Measurement of Biocolloid Collision Efficiencies for Granular Activated Carbon by Use of a Two-Layer Filtration Model

Point-of-use filters containing granular activated carbon (GAC) are an effective method for removing certain chemicals from water, but their ability to remove bacteria and viruses has been relatively untested. Collision efficiencies (α) were determined using clean-bed filtration theory for two bacteria (Raoutella terrigena 33257 and Escherichia coli 25922), a bacteriophage (MS2), and latex microspheres for four GAC samples. These GAC samples had particle size distributions that were bimodal, but only a single particle diameter can be used in the filtration equation. Therefore, consistent with previous reports, we used a particle diameter based on the smallest diameter of the particles (derived from the projected areas of 10% of the smallest particles). The bacterial collision efficiencies calculated using the filtration model were high (0.8 ≤ α ≤ 4.9), indicating that GAC was an effective capture material. Collision efficiencies greater than unity reflect an underestimation of the collision frequency, likely as a result of particle roughness and wide GAC size distributions. The collision efficiencies for microspheres (0.7 ≤ α ≤ 3.5) were similar to those obtained for bacteria, suggesting that the microspheres were a reasonable surrogate for the bacteria. The bacteriophage collision efficiencies ranged from ≥0.2 to ≤0.4. The predicted levels of removal for 1-cm-thick carbon beds ranged from 0.8 to 3 log for the bacteria and from 0.3 to 1.0 log for the phage. These tests demonstrated that GAC can be an effective material for removal of bacteria and phage and that GAC particle size is a more important factor than relative stickiness for effective particle removal.     

The retromer complex influences Wnt secretion by recycling wntless from endosomes to the trans-Golgi network

Secreted Wnt proteins play essential roles in many biological processes during development and diseases. However, little is known about the mechanism(s) controlling Wnt secretion. Recent studies have identified Wntless (Wls) and the retromer complex as essential components involved in Wnt signaling. While Wls has been shown to be essential for Wnt secretion, the function(s) of the retromer complex in Wnt signaling is unknown. Here, we have examined a role of Vps35, an essential retromer subunit, in Wnt signaling in Drosophila and mammalian cells. We provide compelling evidence that the retromer complex is required for Wnt secretion. Importantly, Vps35 colocalizes in endosomes and interacts with Wls. Wls becomes unstable in the absence of retromer activity. Our findings link Wls and retromer functions in the same conserved Wnt secretion pathway. We propose that retromer influences Wnt secretion by recycling Wntless from endosomes to the trans-Golgi network (TGN).     

Enhanced biodegradation of aromatic pollutants in cocultures of anaerobic and aerobic bacterial consortia

Toxic aromatic pollutants, concentrated in industrial wastes and contaminated sites, can potentially be eliminated by low cost bioremediation systems. Most commonly, the goal of these treatment systems is directed at providing optimum environmental conditions for the mineralization of the pollutants by naturally occurring microflora. Electrophilic aromatic pollutants with multiple chloro, nitro and azo groups have proven to be persistent to biodegradation by aerobic bacteria. These compounds are readily reduced by anaerobic consortia to lower chlorinated aromatics or aromatic amines but are not mineralized further. The reduction increases the susceptibility of the aromatic molecule for oxygenolytic attack. Sequencing anaerobic and aerobic biotreatment steps provide enhanced mineralization of many electrophilic aromatic pollutants. The combined activity of anaerobic and aerobic bacteria can also be obtained in a single treatment step if the bacteria are immobilized in particulate matrices (e.g. biofilm, soil aggregate, etc.). Due to the rapid uptake of oxygen by aerobes and facultative bacteria compared to the slow diffusion of oxygen, oxygen penetration into active biofilms seldom exceeds several hundred micrometers. The anaerobic microniches established inside the biofilms can be applied to the reduction of electron withdrawing functional groups in order to prepare recalcitrant aromatic compounds for further mineralization in the aerobic outer layer of the biofilm.Aside from mineralization, polyhydroxylated and chlorinated phenols as well as nitroaromatics and aromatic amines are susceptible to polymerization in aerobic environments. Consequently, an alternative approach for bioremediation systems can be directed towards incorporating these aromatic pollutants into detoxified humic-like substances. The activation of aromatic pollutants for polymerization can potentially be encouraged by an anaerobic pretreatment step prior to oxidation. Anaerobic bacteria can modify aromatic pollutants by demethylating methoxy groups and reducing nitro groups. The resulting phenols and aromatic amines are readily polymerized in a subsequent aerobic step.     

Protein kinase C is regulated in vivo by three functionally distinct phosphorylations

Background: Protein kinase Cs are a family of enzymes that transduce the plethora of signals promoting lipid hydrolysis. Here, we show that protein kinase C must first be processed by three distinct phosphorylations before it is competent to respond to second messengers.Results We have identified the positions and functions of the in vivo phosphorylation sites of protein kinase C by mass spectrometry and peptide sequencing of native and phosphatase-treated kinase from the detergent-soluble fraction of cells. Specifically, the threonine at position 500 (T500) on the activation loop, and T641 and S660 on the carboxyl terminus of protein kinase C βII are phosphorylated in vivo. T500 and S660 are selectively dephosphorylated in vitro by protein phosphatase 2A to yield an enzyme that is still capable of lipid-dependent activation, whereas all three residues are dephosphorylated by protein phosphatase 1 to yield an inactive enzyme. Biochemical analysis reveals that protein kinase C autophosphorylates on S660, that autophosphorylation on S660 follows T641 autophosphorylation, that autophosphorylation on S660 is accompanied by the release of protein kinase C into the cytosol, and that T500 is not an autophosphorylation site.Conclusion Structural and biochemical analyses of native and phosphatase-treated protein kinase C indicate that protein kinase C is processed by three phosphorylations. Firstly, trans-phosphorylation on the activation loop (T500) renders it catalytically competent to autophosphorylate. Secondly, a subsequent autophosphorylation on the carboxyl terminus (T641) maintains catalytic competence. Thirdly, a second autophosphorylation on the carboxyl terminus (S660) regulates the enzyme's subcellular localization. The conservation of each of these residues (or an acidic residue) in conventional, novel and atypical protein kinase Cs underscores the essential role for each in regulating the protein kinase C family.     

Apolipoprotein A-I alpha -helices 7 and 8 modulate high density lipoprotein subclass distribution.

Mice have a monodisperse high density lipoprotein (HDL) profile, whereas humans have two major subfractions designated HDL(2) and HDL(3). Human apoA-I transgenic mice exhibit a human-like HDL profile, indicating that the amino acid sequence of apoA-I is a determinant of the HDL profile. Comparison of the primary sequence of mouse and human apoA-I and the previously designated "hinge" domain of apoA-I led us to hypothesize that alpha-helices 7 and 8 (7/8) are determinants of HDL subclass distribution. The following proteins were expressed in Escherichia coli: human apoA-I, T7-hAI; mouse apoA-I, T7-mAI; chimeric human apoA-I containing murine helices 7/8 in place of human helices 7/8, T7-hAI(m7/8); and the reciprocal chimera, T7-mAI(h7/8). The recombinant proteins were examined for their association with human plasma HDL subclasses. The results demonstrated that T7-hAI bound HDL(2) and HDL(3) equally well, whereas T7-mAI bound to HDL(2) preferentially. T7-hAI(m7/8) behaved like T7-mAI, and T7-mAI(h7/8) behaved like T7-hAI. Thus, alpha-helices 7/8 are strong contributors to the pattern of HDL subclass association. Self-association, alpha-helicity, cholesterol efflux, and lecithin-cholesterol acyltransferase activity of the recombinant proteins were also assessed. Human apoA-I self-associates more and activates human lecithin-cholesterol acyltransferase better than mouse apoA-I. These differential characteristics of human and mouse apoA-I are not dependent on helices 7/8.     

Random amplified polymorphic DNA markers reveal genetic variation in the symbiotic fungus of leaf-cutting ants

RAPD markers were used to examine the degree of genetic variation within the putatively asexual basidiomycete fungus (Lepiotaceae: provisionally named Leucoagaricus gongylophorus) associated with the leaf-cutting ant species Atta cephalotes. We analyzed fungal isolates from ant nests in two geographically distant sites, two isolates from Panama and five isolates from Trinidad. Ten decamer primers were used to amplify total DNA from these seven fungal isolates, and RAPD banding patterns were compared. Genetic similarity among isolates was determined by pair-wise comparisons of the shared number of DNA bands on an agarose gel. There was considerable genetic variation among isolates of the symbiotic fungus even within sites. Pairs of fungal isolates from the two different sites shared an average of only 36% of the bands in their RAPD profiles, while pairs from the within sites shared an average of 72% of the bands. RAPD markers may be useful for further investigation of the genetic structure of the fungal symbiont within species of leaf-cutting ants.     

Isolation of an actin polymerization stimulator from bovine thyroid plasma membranes

An actin polymerization stimulator was purified from bovine thyroid plasma membranes by DNase I affinity column chromatography. Although the molecular weight of the protein was about 42,000 (42K) by sodium dodecyl sulfate polyacrylamide gel electrophoresis, it did not comigrate with actin. In the presence of 30 mM KCl, the 42K protein facilitated formation of actin filaments when analyzed by a centrifugation method, accelerated the initial phase of actin polymerization as measured in an Ostwald viscometer and increased the length of filaments as shown by electron microscopy. The 42K protein also accelerated the initial phase of actin polymerization in the presence of 100 mM KCl and 2 mM MgCl₂ but did not affect the final viscosity. The effect of the 42K protein was diminished by 5 uM cytochalasin B or 1 uM cytochalasin D. This 42K protein may anchor actin filaments onto the thyroid plasma membrane.