RAISE: a simple and novel method of generating random insertion and deletion mutations

Although proteins may be artificially improved by random insertion and deletion mutagenesis methods, these procedures are technically difficult, and the mutations introduced are no more variable than those introduced by the introduction of random point mutations. We describe here a three-step method called RAISE, which is based on gene shuffling and can introduce a wide variety of insertions, deletions and substitutions. To test the efficacy of this method, we used it to mutate TEM β-lactamase to generate improved antibiotic resistance. Some unique insertion or deletion mutations were observed in the improved mutants, some of which caused higher activities than point mutations. Our findings indicate that the RAISE method can yield unique mutants and may be a powerful technique of protein engineering.     

Molecular cloning of pigGnT-I and I.2: an application to xenotransplantation

Xenotransplantation is one of the most attractive solutions for the current worldwide shortage of organs. The knocking out of alpha1,3-galactosyltransferase in pigs resulted in a drastic reduction in xenoantigenicity. However, more recent studies indicate that other xeno-antigens, so-called non-Gal antigens, will also need to be downregulated. In this study, pig N-acetylglucosaminyltransferase I (GnT-I), a key enzyme that initiates the biosynthesis of hybrid- and complex-type N-linked sugar chains, was isolated and the pigGnT-I.2 specific for the O-linked sugar chain was also isolated. Point mutants, pigGnT-I(123) and pigGnT-I(320), were subsequently constructed. While pigGnT-I(123) shows an indistinct dominant negative effect for endogenous GnT-I in pig cells, pigGnT-I(320) had a drastic effect. In addition, in the case of pig cell transfectants with pigGnT-I(320), cell surface carbohydrate structures were significantly altered and its antigenicity to human serum was reduced. Consequently, pigGnT-I(320) appears to be potentially useful in xenotransplantation by remodeling the carbohydrate structures on pig cells.     

Distinct contributions of vacuolar Qabc- and R-SNARE proteins to membrane fusion specificity

In eukaryotic endomembrane systems, Qabc-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) on one membrane and R-SNARE on the opposing membrane assemble into a trans-QabcR-SNARE complex to drive membrane fusion. However, it remains ambiguous whether pairing of Qabc- and R-SNAREs mediates membrane fusion specificity. Here, we explored the fusion specificity of reconstituted proteoliposomes bearing purified SNAREs in yeast vacuoles and other organelles. We found that not only vacuolar R-SNARE Nyv1p but also the non-cognate R-SNAREs, endosomal Snc2p, and endoplasmic reticulum-Golgi Sec22p caused efficient fusion with vacuolar Qabc-SNAREs. In contrast, their fusion is blocked completely by replacing vacuolar Qc-SNARE Vam7p with the non-cognate endosomal Tlg1p and Syn8p, although these endosomal Qc-SNAREs fully retained the ability to form cis-SNARE complexes with vacuolar SNAREs in solution and on membranes. Thus, our current study establishes that an appropriate assembly of Qabc-SNAREs is crucial for regulating fusion specificity, whereas R-SNARE itself has little contribution to specificity.     

Clostridium perfringens alpha-toxin induces the release of IL-8 through a dual pathway via TrkA in A549 cells

A characteristic feature of gas gangrene with Clostridium perfringens (C. perfringens) is the absence of neutrophils within the infected area and the massive accumulation of neutrophils at the vascular endothelium around the margins of the necrotic region. Intravenous injection of C. perfringens alpha-toxin into mice resulted in the accumulation of neutrophils at the vascular endothelium in lung and liver, and release of GRO/KC, a member of the CXC chemokine family with homology to human interleukin-8 (IL-8). Alpha-toxin triggered activation of signal transduction pathways causing mRNA expression and production of IL-8, which activates migration and binding of neutrophils, in A549 cells. K252a, a tyrosine kinase A (TrkA) inhibitor, and siRNA for TrkA inhibited the toxin-induced phosphorylation of TrkA and production of IL-8. In addition, K252a inhibited the toxin-induced phosphorylation of extracellular regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). PD98059, an ERK1/2 inhibitor, depressed phosphorylation of ERK1/2 and nuclear translocation of nuclear factor kappa B (NF-κB) p65, but SB203580, a p38 MAPK inhibitor, did not. On the other hand, PD98059 and SB203580 suppressed the toxin-induced production of IL-8. Treatment of the cells with PD98059 resulted in inhibition of IL-8 mRNA expression induced by the toxin and that with SB203580 led to a decrease in the stabilization of IL-8 mRNA. These results suggest that alpha-toxin induces production of IL-8 through the activation of two separate pathways, the ERK1/2/NF-κB and p38 MAPK pathways.     

Enhanced accumulation of adipocytes in bone marrow stromal cells in the presence of increased extracellular and intracellular [Ca²⁺

The bone marrow stroma contains osteoblasts and adipocytes that have a common precursor: the pluripotent mesenchymal stem cell found in bone marrow stromal cells (BMSCs). Local bone marrow Ca(2+) levels can reach high concentrations due to bone resorption, which is one of the notable features of the bone marrow stroma. Here, we describe the effects of high [Ca(2+)](o) on the accumulation of adipocytes in the bone marrow stroma. Using primary mouse BMSCs, we evaluated the level of adipocyte accumulation by measuring Oil Red O staining and glycerol-3-phosphate dehydrogenase (GPDH) activity. High [Ca(2+)](o) enhanced the accumulation of adipocytes following treatment with both insulin and dexamethasone together but not in the absence of this treatment. This enhanced accumulation was the result of both the accelerated proliferation of BMSCs and their differentiation into adipocytes. Using the fura-2 method, we also showed that high [Ca(2+)](o) induces an increase in [Ca(2+)](i). An intracellular Ca(2+) chelator suppressed the enhancement in adipocyte accumulation due to increased [Ca(2+)](o) in BMSCs. These data suggest a new role for extracellular Ca(2+) in the bone marrow stroma: increased [Ca(2+)](o) induces an increase in [Ca(2+)](i) levels, which in turn enhances the accumulation of adipocytes under certain conditions.     

Mapping ultra-weak protein-protein interactions between heme transporters of Staphylococcus aureus

Iron is an essential nutrient for the proliferation of Staphylococcus aureus during bacterial infections. The iron-regulated surface determinant (Isd) system of S. aureus transports and metabolizes iron porphyrin (heme) captured from the host organism. Transportation of heme across the thick cell wall of this bacterium requires multiple relay points. The mechanism by which heme is physically transferred between Isd transporters is largely unknown because of the transient nature of the interactions involved. Herein, we show that the IsdC transporter not only passes heme ligand to another class of Isd transporter, as previously known, but can also perform self-transfer reactions. IsdA shows a similar ability. A genetically encoded photoreactive probe was used to survey the regions of IsdC involved in self-dimerization. We propose an updated model that explicitly considers self-transfer reactions to explain heme delivery across the cell wall. An analogous photo-cross-linking strategy was employed to map transient interactions between IsdC and IsdE transporters. These experiments identified a key structural element involved in the rapid and specific transfer of heme from IsdC to IsdE. The resulting structural model was validated with a chimeric version of the homologous transporter IsdA. Overall, our results show that the ultra-weak interactions between Isd transporters are governed by bona fide protein structural motifs.     

Facile synthesis of 4-O-β-N-Acetylchitooligosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone based on the transformation of chitooligosaccharide and its suppressive effects against the furylfuramide-induced SOS response

A facile synthesis method is described for transforming the reducing-end residue of chitooligosaccharides (DP 2-4) into lactone. The desired 4-O-β-N-acetylchitooligosyl lactones (GN(n)L) were conveniently prepared from chitooligosaccharides by consecutive dehydration and oxidation reactions to afford 4-O-β-tri-N-acetylchitotriosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone (GN(3)L), 4-O-β-di-N-acetylchitobiosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone (GN(2)L), and 4-O-β-2-acetamido-2-deoxy-D-glucopyranosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone (GNL). The resulting lactone derivatives exhibited considerable suppression (42.6-54.3% at a concentration of 400 μM) in umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamido (AF-2). Lactonization of the chitooligosaccharides was found to be essential for their suppression of the SOS-inducing activity.     

Quality evaluation of rice crackers based on physicochemical measurements

The processing suitability as a material for rice crackers was characterized in the present study, based on physicochemical measurements and sensory testing of high-quality premium rice, low-amylose rice, Japonica-Indica hybrid rice, and red rice as the rice cultivar samples. Puffed rice crackers were prepared and the relationship between the physicochemical properties of the rice grains and the quality of the resulting products was investigated. It was possible to estimate the physical properties of a rice cracker by using multiple-regression analysis based on the chemical components, pasting properties and physical properties of the constituent rice. A formula for estimating the amylose content of the constituent rice was developed from the results of physicochemical measurements of the rice crackers. We assayed the quality of commercial rice crackers and examined the deterioration during the storage by measuring the physicochemical properties. The hardness and fat acidity of crackers increased markedly during storage for 20 d at 35 °C. The novel method of a one-bite test with a Tensipresser was useful to assay the quality of a rice cracker and made it possible to evaluate the quality deterioration of the rice cracker during storage.     

Principles of branch dynamics governing shape characteristics of cerebellar Purkinje cell dendrites

Neurons develop dendritic arbors in cell type-specific patterns. Using growing Purkinje cells in culture as a model, we performed a long-term time-lapse observation of dendrite branch dynamics to understand the rules that govern the characteristic space-filling dendrites. We found that dendrite architecture was sculpted by a combination of reproducible dynamic processes, including constant tip elongation, stochastic terminal branching, and retraction triggered by contacts between growing dendrites. Inhibition of protein kinase C/protein kinase D signaling prevented branch retraction and significantly altered the characteristic morphology of long proximal segments. A computer simulation of dendrite branch dynamics using simple parameters from experimental measurements reproduced the time-dependent changes in the dendrite configuration in live Purkinje cells. Furthermore, perturbation analysis to parameters in silico validated the important contribution of dendritic retraction in the formation of the characteristic morphology. We present an approach using live imaging and computer simulations to clarify the fundamental mechanisms of dendrite patterning in the developing brain.