Therapeutic efficacy of new botulinum toxin identified in CCUG 7968 strain.

Botulinum neurotoxin type A (BoNT/A) induces muscle atrophy by cleaving synaptosomal-associated protein 25. Thus, BoNT/A has been actively utilized for the treatment of masseter and gastrocnemius hypertrophy. In this study, INI101 toxin was newly identified from the CCUG 7968 strain, and its therapeutic efficacy was evaluated both in vitro and in vivo. The INI101 toxin showed identical genetic sequence, amino acid sequence, and protein subunit composition to BoNT/A produced from strain Hall A. Electromyography (EMG), and immunofluorescence staining demonstrated that INI101 (at 2 ~ 8 U/rat) effectively blocked the neuromuscular junction with no toxicity in a rat model. The EMG results showed INI101 toxin-induced weight loss and volume reduction of the gastrocnemius, similar to the effects of Botox® (BTX). Histological and immunofluorescence staining was consistent with this EMG result, showing that INI101 toxin caused muscle fiber reduction in the gastrocnemius. Notably, INI101 toxin diffused less into adjacent muscle tissue than BTX, indicating that INI101 toxin may reduce potential side effects due to diffusion into normal tissues. INI101 toxin isolated from the novel strain CCUG 7968 is a newly identified meaningful biopharmaceutical comparable to the conventional BoNT/A in the medical field. KEY POINTS: • Botulinum neurotoxin type A (BoNT/A, INI101) was identified from the CCUG 7968 strain. • INI101 toxin showed similar safety and therapeutic efficacy comparable to conventional BoNT/A both in vitro and in vivo. • INI101 toxin is a meaningful biopharmaceutical comparable to the conventional BoNT/A in the medical field.

The Effect of Intragastric Administration of Botulinum Toxin Type A on Reducing Adiposity in a Rat Model of Obesity Using Micro-CT and Histological Examinations.

BACKGROUND/AIMS: Botulinum toxin type A (BTX), a long-acting inhibitor of muscular contraction in both striated and smooth muscles, is responsible for gastric motility. The aim of this study was to investigate the effects of an endoscopic intragastric BTX injection on weight loss, body fat accumulation, and gastric emptying time. METHODS: The BTX group consisted of 15 obese rats in which 20 U of BTX were injected into the gastric antrum. The saline group consisted of 15 obese rats injected with 20 U of saline, and the control group included 10 obese rats that did not receive a surgical intervention. The gastric emptying time, biochemical parameters, and body fat volume were evaluated using micro-computed tomography (micro-CT) and histologic evaluations. RESULTS: The postoperative body weight of the BTX group was significantly lower than those of the other groups (p<0.001) at 6 weeks after the operation. The gastric emptying time (156±54 minutes) was significantly delayed in the BTX group. The BTX group showed significantly lower lipid levels than the other groups. A reduction in body fat volume was observed in the BTX group using micro-CT and histological evaluations. CONCLUSIONS: BTX application to the gastric antrum represents a potentially effective treatment for obesity and may help improve the lipid profile by increasing the gastric emptying time.

Comparative trial of a novel botulinum neurotoxin type A versus onabotulinumtoxinA in the treatment of glabellar lines: a multicenter, randomized, double-blind, active-controlled study.

A novel botulinum neurotoxin type A (DWP450; Daewoong Pharmaceutical, Seoul, Korea) has recently been introduced for the treatment of facial wrinkles. The efficacy of this agent has previously been demonstrated in an in vivo study using an electrophysiological protocol in a rat model. To compare the efficacy and safety of DWP450 with onabotulinumtoxinA (OBoNT) for use in the treatment of glabellar lines, we performed a multicenter, double-blind, randomized, active-controlled trial comparing DWP450 and OBoNT (Allergan Inc., Irvine, CA, USA). A total of 268 subjects with moderate to severe glabellar lines were randomized at a 1:1 ratio. Each patient received treatment with 20 U of study medication. Maximum frown responder rates at week 4 were measured to analyze the primary efficacy endpoint. To evaluate secondary efficacy endpoints, response rates were measured at weeks 8, 12, and 16, at maximum frown and rest. Specifically, responder rates at both maximum frown and at rest were assessed based on clinical photography. Subject degree of satisfaction and self-assessed rate of response were also measured. Adverse events (AEs) were documented to evaluate safety. Responder rate by physician-rating severity at maximal contraction at week 4 was 93.89% in the DWP450 group and 88.64% in OBoNT group. As the lower limit of the 97.5% one-sided confidence interval (-1.53%) surpassed the -15% threshold, we determined that DWP450 was not inferior to OBoNT. For the secondary efficacy endpoint analyses, no significant differences were observed between the two groups for any variable at any point in time. The incidences of AEs were similar for the two groups. Most of AEs were considered mild. DWP450 and OBoNT were comparable in efficacy and safety in the treatment of glabellar lines.

Multimodal electrochemical sensing of transcription factor-operator complexes.

Interactions of proteins with nucleic acids arise at all levels of cellular function, from chromosomal packing to biological regulation. These interactions can be analyzed in a high-throughput fashion by immobilizing the DNA sequences of interest, possibly numbering in the thousands, at discrete locations on a solid support and identifying those sequences that a protein analyte binds. Ideally, such surface assays would use unlabeled analyte to simplify protocols and avoid the possibility of perturbing the protein/DNA interaction. The present study compares three electrochemical modalities for simultaneously detecting binding of unlabeled transcription factor proteins to immobilized DNA duplexes based on (i) changes in the duplex diffusive motions, (ii) variations in the surface potential, and (iii) variations in the interfacial charging impedance, all of which can be conveniently derived from AC voltammetry traces. Cro protein from bacteriophage lambda is used as a model transcription factor. Specific binding of protein was successfully detected through modalities (i) and (ii), but not (iii). The effectiveness of these techniques is compared as a function of sampling frequency and protein concentration. Binding of 15 kDa Cro slowed down rotational diffusion of immobilized duplexes approximately 3-fold, and induced up to 5 mV changes in the surface potential. Moreover, by assessing Cro binding to bacteriophage operators of variable affinity, the study illustrates how contrast between specific and nonspecific interactions impacts detection.

A strategy for designing a peptide probe for detection of ß-amyloid oligomers.

Aggregation of ß-amyloid (Aß) is implicated in the pathology of Alzheimer's disease. Development of a robust strategy to detect Aß oligomeric intermediates, which have been identified as significant toxic agents, would be highly beneficial in the screening of drug candidates as well as enhancing our understanding of Aß oligomerization. Rapid, specific and quantitative detection, currently unavailable, would be highly preferred for accurate and reliable probing of transient Aß oligomers. Here, we report the development of a novel peptide probe, PG46, based on the nature of Aß self-assembly and the conformation-sensitive fluorescence of the biarsenical dye, FlAsH. PG46 was found to bind to Aß oligomers and displayed an increase in FlAsH fluorescence upon binding. No such event was observed when PG46 was co-incubated with Aß low-molecular-weight species or Aß fibrils. Aß oligomer detection was fast, and occurred within one hour without any additional sample incubation or preparation. We anticipate that the development of a strategy for detection of amyloid oligomers described in this study will be directly relevant to a host of other amyloidogenic proteins.

Enzyme stabilization by domain insertion into a thermophilic protein.

Insufficient kinetic stability of exoinulinase (EI) restricts its application in many areas including enzymatic transformation of inulin for production of ultra-high fructose syrup and oligofructan, as well as fermentation of inulin into bioethanol. The conventional method for enzyme stabilization involves mutagenesis and therefore risks alteration of an enzyme's desired properties, such as activity. Here, we report a novel method for stabilization of EI without any modification of its primary sequence. Our method employs domain insertion of an entire EI domain into a thermophilic scaffold protein. Insertion of EI into a loop of a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) resulted in improvement of kinetic stability (the duration over which an enzyme remains active) at 37 degrees C without any compromise in EI activity. Our analysis suggests that the improved kinetic stability at 37 degrees C might originate from a raised kinetic barrier for irreversible conversion of unfolded intermediates to completely inactivated species, rather than an increased energy difference between the folded and unfolded forms.

Catalytic mechanism of inulinase from Arthrobacter sp. S37.

Detailed catalytic roles of the conserved Glu323, Asp460, and Glu519 of Arthrobacter sp. S37 inulinase (EnIA), a member of the glycoside hydrolase family 32, were investigated by site-directed mutagenesis and pH-dependence studies of the enzyme efficiency and homology modeling were carried out for EnIA and for D460E mutant. The enzyme efficiency (k(cat)/K(m)) of the E323A and E519A mutants was significantly lower than that of the wild-type due to a substantial decrease in k(cat), but not due to variations in K(m), consistent with their putative roles as nucleophile and acid/base catalyst, respectively. The D460A mutant was totally inactive, whereas the D460E and D460N mutants were active to some extent, revealing Asp460 as a catalytic residue and demonstrating that the presence of a carboxylate group in this position is a prerequisite for catalysis. The pH-dependence studies indicated that the pK(a) of the acid/base catalyst decreased from 9.2 for the wild-type enzyme to 7.0 for the D460E mutant, implicating Asp460 as the residue that interacts with the acid/base catalyst Glu519 and elevates its pK(a). Homology modeling and molecular dynamics simulation of the wild-type enzyme and the D460E mutant shed light on the structural roles of Glu323, Asp460, and Glu519 in the catalytic activity of the enzyme.

Cloning, expression, and characterization of Bacillus sp. snu-7 inulin fructotransferase.

A gene encoding inulin fructotransferase (di-D-fructofuranose 1,2': 2,3' dianhydride [DFA III]-producing IFTase, EC 4.2.2.18) from Bacillus sp. snu-7 was cloned. This gene was composed of a single, 1,353-bp open reading frame encoding a protein composed of a 40-amino acid signal peptide and a 410-amino acid mature protein. The deduced amino acid sequence was 98% identical to Arthrobacter globiformis C11-1 IFTase (DFA III-producing). The enzyme was successfully expressed in E. coli as a functionally active, His-tagged protein, and it was purified in a single step using immobilized metal affinity chromatography. The purified enzyme showed much higher specific activity (1,276units/mg protein) than other DFA III-producing IFTases. The recombinant and native enzymes were optimally active in very similar pH and temperature conditions. With a 103-min half-life at 60 degrees C, the recombinant enzyme was as stable as the native enzyme. Acidic residues and cysteines potentially involved in the catalytic mechanism are proposed based on an alignment with other IFTases and a DFA IIIase.

Structural and functional insights into intramolecular fructosyl transfer by inulin fructotransferase.

Inulin fructotransferase (IFTase), a member of glycoside hydrolase family 91, catalyzes depolymerization of beta-2,1-fructans inulin by successively removing the terminal difructosaccharide units as cyclic anhydrides via intramolecular fructosyl transfer. The crystal structures of IFTase and its substrate-bound complex reveal that IFTase is a trimeric enzyme, and each monomer folds into a right-handed parallel beta-helix. Despite variation in the number and conformation of its beta-strands, the IFTase beta-helix has a structure that is largely reminiscent of other beta-helix structures but is unprecedented in that trimerization is a prerequisite for catalytic activity, and the active site is located at the monomer-monomer interface. Results from crystallographic studies and site-directed mutagenesis provide a structural basis for the exolytic-type activity of IFTase and a functional resemblance to inverting-type glycosyltransferases.

Cloning, mutagenesis, and expression of the acetylcholinesterase gene from a strain of Musca domestica; the change from a drug-resistant to a sensitive enzyme.

Insect acetylcholinesterase (AChE) is known to be a primary target of organophosphorus and carbamate insecticides. However chronic exposure to these chemicals has led to resistance to applied insecticides, due usually to mutation of the AChE gene. Analysis of the AChE gene (hm) of Musca domestica (the housefly), which is cloned in this report, reveals the relationship between mutation and insecticide resistance. The 2,076 bp hm encodes a mature protein of 612 amino acids (67 kDa), and an 80 residue signal peptide. Unlike the enzyme of 'sensitive' strains, the AChE used in this study was resistant to the organophosphorus insecticide, trichlorphon. DNA sequencing showed that this AChE is identical to that of the sensitive strains with the exception of three amino acids Met-82, Ala-262, and Tyr-327. Site-directed mutagenesis of the Ala-262 and Tyr-327 residues largely restored sensitivity to the insecticide, suggesting that these two residues are the key structural elements controlling sensitivity. In addition to these residues, Glu-234 and Ala-236 in the conserved sequence FGESAG are thought to play a role in modulating sensitivity to organophosphorus insecticides.