alpha-N-acetylgalactosamine-capping of chondroitin sulfate core region oligosaccharides primed on xylosides

We previously reported that cultured mammalian cells incubated with 4- methylumbelliferyl (MU) or p -nitrophenyl (pNP) beta-xyloside synthesize an alpha-GalNAc-terminated pentasaccharide resembling the glycosaminoglycan-core protein linkage region. Here we show that human melanoma M21 cells and human neuroblastoma cells incubated with Xylbeta- MU/pNP also make an alpha-GalNAc-terminated heptasaccharide containing one chondroitin disaccharide repeat. High performance liquid chromatography and matrix-assisted laser desorption ionization mass spectrometry analysis of intact or glycosidase-digested xyloside showed the structure as: GalNAcalphaGlcAbeta1,3GalNAcbeta1,4GlcAbeta1,3Galbe ta1,3Galbeta1, 4Xylbeta-MU/pNP. The alpha-GalNAc-terminated xylosides can account for approximately 10% of the total Xylbeta-MU/pNP products ( approximately 1.5 nmol/h/mg). These results show that GalNAcalphaGlcAbeta-modification is relatively abundant, but not unique to the GAG-linkage tetrasaccharide. alpha-GalNAc addition to the GlcA residue does not appear to be an extension of general phase II detoxification of xenobiotics that involve glucuronidation, since M21 cells incubated with MU synthesize only 0.3 pmol GlcAbeta-MU/h/mg protein, and undetectable amount of GalNAcalphaGlcAbeta-MU (<40 fmol/h/mg). Further, subcellular fractionation shows that the alpha- N- acetylgalactosaminyltransferase activity colocalizes in the Golgi with other glycosyl transferases and not in the ER, where xenobiotic detoxification glucuronosyltransferases are found. Although GalNAcalphaGlcAbeta-terminal modification has not been detected on naturally occurring GAG chains, the substantial amount of alpha-GalNAc transferase activity suggests that the alpha-GalNAc transferase could utilize other GlcA-containing glycoconjugates as acceptors.      

Phosphatase activity as an early warning indicator of wetland eutrophication: problems and prospects

A phosphorus (P) loading experiment conducted in the oligotrophic P-limited Everglades was used to assess the utility of phosphatase activity (PA) of periphyton as an early warning (EW) indicator of wetland eutrophication. Phosphorus loads of 0, 0.4, 0.8, 1.6, 3.2, 6.4 and 12.8 g P m^–2 yr^–1 were applied to mesocosms placed in a slough community consisting of Cladium jamaicense Crantz, Eleocharis spp. and calcareous periphyton mats. Phosphatase activity, expressed on a biomass-specific basis, was not a sensitive indicator of P enrichment for epiphytic periphyton growing on acrylic dowels or floating mat periphyton. However, surface-area-specific PA was a sensitive indicator of P enrichment, responding within 2–3 weeks of the initiation of dosing. Surface-area-specific PA of unenriched periphyton ranged from 0.42 to 0.7 nmol cm^–2 min^–1, while PA of periphyton growing in the highest load (12.8 g P m^–2 yr^–1), ranged from 0.11 to 0.29 nmol cm^–2 min^–1. Conclusions drawn from PA analyses were consistent with those obtained from periphyton primary productivity and P content. Phosphatase activity is a potentially valuable EW indicator when used in conjunction with other complementary indicators.     

Functional inactivity and mutations of p53 differentially affect sensitivity to 5-fluorouracil and antifolate inhibitors of thymidylate synthase (TS) by altering TS levels in colorectal cancer cells

The role of p53 in altering TS expression and chemosensitivity was studied in colorectal cancer cells with wildtype, mutated, or functionally inactive p53. Cytotoxicity of TS inhibitors was studied by MTT, while PCR, Western blot, and activity assays assessed whether p53 status influenced TS expression. Lovo-175X2 cells showed increased resistance to TS inhibitors and significantly greater than wildtype expression and activity of TS. In contrast, Lovo-273X17 and Lovo-li were more sensitive to TS inhibitors and had reduced TS expression, due either to reduced TS mRNA or altered regulation of TS activity. Thus, functional inactivity and mutations of p53 differentially affect TS, potentially influencing response to TS inhibitor-based treatment.     

Correlations of cibarial muscle activities of Homalodisca spp. sharpshooters (Hemiptera: Cicadellidae) with EPG ingestion waveform and excretion

Fluid flow into and out of the stylets of xylem-ingesting sharpshooters (Hemiptera: Cicadellidae: Cicadellinae) is powered by muscles of the cibarial pump. Such fluid flow is crucial for transmission of Xylella fastidiosa, the Pierce’s Disease bacterium, yet has not been rigorously studied via electrical penetration graph (EPG) technology. We correlated EPG waveforms with electromyographically (EMG) recorded muscle potentials from the cibarial dilator muscles, which power the piston-like cibarial diaphragm. There was a 1:1 correspondence of each cycle of cibarial muscle contraction/relaxation with each plateau of EPG waveform C. Results definitively showed that the C waveform represents active ingestion, i.e. fluid flow is propelled by cibarial muscle contraction. Moreover, each C waveform episode represents muscular diaphragm uplift, probably combined with a “bounce” from cuticular elasticity, to provide the suction that pulls fluid into the stylets. Fine structure of the EPG ingestion waveform represents directionality of fluid flow, supporting the primary role of streaming potentials as the electrical origin of the C waveform. Rhythmic bouts of cibarial pumping were generally correlated with sustained production of excretory droplets. However, neither the onset nor cessation of ingestion was correlated with onset or cessation of excretion, respectively. Volume of excreta is an inexact measure of ingestion. Implications for using EPG to understand the mechanism of X. fastidiosa transmission are discussed.     

Novel human monoclonal antibody combination effectively neutralizing natural rabies virus variants and individual in vitro escape mutants

The need to replace rabies immune globulin (RIG) as an essential component of rabies postexposure prophylaxis is widely acknowledged. We set out to discover a unique combination of human monoclonal antibodies (MAbs) able to replace RIG. Stringent criteria concerning neutralizing potency, affinity, breadth of neutralization, and coverage of natural rabies virus (RV) isolates and in vitro escape mutants were set for each individual antibody, and the complementarities of the two MAbs were defined at the onset. First, we identified and characterized one human MAb (CR57) with high in vitro and in vivo neutralizing potency and a broad neutralization spectrum. The linear antibody binding site was mapped on the RV glycoprotein as antigenic site I by characterizing CR57 escape mutants. Secondly, we selected using phage display a complementing antibody (CR4098) that recognized a distinct, nonoverlapping epitope (antigenic site III), showed similar neutralizing potency and breadth as CR57, and neutralized CR57 escape mutants. Reciprocally, CR57 neutralized RV variants escaping CR4098. Analysis of glycoprotein sequences of natural RV isolates revealed that the majority of strains contain both intact epitopes, and the few remaining strains contain at least one of the two. In vitro exposure of RV to the combination of CR57 and CR4098 yielded no escape mutants. In conclusion, a novel combination of human MAbs was discovered suitable to replace RIG.     

Uptake,flow, and digestion of casein and green fluorescent protein in the digestive system of Lygus hesperus Knight

Selected compounds were used to study physiological processes associated with digestion in the western tarnished plant bug, Lygus hesperus Knight. Durations of passage and rates of absorption, digestion, and excretion were determined for a digestible protein (casein), a non-digestible protein (green fluorescent protein, GFP), and a non-digestible carbohydrate (dextran). Dextran was used as a control to monitor the non-absorptive flow rate of ingesta through the digestive system. Fluorescent tracking of FITC-conjugates of casein and dextran, as well as immunoblotting and immunofluorescent staining of casein and GFP, were used to monitor the degradation (in vitro) and ingestion, digestion, and distribution (in vivo) of the respective compounds. Under our experimental conditions, L. hesperus took discrete meals, feeding and excreting at 2-3 h intervals. Rate of food passage was variable. FITC-dextran was found in the fecal material of most insects by 6-8 h after treatment initiation; by 12 h, 95% of ingested FITC-dextran was recovered from all insects. FITC-casein was digested extensively in in vitro homogenates of gut, hemolymph, and salivary gland. In vivo, FITC-casein was ingested and partially absorbed as a holoprotein into the hemolymph. Ingested FITC-casein was partially degraded in the gut and hemolymph within 2 h of ingestion, and no holoprotein was found after 12 h. In contrast, there was no detectable degradation of GFP in hemolymph, gut, and salivary gland homogenates after 24 h of incubation. Ingested GFP was not degraded in gut or hemolymph up to 8 h after treatment initiation, but did transfer to the hemolymph as a holoprotein. Analysis of immunohistological images confirmed that GFP bound to gut epithelial cell brush-border membranes. However, the mechanism by which GFP and casein pass as holoproteins into the hemolymph remains unknown.     

Spatial and seasonal patterns of periphyton biomass and productivity in the northern Everglades,Florida, U.S.A.

We sampled periphyton in dominant habitats at oligotrophic and eutrophic sites in the northern Everglades during the wet and the dryseasons to determine the effects of nutrient enrichment on periphytonbiomass, taxonomic composition, productivity, and phosphorus storage. Arealbiomass was high (100–1600 g ash-free dry mass [AFDM]m⁻²) in oligotrophic sloughs and in stands of the emergentmacrophyte Eleocharis cellulosa, but was low in adjacent stands of sawgrass,Cladium jamaicense (7–52 g AFDM m⁻²). Epipelon biomasswas high throughout the year at oligotrophic sites whereas epiphyton andmetaphyton biomass varied seasonally and peaked during the wet season.Periphyton biomass was low (3–68 g AFDM m⁻²) and limitedto epiphyton and metaphyton in open-water habitats at eutrophic sites andwas undetectable in cattail stands (Typha domingensis) that covered morethan 90% of the marsh in these areas. Oligotrophic periphytonassemblages exhibited strong seasonal shifts in species composition and weredominated by cyanobacteria (e.g., Chroococcus turgidus, Scytonema hofmannii)during the wet season and diatoms (e.g. Amphora lineolata, Mastogloiasmithii) during the dry season. Eutrophic assemblages were dominated byCyanobacteria (e.g., Oscillatoria princeps) and green algae (e.g., Spirogyraspp.) and exhibited comparatively little seasonality. Biomass-specific grossprimary productivity (GPP) of periphyton assemblages in eutrophic openwaters was higher than for comparable slough assemblages, but areal GPP wassimilar in these eutrophic (0.9–9.1 g C m⁻²d⁻¹) and oligotrophic (1.75–11.49 g C m⁻²d⁻¹) habitats. On a habitat-weighted basis, areal periphytonGPP was 6- to 30-fold lower in eutrophic areas of the marsh due to extensiveTypha stands that were devoid of periphyton. Periphyton at eutrophic siteshad higher P content and uptake rates than the oligotrophic assemblage, butstored only 5% as much P because of the lower areal biomass.Eutrophication in the Everglades has resulted in a decrease in periphytonbiomass and its contribution to marsh primary productivity. These changesmay have important implications for efforts to manage this wetland in asustainable manner. This revised version was published online in July 2006 with corrections to the Cover Date.     

Mechanical and salivary aspects of potato leafhopper probing in alfalfa stems

Studies were conducted to separate the salivary and mechanical aspects of probing damage by the potato leafhopper,Empoasca fabae (Harris) (Homoptera: Cicadellidae), to stem vascular tissue of alfalfa,Medicago sativa L. Implantation of salivary gland tissue or fed-upon artificial diet under the stem epidermis yielded no evidence, three days later, of hopperburn-associated, anatomical changes. Mechanical puncturing of stems with implements approximating the size and shape of leafhopper stylets caused some anatomical changes, three days later, similar to those underlying hopperburn, i.e. tracks of necrosis, chlorosis, cell enlargement, and cell division. These changes, however, were much less severe than those observed in tissues three days after potato leafhopper probing. In contrast, puncturing through salivary gland or Malpighian tubule tissues produced extreme hyperplasia and other symptoms of wounding in cells near the puncture. This was similar to but more severe than effects from leafhopper probing, and was probably caused by leafhopper structural tissues or larger amounts of saliva being conveyed into the plant than normally occurs during leafhopper probing. We conclude that both salivation and mechanical wounding by leafhopper stylets are probably necessary to cause hopperburn-associated anatomical changes to vascular tissue in stems of alfalfa. This conclusion supports our hypothesis that hopperburn is a saliva-enhanced wound response.     

Apolipoprotein B mRNA sequences 3'' of the editing site are necessary and sufficient for editing and editosome assembly.

Apolipoprotein B (apoB) mRNA is edited in rat liver and intestine through the direct conversion of cytidine to uridine at nucleotide 6666. Recently, we have proposed the 'Mooring Sequence' model, in which editing complexes (editosomes) assemble on specific apoB mRNA flanking sequences to direct this site-specific editing event. To test this model, apoB mRNA deletion and translocation mutants were constructed and analyzed. Specific sequences 3' of the editing site were absolutely required for editing, while specific sequences and bulk RNA 5' of the editing site were required for efficient editing. Translocation of apoB 3' flanking sequences induced editing of an upstream cytidine, demonstrating that 3' sequences are necessary and sufficient to direct editing in vitro. 3' flanking sequences were also shown to be necessary and sufficient for editosome complex assembly. These data provide strong support for a 'Mooring Sequence' model in which 3' apoB flanking sequences direct editosome assembly and subsequent editing in vitro, while 5' flanking sequences enhance these functions.     

Quantitation of endogenous liver apolipoprotein B mRNA editing

The mRNA for apolipoprotein B is translated into either a high molecular weight (apo BH) or low molecular weight (apo BL) form of the protein depending on a novel form of RNA processing known as RNA editing. Apo BH mRNA editing is both tissue-specific and hormonally regulated and involves transition of cytidine to uridine at codon 2153 thereby converting a glutamine codon (CAA) to a translational stop codon (UAA). Three methods for quantitating the endogenous levels of liver apo B mRNA editing were compared: (1) Southern blot hybridization with discriminative thermal washes, (2) competimer-hybridization with discriminative thermal washes and (3) competimer-polymerase chain reaction (competimer-PCR). The data suggest that hybridization and PCR can yield similar quantitation when competing oligonucleotides are used. Based on competimer-PCR it is proposed that 40% and 85% of normal rat liver and small intestine apo B mRNA (respectively) are edited.