Bridging the gap: heparan sulfate and Scube2 assemble Sonic hedgehog release complexes at the surface of producing cells.

Decision making in cellular ensembles requires the dynamic release of signaling molecules from the producing cells into the extracellular compartment. One important example of molecules that require regulated release in order to signal over several cell diameters is the Hedgehog (Hh) family, because all Hhs are synthesized as dual-lipidated proteins that firmly tether to the outer membrane leaflet of the cell that produces them. Factors for the release of the vertebrate Hh family member Sonic Hedgehog (Shh) include cell-surface sheddases that remove the lipidated terminal peptides, as well as the soluble glycoprotein Scube2 that cell-nonautonomously enhances this process. This raises the question of how soluble Scube2 is recruited to cell-bound Shh substrates to regulate their turnover. We hypothesized that heparan sulfate (HS) proteoglycans (HSPGs) on the producing cell surface may play this role. In this work, we confirm that HSPGs enrich Scube2 at the surface of Shh-producing cells and that Scube2-regulated proteolytic Shh processing and release depends on specific HS. This finding indicates that HSPGs act as cell-surface assembly and storage platforms for Shh substrates and for protein factors required for their release, making HSPGs critical decision makers for Scube2-dependent Shh signaling from the surface of producing cells.

Comparison of cost- and energy-efficient signal modulations for next generation passive optical networks.

Extensive numerical investigations are undertaken to analyze and compare, for the first time, the performance, techno-economy, and power consumption of three-level electrical Duobinary, optical Duobinary, and PAM-4 modulation formats as candidates for high-speed next-generation PONs supporting downstream 40 Gb/s per wavelength signal transmission over standard SMFs in C-band. Optimization of transceiver bandwidths are undertaken to show the feasibility of utilizing low-cost and band-limited components to support next-generation PON transmissions. The effect of electro-absorption modulator chirp is examined for electrical Duobinary and PAM-4. Electrical Duobinary and optical Duobinary are power-efficient schemes for smaller transmission distances of 10 km SMFs and optical Duobinary offers the best receiver sensitivity albeit with a relatively high transceiver cost. PAM-4 shows the best power budget and cost-efficiency for larger distances of around 20 km, although it consumes more power. Electrical Duobinary shows the best trade-off between performance, cost and power dissipation.

Sulfotransferase Ndst1 is needed for mandibular and TMJ development.

Heparan sulfate proteoglycans (HS-PGs) regulate several developmental processes, but their possible roles in mandibular and TMJ formation are largely unclear. To uncover such roles, we generated mice lacking Golgi-associated N-sulfotransferase 1 (Ndst1) that catalyzes sulfation of HS-PG glycosaminoglycan chains. Ndst1-null mouse embryos exhibited different degrees of phenotypic penetrance. Severely affected mutants lacked the temporomandibular joint and condyle, but had a mandibular remnant that displayed abnormal tooth germs, substandard angiogenesis, and enhanced apoptosis. In mildly affected mutants, the condylar growth plate was dysfunctional and exhibited thicker superficial and polymorphic cell zones, a much wider distribution of Indian hedgehog signaling activity, and ectopic ossification along its lateral border. Interestingly, mildly affected mutants also exhibited facial asymmetry resembling that seen in individuals with hemifacial microsomia. Our findings indicate that Ndst1-dependent HS sulfation is critical for mandibular and TMJ development and allows HS-PGs to exert their roles via regulation of Ihh signaling topography and action.

Role of dose concentration in biocide efficacy against Pseudomonas aeruginosa biofilms.

Pseudomonas aeruginosa entrapped in alginate gel beads to form artificial biofilms resisted killing by chlorine, glutaraldehyde, 2,2-dibromo-3-nitrilopropionamide (DBNPA), and an alkyl dimethyl benzyl ammonium compound (ADBAC). The degree of resistance was quantified by a resistance factor that compared killing times for biofilm and planktonic cells in response to the same concentration of antimicrobial agent. Resistance factors averaged 120 for chlorine, 34 for glutaraldehyde, 29 for DBNPA, and 1900 for ADBAC. In every case, resistance factors decreased with increasing concentration of the antimicrobial agent. An independent analysis of the concentration dependence of the apparent rates of killing of planktonic and biofilm bacteria showed that elevating the treatment concentration increased bacterial killing more in the biofilm than it did in a suspension culture. Calculation of a transport modulus comparing the rates of biocide reaction and diffusion suggested that at least part of the biofilm resistance to chlorine, glutaraldehdye, and DBNPA could be attributed to incomplete or slow penetration of these agents into the biofilm. Time-kill curves were nonlinear for biofilm bacteria in some cases. The shapes of these curves implicated retarded antimicrobial penetration for chlorine and glutaraldehyde and the presence of a tolerant subpopulation for DBNPA and ADBAC. The results indicate that treating biofilms with a concentrated dose of biocide is more effective than using prolonged doses of a lower concentration.

Multiple isozymes of heparan sulfate/heparin GlcNAc N-deacetylase/GlcN N-sulfotransferase. Structure and activity of the fourth member, NDST4.

We report the cloning and partial characterization of the fourth member of the vertebrate heparan sulfate/heparin: GlcNAc N-deacetylase/GlcN N-sulfotransferase family, which we designate NDST4. Full-length cDNA clones containing the entire coding region of 872 amino acids were obtained from human and mouse cDNA libraries. The deduced amino acid sequence of NDST4 showed high sequence identity to NDST1, NDST2, and NDST3 in both species. NDST4 maps to human chromosome 4q25-26, very close to NDST3, located at 4q26-27. These observations, taken together with phylogenetic data, suggest that the four NDSTs evolved from a common ancestral gene, which diverged to give rise to two subtypes, NDST3/4 and NDST1/2. Reverse transcription-polymerase chain reaction analysis of various mouse tissues revealed a restricted pattern of NDST4 mRNA expression when compared with NDST1 and NDST2, which are abundantly and ubiquitously expressed. Comparison of the enzymatic properties of the four murine NDSTs revealed striking differences in N-deacetylation and N-sulfation activities; NDST4 had weak deacetylase activity but high sulfotransferase, whereas NDST3 had the opposite properties. Molecular modeling of the sulfotransferase domains of the murine and human NDSTs showed varying surface charge distributions within the substrate binding cleft, suggesting that the differences in activity may reflect preferences for different substrates. An iterative model of heparan sulfate biosynthesis is suggested in which some NDST isozymes initiate the N-deacetylation and N-sulfation of the chains, whereas others bind to previously modified segments to fill in or extend the section of modified residues.

Modeling biofilm antimicrobial resistance.

A computer model capable of integrating mechanisms of biofilm resistance to disinfection by antimicrobial agents was developed. Resistance mechanisms considered included retarded penetration due to a stoichiometric reaction between the antimicrobial agent and biomass, incomplete penetration due to a catalytic reaction between the antimicrobial agent and the biomass, and the existence of a fraction of the cells in a resistant phenotypic state. Mathematical models of these processes were derived and solved in the computer simulation package MATLAB. Four sets of fitted experimental data on the disinfection of Pseudomonas aeruginosa biofilms were fit to each of the three models. No one model fit all of the data sets adequately. Killing of a 2-day old biofilm by tobramycin was best described by the physiological limitation model. Killing by hypochlorite was best described by the stoichiometric transport model. Killing by hydrogen peroxide was best simulated by the catalytic transport model. These results suggest that multiple mechanisms of biofilm reduced susceptibility are manifested even in biofilms of the same species and that the particular resistance mechanism depends on the biofilm age, antimicrobial agent, and biofilm thickness. The models presented in this article may be useful for diagnosing mechanisms of biofilm resistance from experimental data.

Grass group I allergens (beta-expansins) are novel, papain-related proteinases.

Expansins are a family of proteins that catalyse long-term extension of isolated plant cell walls due to an as yet unknown biochemical mechanism. They are divided into two groups, the alpha-expansins and beta-expansins, the latter group consisting of grass group I allergens and their vegetative homologs. These grass group I allergens, to which more than 95% of patients allergic to grass pollen possess IgE antibodies, are highly immunologically crossreactive glycoproteins exclusively expressed in pollen of all grasses. Alignments of the amino-acid sequences of grass group I allergens derived from diverse grass species reveal up to 95% homology. It is therefore likely that these molecules share a similar biological function. The major grass group I allergen from timothy grass (Phleum pratense), Phl p 1, was chosen as a model glycoprotein and expressed in the methylotrophic yeast Pichia pastoris to obtain a post-translationally modified and functionally active allergen. The recombinant allergen exhibited proteolytic activity when assayed with various test systems and substrates, which was also subsequently demonstrated with the natural protein, nPhl p 1. These observations are confirmed by amino-acid alignments of Phl p 1 with three functionally important sequence motifs surrounding the active-site amino acids of the C1 (papain-like) family of cysteine proteinases. Moreover, the significantly homologous alpha-expansins mostly share the functionally important C1 sequence motifs. This leads us to propose a C1 cysteine proteinase function for grass group I allergens, which may mediate plant cell wall growth and possibly contributes to the allergenicity of the molecule.

Implications of the grass group I allergens on the sensitization and provocation process.

Grass pollen allergens of group I are particularly important because of their high IgE prevalence and occurrence in all grass species. Four independent IgE-binding regions and one continous epitope were identified. The posttranslational modifications on the molecule increased allergenicity. Phl p 1 is a cysteine protease, as determined by specific substrates, inhibitors and consensus sequence motifs. In analogy to other allergens and/or proteases, we deduce that Phl p 1 might enhance the permeability of the epithelium, influence T helper cells to bias Th2, and increase the IgE production of plasma cells. Thus, the group I allergens seem to be the crucial components in a pollen extract which can mediate sensitization and enhance the triggering of symptoms leading to the persistence of a grass pollen allergy.

Enzymatic and molecular properties of the Clostridium tertium sialidase.

Clostridium tertium metabolizes sialoglycoconjugates via a secreted sialidase [EC 3.2.1.18] and an intracellular acylneuraminate pyruvate lyase [EC 4.1.3.3]. The sialidase was enriched 1,900-fold from the culture medium with a specific activity of 0.7 U per mg protein. It exhibits a temperature optimum of 50 degreesC and tolerates mercury ions at relatively high concentrations (50% inhibition at 5.2 mM Hg2+). The sialidase gene was detected on two restriction fragments (HincII, HindIII) of chromosomal DNA and their correct recombination resulted in an active enzyme expressed by Escherichia coli. The structural gene is represented by 2,319 bp encoding a protein of 773 amino acids with a molecular mass of 85.4 kDa. The first 28 amino acids possess the character of a signal peptide. The protein reveals a FRIP-region and four Asp-boxes common in all bacterial sialidases. It has 42.6% identical amino acids when compared with the sialidase of Clostridium septicum and 64.8% with a sialidase gene amplified from Macrobdella decora. A further open reading frame was detected 30 bp downstream from the sialidase gene, which exhibits significant homology with acylneuraminate pyruvate lyases. For the first time, both genes were found in close proximity on a bacterial chromosome, probably being part of one operon.

Comparison of natural and recombinant isoforms of grass pollen allergens.

More than 95% of grass pollen allergic patients possess IgE antibodies against grass group I, a heterogeneous group of glycoproteins found in all temperate grasses. We studied the structural variability of the group I allergens in single species and among different grasses. By 2-DE blotting using patients' IgE and monoclonal antibodies, we detected IgE-reactive isoforms with molecular masses between 32 and 37 kDa and focusing in a wide pI ranging from 4.7 to 7.6. While the group I allergens of timothy grass (Phl p 1) were composed of 37 and 35 kDa components, only single isoforms were found for ryegrass (Lol p 1) and velvet grass (Hol l 1): 32 and 34 kDa, respectively. By N-terminal microsequencing we determined single amino acid substitutions in different-sized group I allergens. The post-translational modifications (one N-glycosylation site, two hydroxylated proline residues and seven cysteine residues for potential disulfide formations), which contribute to IgE reactivity, were identical in all. From the cDNA sequences we deduced protein sequence homologies > 90%, a result which might explain the high IgE cross-reactivity among the grasses. In order to test whether recombinant group I grass allergens can act as substitutes for the natural forms, we expressed rPhl p 1 in E. coli and in P. pasteuris. 2-DE immunoblotting again demonstrated a microheterogeneity in molecular mass and pI. While the E. coli products were free from post-translational modifications, rPhl p 1 from Pichia is a heterogeneous glycoprotein fraction with a carbohydrate content of about 15%. This rPhl p 1 is hyperglycosylated compared to the nPhl p 1, which only has a 5% carbohydrate content.