A novel, 11 nucleotide variant of chi, chi*: one of a class of sequences defining the Escherichia coli recombination hotspot chi

In wild-type Escherichia coli, recognition of the recombination hotspot, chi (5'-GCTGGTGG-3'), by the RecBCD enzyme is central to homologous recombination. However, in the recC* class of RecBCD mutants, stimulation of recombination by the canonical chi sequence is not detectable, but the levels of homologous recombination are nearly wild-type. In vivo studies demonstrate that a member of this class of mutants, the recC1004 allele, encodes an enzyme that responds to a novel variant of chi, termed chi* (5'-GCTGGTGCTCG-3'). Here, we establish that, in vitro, the chi* sequence is recognized more efficiently by the RecBC(1004)D enzyme than is the wild-type chi. This is manifest by both a greater modification of nuclease activity and a higher stimulation of RecA protein-mediated joint molecule formation at chi* than at chi. Sequencing of the recC1004 gene revealed that it contains a frameshift mutation, which results in a replacement of nine of the wild-type amino acid residues by eight in the mutant protein, and defines a locus that is important for the specificity of chi-recognition. In addition, we show that this novel, 11 nucleotide chi* sequence also regulates the wild-type RecBCD enzyme, supporting the notion that variants of the canonical chi constitute a class of sequences that regulate the recombination function of RecBCD enzyme.     

Targeting of liposomes carrying recombinant fragments of platelet membrane glycoprotein Ibalpha to immobilized von Willebrand factor under flow conditions

Liposomes with covalently bound recombinant fragments of platelet membrane glycoprotein Ibalpha that retain the von Willebrand factor (vWf)-binding function (rGPIbalpha-liposomes) were prepared. Their interactions with an immobilized vWf surface under flow conditions were evaluated with a recirculating flow chamber, mounted on an epifluorescence microscope, which allows real-time visualization of fluorescence-labeled liposomes interacting with the surface. The interaction of rGPIbalpha-liposomes with the vWf surface was directly related to shear rate. At high densities of rGPIbalpha and vWf, rGPIbalpha-liposomes establishing contact with the vWf surface exhibited continuous displacement with decreased velocity relative to the hydrodynamic flow, depending on receptor density and matrix concentration. At lower densities of rGPIbalpha and vWf, rGPIbalpha-liposomes stopped only transiently, in the millisecond range, on the surface. This is the first study to demonstrate that the targeting of rGPIbalpha-liposomes is specific to the vWf surface under flow conditions.     

High-performance affinity beads for identifying drug receptors

We have developed a method using novel latex beads for rapid identification of drug receptors using affinity purification. Composed of a glycidylmethacrylate (GMA) and styrene copolymer core with a GMA polymer surface, the beads minimize nonspecific protein binding and maximize purification efficiency. We demonstrated their performance by efficiently purifying FK506-binding protein using FK506-conjugated beads, and found that the amount of material needed was significantly reduced compared with previous methods. Using the latex beads, we identified a redox-related factor, Ref-1, as a target protein of an anti-NF-kappaB drug, E3330, demonstrating the existence of a new class of receptors of anti-NF-kappaB drugs. Our results suggest that the latex beads could provide a tool for the identification and analysis of drug receptors and should therefore be useful in drug development.     

A Multicenter,Randomized, Double-Blind,Placebo-Controlled Trial of High-Dose Rebamipide Treatment for Low-Dose Aspirin-Induced Moderate-to-Severe Small Intestinal Damage

Background

Low-dose aspirin (LDA) frequently causes small bowel injury. While some drugs have been reported to be effective in treating LDA-induced small intestinal damage, most studies did not exclude patients with mild damage thought to be clinically insignificant.

Aim

We conducted a multicenter, randomized, double-blind, placebo-controlled trial to assess the efficacy of a high dose of rebamipide, a gastroprotective drug, for LDA-induced moderate-to-severe enteropathy.

Methods

We enrolled patients who received 100 mg of enteric-coated aspirin daily for more than 3 months and were found to have more than 3 mucosal breaks (i.e., erosions or ulcers) in the small intestine by capsule endoscopy. Eligible patients were assigned to receive either rebamipide 300 mg (triple dose) 3 times daily or placebo for 8 weeks in a 2:1 ratio. Capsule endoscopy was then repeated. The primary endpoint was the change in the number of mucosal breaks from baseline to 8 weeks. Secondary endpoints included the complete healing of mucosal breaks at 8 weeks and the change in Lewis score (an endoscopic score assessing damage severity) from baseline to 8 weeks.

Results

The study was completed by 38 patients (rebamipide group: n = 25, placebo group: n = 13). After 8 weeks of treatment, rebamipide, but not placebo, significantly decreased the number of mucosal breaks (p = 0.046). While the difference was not significant (p = 0.13), the rate of complete mucosal break healing in the rebamipide group (32%, 8 of 25) tended to be higher than that in the placebo group (7.7%, 1 of 13). Rebamipide treatment significantly improved intestinal damage severity as assessed by the Lewis score (p = 0.02), whereas placebo did not. The triple dose of rebamipide was well tolerated.

Conclusions

High-dose rebamipide is effective for the treatment of LDA-induced moderate-to-severe enteropathy.

Trial Registration

UMIN Clinical Trials Registry UMIN000003463     

Design and Evaluation of the Highly Concentrated Human IgG Formulation Using Cyclodextrin Polypseudorotaxane Hydrogels

To achieve the potent therapeutic effects of human immunoglobulin G (IgG), highly concentrated formulations are required. However, the stabilization for highly concentrated human IgG is laborious work. In the present study, to investigate the potentials of polypseudorotaxane (PPRX) hydrogels consisting of polyethylene glycol (PEG) and α- or γ-cyclodextrin (α- or γ-CyD) as pharmaceutical materials for highly concentrated human IgG, we designed the PPRX hydrogels including human IgG and evaluated their pharmaceutical properties. The α- and γ-CyDs formed PPRX hydrogels with PEG (M.W. 20,000) even in the presence of highly concentrated human IgG (>100 mg/mL). According to the results of ¹H-NMR, powder X-ray diffraction, and Raman microscopy, the formation of human IgG/CyD PPRX hydrogels was based on physical cross-linking arising from their columnar structures. The release profiles of human IgG from the hydrogels were in accordance with the non-Fickian diffusion model. Importantly, the stabilities of human IgG included into the hydrogels against thermal and shaking stresses were markedly improved. These findings suggest that PEG/CyD PPRX hydrogels are useful to prepare the formulation for highly concentrated human IgG.KEY WORDS: cyclodextrin, human IgG, hydrogel, polypseudorotaxane, stability     

Role of Site-Specific N-Glycans Expressed on GluA2 in the Regulation of Cell Surface Expression of AMPA-Type Glutamate Receptors

The AMPA-type glutamate receptor (AMPAR), which is a tetrameric complex composed of four subunits (GluA1-4) with several combinations, mediates the majority of rapid excitatory synaptic transmissions in the nervous system. Cell surface expression levels of AMPAR modulate synaptic plasticity, which is considered one of the molecular bases for learning and memory formation. To date, a unique trisaccharide (HSO₃-3GlcAβ1-3Galβ1-4GlcNAc), human natural killer-1 (HNK-1) carbohydrate, was found expressed specifically on N-linked glycans of GluA2 and regulated the cell surface expression of AMPAR and the spine maturation process. However, evidence that the HNK-1 epitope on N-glycans of GluA2 directly affects these phenomena is lacking. Moreover, it is thought that other N-glycans on GluA2 also have potential roles in the regulation of AMPAR functions. In the present study, using a series of mutants lacking potential N-glycosylation sites (N256, N370, N406, and N413) within GluA2, we demonstrated that the mutant lacking the N-glycan at N370 strongly suppressed the intracellular trafficking of GluA2 from the endoplasmic reticulum (ER) in HEK293 cells. Cell surface expression of GluA1, which is a major subunit of AMPAR in neurons, was also suppressed by co-expression of the GluA2 N370S mutant. The N370S mutant and wild-type GluA2 were co-immunoprecipitated with GluA1, suggesting that N370S was properly associated with GluA1. Moreover, we found that N413 was the main potential site of the HNK-1 epitope that promoted the interaction of GluA2 with N-cadherin, resulting in enhanced cell surface expression of GluA2. The HNK-1 epitope on N-glycan at the N413 of GluA2 was also involved in the cell surface expression of GluA1. Thus, our data suggested that site-specific N-glycans on GluA2 regulate the intracellular trafficking and cell surface expression of AMPAR.     

Molecular Monitoring and Isolation of Previously Uncultured Bacterial Strains from the Sheep Rumen

To estimate the contribution of uncultured bacterial groups to fiber degradation, we attempted to retrieve both ecological and functional information on uncultured groups in the rumen. Among previously reported uncultured bacteria, fiber-associated groups U2 and U3, belonging to the low-GC Gram-positive bacterial group, were targeted. PCR primers and fluorescence in situ hybridization (FISH) probe targeting 16S rRNA genes or rRNA were designed and used to monitor the distribution of targets. The population size of group U2 in the rumen was as high as 1.87%, while that of group U3 was only 0.03%. Strong fluorescence signals were observed from group U2 cells attached to plant fibers in the rumen. These findings indicate the ecological significance of group U2 in the rumen. We succeeded in enriching group U2 using rumen-incubated rice straw as the inoculum followed by incubation in an appropriate medium with an agent inhibitory for Gram-negative bacteria. Consequently, we successfully isolated two strains, designated B76 and R-25, belonging to group U2. Both strains were Gram-positive short rods or cocci that were 0.5 to 0.8 μm in size. Strain B76 possessed xylanase and α-l-arabinofuranosidase activity. In particular, the xylanase activity of strain B76 was higher than that of xylanolytic Butyrivibrio fibrisolvens H17c grown on cellobiose. Strain R-25 showed an α-l-arabinofuranosidase activity higher than that of strain B76. These results suggest that strains B76 and R-25 contribute to hemicellulose degradation in the rumen.Ruminants can utilize plant fiber as an energy source with the aid of a symbiotic relationship with microbes in the rumen. The rumen is a complex microbial ecosystem comprised of bacteria (10¹⁰ to 10¹¹ per ml), protozoa (10⁴ to 10⁶ per ml), and fungi (10³ to 10⁶ per ml) (8, 23, 39). Of the rumen microbes, bacteria are considered to be primarily responsible for the biological degradation of plant fiber, due to their high fibrolytic activity and large biomass in the rumen. In order to determine the mechanism of plant fiber degradation in the rumen, numerous studies have been performed on both the physiological and ecological characteristics of rumen bacteria (16, 27, 36). In particular, various aspects of bacterial attachment to feed particles have been investigated (19, 21, 25), because attachment to plant fiber is a critical step in initiating fiber degradation (20).Recent advances in molecular techniques have allowed recognition of a predominance of uncultured bacteria in the rumen (6, 24, 33). The majority (77%) of fiber-associated community members are uncultured bacteria, although 17% of cloned bacterial 16S rRNA gene sequences were classified as known fibrolytic species, such as Fibrobacter succinogenes and Butyrivibrio fibrisolvens (12). These findings clearly indicate the possibility for involvement of uncultured bacteria in ruminal fiber degradation. Through the phylogenetic analysis of fiber-associated community members, the unidentified bacterial groups were detected and designated uncultured group 2 (U2) and uncultured group 3 (U3). However, their roles in plant fiber digestion have yet to be determined.The predominance of uncultured bacteria has also been pointed out in other environments (26). Recently, new strategies for cultivation have been introduced to resolve the problem of the bacteria being unculturable. Sait et al. (28) reported that culturing with a polymeric growth substrate and longer incubation time was effective for the isolation of previously uncultured bacteria from soil. Cultivation on low-nutrient medium, using increased incubation times, with simulated natural environments or using a membrane as a solid support for growth has apparently led to improvements in bacterial cultivation (7, 31). On the other hand, the majority of rumen bacteria have yet to be isolated (10) despite great efforts toward the isolation of rumen bacterial strains over the past 50 years. Considering the ecological significance of uncultured rumen bacteria, it is important to cultivate and characterize these bacteria to fully understand the ecology of fiber digestion.In the present study, molecular monitoring tools were developed to obtain ecological information on target uncultured bacterial groups in the rumen. Previously uncultured rumen bacteria were then isolated and characterized to retrieve functional information.