Substrate specificities of recombinant murine Golgi alpha1, 2- mannosidases IA and IB and comparison with endoplasmic reticulum and Golgi processing alpha1,2-mannosidases

The catalytic domains of murine Golgi alpha1,2-mannosidases IA and IB that are involved in N-glycan processing were expressed as secreted proteins in P.pastoris . Recombinant mannosidases IA and IB both required divalent cations for activity, were inhibited by deoxymannojirimycin and kifunensine, and exhibited similar catalytic constants using Manalpha1,2Manalpha-O-CH3as substrate. Mannosidase IA was purified as a 50 kDa catalytically active soluble fragment and shown to be an inverting glycosidase. Recombinant mannosidases IA and IB were used to cleave Man9GlcNAc and the isomers produced were identified by high performance liquid chromatography and proton-nuclear magnetic resonance spectroscopy. Man9GlcNAc was rapidly cleaved by both enzymes to Man6GlcNAc, followed by a much slower conversion to Man5GlcNAc. The same isomers of Man7GlcNAc and Man6GlcNAc were produced by both enzymes but different isomers of Man8GlcNAc were formed. When Man8GlcNAc (Man8B isomer) was used as substrate, rapid conversion to Man5GlcNAc was observed, and the same oligosaccharide isomer intermediates were formed by both enzymes. These results combined with proton-nuclear magnetic resonance spectroscopy data demonstrate that it is the terminal alpha1, 2-mannose residue missing in the Man8B isomer that is cleaved from Man9GlcNAc at a much slower rate. When rat liver endoplasmic reticulum membrane extracts were incubated with Man9GlcNAc2, Man8GlcNAc2was the major product and Man8B was the major isomer. In contrast, rat liver Golgi membranes rapidly cleaved Man9GlcNAc2to Man6GlcNAc2and more slowly to Man5GlcNAc2. In this case all three isomers of Man8GlcNAc2were formed as intermediates, but a distinctive isomer, Man8A, was predominant. Antiserum to recombinant mannosidase IA immunoprecipitated an enzyme from Golgi extracts with the same specificity as recombinant mannosidase IA. These immunodepleted membranes were enriched in a Man9GlcNAc2to Man8GlcNAc2- cleaving activity forming predominantly the Man8B isomer. These results suggest that mannosidases IA and IB in Golgi membranes prefer the Man8B isomer generated by a complementary mannosidase that removes a single mannose from Man9GlcNAc2.      

Neutralizing antibody responses to Japanese encephalitis vaccine in children

Two shots of the current Japanese encephalitis (JE) vaccine were given to children and their immune responses to the Nakayama strain (the vaccine strain) and two wild strains (JaGAr-01 and E-50) of JE virus were examined by neutralizing (N) antibody titrations. Seventy vaccinees had no N antibody to JE virus before the first vaccination and were bled one month after the second vaccination. The N antibody responses to the JaGAr-01 and E-50 strains were found to be similar and to be less than that to the Nakayama strain after the second vaccination: the geometric mean titers (GMT) of N antibodies to the JaGAr-01 and E-50 strains (as logarithms) were 1.87 and 1.75, respectively, while the GMT to the Nakayama strain was 2.89. The seroconversion rates to the Nakayama, JaGAr-01 and E-50 strains were 70/70 (100%), 69/70 (99%) and 68/70 (97%), respectively, after the second vaccination. Twenty-seven of the 70 vacciness were also bled before the second vaccination. Most of them showed a considerably high N antibody response against the Nakayama strain and only one vaccinee failed to show seroconversion after the first vaccination. However, the antibody response to the E-50 strain appeared to be rather low and 9 of 25 vaccinees did not show any seroconversion. Similarly 3 of 25 failed to show any seroconversion against the JaGAr-01 strain. These results indicate that at the initial immunization two shots, at least, of the current JE vaccine are necessary to stimulate effective immune responses to wild strains of JE virus.     

A blood-brain barrier without tight junctions in the fly central nervous system in the early postembryonic stage

Summary The anatomical basis of the vertebrate blood-brain barrier is a series of tight junctions between endothelial cells of capillaries in the central nervous system. Over two decades ago, tight junctions were also proposed as the basis of the blood-brain barrier in insects. Currently there is a growing understanding that septate junctions might possess barrier properties in various invertebrate epithelial cells. We now examine these two views by studying the blood-brain barrier properties of the early postembryonic larva of a dipteran fly (Delia platura) by transmission electron microscopy. Newly hatched larvae possess a functioning blood-brain barrier that excludes the extracellular tracer, ionic lanthanum. This barrier is intact throughout the second instar stage as well. The ultrastructural correlate of this barrier is a series of extensive septate junctions that pervade the intercellular space between adjacent perineurial cells. No tight junctions were located in either nerve, glial or perineurial cell layers. We suggest that the overall barrier might involve septate junctions within extensive, meandering intercellular clefts.     

Separation and purification of enzymes by continuous pH-parametric pumping

Trypsin and chymotrypsin were separated from porcine pancreas extract by continuous pH-parametric pumping. CHOM (chicken ovomucoid) was convalently bound to laboratory-prepared crab chitin with glutaraldehyde to form an affinity adsorbent of trypsin. The pH levels of top and bottom feeds were 8.0 and 2.5, respectively. Similar inhibitor, DKOM (duck ovomucoid), and pH levels 8.0 and 2.0 for top and bottom feeds, respectively, were used for separation and purification of chymotrypsin. epsilon-Amino caproyl-D-tryptophan methyl ester was coupled to chitosan to form an affinity adsorbent for stem bromelain. The pH levels were 8.7 and 3.0. Separation continued fairly well with high yield, e.g., 95% recovery of trypsin after continuous pumping of 10 cycles. Optimum operational conditions for concentration and purification of these enzymes were investigated. The results showed that the continuous pH-parametric pumping coupled with affinity chromatography is effective for concentration and purification of enzymes.     

The C. elegans cGMP-Dependent Protein Kinase EGL-4 Regulates Nociceptive Behavioral Sensitivity

Signaling levels within sensory neurons must be tightly regulated to allow cells to integrate information from multiple signaling inputs and to respond to new stimuli. Herein we report a new role for the cGMP-dependent protein kinase EGL-4 in the negative regulation of G protein-coupled nociceptive chemosensory signaling. C. elegans lacking EGL-4 function are hypersensitive in their behavioral response to low concentrations of the bitter tastant quinine and exhibit an elevated calcium flux in the ASH sensory neurons in response to quinine. We provide the first direct evidence for cGMP/PKG function in ASH and propose that ODR-1, GCY-27, GCY-33 and GCY-34 act in a non-cell-autonomous manner to provide cGMP for EGL-4 function in ASH. Our data suggest that activated EGL-4 dampens quinine sensitivity via phosphorylation and activation of the regulator of G protein signaling (RGS) proteins RGS-2 and RGS-3, which in turn downregulate Gα signaling and behavioral sensitivity.     

Strains of internal biofilm in aerobic granular membrane bioreactors

This study isolated strains in suspended liquor, the surface fouling layer, and biofilm inside hollow-fiber membranes of a membrane bioreactor (MBR); analyzed their distributions, sizes, surface charges, and growth behaviors; and determined the quantities of extracellular polymeric substances (EPS) secreted by these strains under different organic loadings. Three strains, which may penetrate the microfiltration membranes, were close relatives of the Ralstonia mannitolilytica strain SDV (GenBank Accession No. GU451066), Arthrobacter sp. BJQ-2 (GenBank Accession No. GU451067), and Actinobacterium DS3 (GenBank Accession No. GU451068). Among these three strains, only Arthrobacter sp. developed an internal biofilm. The relatively short length of Arthrobacter sp. minimizes resistance to cells moving through the membrane matrix, thereby enhancing its ability to build a biofilm in the interior surface of membranes.     

Phenotypic and functional abnormalities of bone marrow-derived dendritic cells in systemic lupus erythematosus

Introduction  

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoreactive T and B cells, which are believed to be secondary to deficient dendritic cells (DCs). However, whether DC abnormalities occur during their development in the bone marrow (BM) or in the periphery is not known.     

Nonviral gene carriers based on diblock copolymers of poly(2-ethyl-2-oxazoline) and linear polyethylenimine

Diblock copolymers that consist of poly(2-ethyl-2-oxazoline) (PEOz) and linear polyethylenimine (LPEI) were prepared for use as nonviral gene carriers. The PEOz-b-LPEI copolymers were synthesized by coupling PEOz with LPEI in a thiol-disulfide exchange reaction between the sulfhydryl and pyridyl disulfide terminal groups. A polymer/DNA weight ratio (P/D) of over 12 was required to enable PEOz-b-LPEI to condense DNA completely. The DNA-condensing capability of the diblock copolymers was increased with increasing the hydrolytic degrees of the LPEI segment. The PEOz-b-LPEI polyplexes were stable in 150 mM NaCl aqueous solution and had a mean diameter around 190 nm, whereas BPEI and LPEI polyplexes formed large aggregates in the range 300-500 nm. In addition, these polyplexes exhibited the sensitivity to solution pH and were dissociated in the acidic buffers (pH < or = 5.5). The results of in vitro cell viability and luciferase assay indicated that PEOz-b-LPEI showed not only low cytotoxicity but also high transfection efficiency in gene expression.     

Preparation of monoclonal antibody bank against whole water-soluble proteins from rapid-growing bamboo shoots

An antibody bank against the whole proteins in a proteome is a useful tool for biological research. Using the standard cell fusion method, and a modified screening protocol, we produced an mAb bank against the total water-soluble proteins extracted from the rapid-growing green bamboo shoots. An improved two-stage strategy was employed to enrich those poor immunogenic or lower expressed proteins. Totally, we obtained a bank of 192 mAb which were identified as distinctive to each other by 2-DE and immunostaining.