde traffic, inhibition of transport through VTCs correlates with the ability of the antibody to block recruitment of COPI coats to the p53/58 cytoplasmic tail and to p53/58-containing membranes. We suggest that p53/58 function may be required for the coupled exchange of COPII for COPI coats during segregation of anterograde and retrograde transported proteins.     

Demonstration of NK cell-mediated lysis of varicella-zoster virus (VZV)-infected cells: characterization of the effector cells

Infection with varicella-zoster virus (VZV) rendered RAJI cells more susceptible to lysis by non-adherent blood lymphocytes. At an effector to target ratio of 80:1 the mean percentage of 51Cr release of VZV-infected RAJI cells was 41 +/- 12%, whereas that of uninfected RAJI cells was 15 +/- 6%. The increased susceptibility to lysis was associated with increased effector to target conjugate formation in immunofluorescence binding assays. The effector cells cytotoxic for VZV-infected RAJI cells were predominantly Leu-11a+ Leu-4- granular lymphocytes as demonstrated by fluorescence-activated cell sorting. The effector cell active against VZV-infected RAJI cells appeared similar to those active against herpes simplex virus (HSV)-infected cells, because in cold target competition experiments the lysis of 51Cr-labeled VZV-infected RAJI cells was efficiently inhibited by either unlabeled VZV-infected RAJI cells (mean 71% inhibition, 2:1 ratio unlabeled to labeled target) or HSV-infected RAJI cells (mean 69% inhibition) but not by uninfected RAJI cells (mean 10% inhibition). In contrast, competition experiments revealed donor heterogeneity in the overlap between effector cells for VZV- or HSV-infected RAJI vs K-562 cells.     

Multiple forms of sulfmyoglobin as detected by 1H nuclear magnetic resonance spectroscopy

The proton nuclear magnetic resonance spectrum of sulfmyoglobin prepared in standard fashion reveals the presence of three forms, A, B, and C, with different chemical reactivity. Conditions for some interconversions of these forms are given. The 1H NMR spectra of the different forms show similar patterns. It appears that the differences between forms involve chemical modification on the porphyrin periphery. The altered heme can be extracted from FeIII(CN) sulfmyoglobin C to give a stable green substance.