Identification of a wheat allergen,Tri a Bd 36K,as a peroxidase

A 36-kDa allergen, Tri a Bd 36K, was purified from wheat albumin and characterized. The protein was similar to barley peroxidase BP-1 both in its amino acid sequence and peroxidase activity. The enzyme seemed to contain L-fucose and D-mannose and the glycan moiety reacted with IgE antibodies in a patient's serum.     

Engineering of tissue inhibitor of metalloproteinases mutants as potential therapeutics

Matrix metalloproteinases (MMPs) play a central role in many biological processes such as development, morphogenesis and wound healing, but their unbalanced activities are implicated in numerous disease processes such as arthritis, cancer metastasis, atherosclerosis, nephritis and fibrosis. One of the key mechanisms to control MMP activities is inhibition by endogenous inhibitors called tissue inhibitors of metalloproteinases (TIMPs). This review highlights the structures and inhibition mechanism of TIMPs, the biological activities of TIMPs, the unique properties of TIMP-3, and the altered specificity towards MMPs achieved by mutagenesis. A potential therapeutic use of TIMP variants is discussed.     

Induction of intratumoral tumor necrosis factor by a synthetic lipid A analog, ONO-4007, with less tolerance in repeated administration and its implication in potent antitumor effects with low toxicity

To evaluate the anti-tumor characteristics of ONO-4007, a synthetic analog of lipid A, the authors examined its acute toxicity and anti-tumor activity in a mouse MM46 mammary tumor system in comparison with LA-15-PP, an E. coli-type synthetic lipid A and LPS. Systemic and local (tumor site) induction of tumor necrosis factor (TNF) by a single i.v. shot of ONO-4007 and LA-15-PP correlated with manifestation of their toxicity, showing that ONO-4007 is 100-fold less effective than LA-15-PP. However, a protocol of repeated administration (3 shots twice a week) exhibited about 10 times more therapeutic potency of ONO-4007 for cancer therapy than expected in the above experiments. In a dose inducing submaximal systemic and intratumoral TNF production, repeated injections (twice a week) of ONO-4007 (10 mg/kg), LA-15-PP (0.1 mg/kg) and LPS (0.1 mg/kg) commonly generated a tolerant state in the systemic response (serum and liver) to subsequent stimulation. The intratumoral response was retained with this repeated administration of ONO-4007, but was not with LA-15-PP or LPS. TIM (tumor-infiltrating macrophages) isolated from mice pre-injected with ONO-4007 and LA-15-PP were found to lose their response to both substances, but the response was rapidly recovered until 72 h after injection and virtually no difference was observed in their response to either drug. The in vitro treatment of naive TIM with ONO-4007 or LA-15-PP for 2 h depressed the response to both substances and the depression continued for 72 h even in culture with fresh medium. The relatively high efficacy of ONO-4007 in cancer therapy likely depends on the retraction of the tolerant state, especially at the tumor site where the response to ONO-4007 is recovered much more efficiently than that to lipid A. While constant recruitment of macrophages to tumor tissue might be involved in the difference of tolerance recovery between this region and others, selective response to ONO-4007 may not be explained simply by the sensitivity of recruited TIM. Pharmacokinetical experiments revealed that repeated injections of LA-15-PP enhanced its clearance from blood circulation, while the clearance of ONO-4007 was stable after repeated injections. Thus, pharmacokinetical properties of ONO-4007 may also possibly be implicated in this event.     

A study of conformational stability of poly(L-alanine), poly(L-valine), and poly(L-alanine)/poly(L-valine) blends in the solid state by (13)C cross-polarization/magic angle spinning NMR

13C cross-polarization/magic angle spinning (CP/MAS) NMR and (1)H T(1rho) experiments of poly(L-alanine) (PLA), poly(L-valine) (PLV), and PLA/PLV blends have been carried out in order to elucidate the conformational stability of the polypeptides in the solid state. These were prepared by adding a trifluoroacetic acid (TFA) solution of the polymer with a 2.0 wt/wt % of sulfuric acid (H(2)SO(4)) to alkaline water. From these experimental results, it is clarified that the conformations of PLA and PLV in their blends are strongly influenced by intermolecular hydrogen-bonding interactions that cause their miscibility at the molecular level.     

E. coli expression of TIMP-4 and comparative kinetic studies with TIMP-1 and TIMP-2: insights into the interactions of TIMPs and matrix metalloproteinase 2 (gelatinase A)

The inhibitory properties of TIMP-4 for matrix metalloproteinases (MMPs) were compared to those of TIMP-1 and TIMP-2. Full-length human TIMP-4 was expressed in E. coli, folded from inclusion bodies, and the active component was purified by MMP-1 affinity chromatography. Progress curve analysis of MMP inhibition by TIMP-4 indicated that association rate constants (k(on)) and inhibition constants (K(i)) were similar to those for other TIMPs ( approximately 10(5) M(-)(1) s(-)(1) and 10(-)(9)-10(-)(12) M, respectively). Dissociation rate constants (k(off)) for MMP-1 and MMP-3 determined using alpha(2)-macroglobulin to capture MMP dissociating from MMP-TIMP complexes were in good agreement with values deduced from progress curves ( approximately 10(-)(4) s(-)(1)). K(i) and k(on) for the interactions of TIMP-1, -2, and -4 with MMP-1 and -3 were shown to be pH dependent. TIMP-4 retained higher reactivity with MMPs at more acidic conditions than either TIMP-1 or TIMP-2. Molecular interactions of TIMPs and MMPs investigated by IAsys biosensor analysis highlighted different modes of interaction between proMMP-2-TIMP-2 (or TIMP-4) and active MMP-2-TIMP-2 (or TIMP-4) complexes. The observation that both active MMP-2 and inactive MMP-2 (with the active site blocked either by the propeptide or a hydroxamate inhibitor) have essentially identical affinities for TIMP-2 suggests that there are two TIMP binding sites on the hemopexin domain of MMP-2: one with high affinity that is involved in proMMP-2 or hydroxamate-inhibited MMP-2; and the other with low affinity involved in formation of the complex of active MMP-2 and TIMP-2. Similar models of interaction may apply to TIMP-4. The latter low-affinity site functions in conjunction with the active site of MMP-2 to generate a tight enzyme-inhibitor complex.     

Insertional mutagenesis in a homologue of a Pi transporter gene confers arsenate resistance on chlamydomonas

An arsenate-resistant mutant AR3 of Chlamydomonas reinhardtii is a recessive mutant generated by random insertional mutagenesis using the ARG7 gene. AR3 shows about 10-fold resistance against arsenate toxicity compared with the wild type. By using a flanking region of an inserted tag as a probe, we cloned the corresponding wild-type allele (PTB1) of a mutated gene, which could completely complement the arsenate-resistance phenotype of AR3. The size of PTB1 cDNA is about 6.0 kb and it encodes a putative protein comprising 1666 amino acid residues. This protein exhibits significant sequence similarity with the yeast Pho89 protein, which is known to be a Na(+)/Pi co-transporter, although the PTB1 protein carries an additional Gln- and Gly-rich large hydrophilic region in the middle of its primary structure. Analyses of arsenic accumulation and release revealed that PTB1-disrupted cells show arsenate resistance due to low arsenate uptake. These results suggest that the PTB1 protein is a factor involved in arsenate (or Pi) uptake. Kinetics of Pi uptake revealed that the activity of high-affinity Pi transport component in AR3 is more activated than that in the wild type.     

Effects of 10-T static magnetic field on human peripheral blood immune cells

The exposure of peripheral blood mononuclear cells (PBMCs) was performed in a 10-T static magnetic field. Without lymphocyte stimulation, there were no significant differences in the viability of the exposed and unexposed CD4(+) T cells, CD8(+) T cells, B cells, and natural killer (NK) cells. The expression of Th1 type chemokine receptor, CXCR3, and Th2 type receptor, CCR3, was unaltered after magnetic-field exposure. No differences were observed in the naive T cells and memory T-cell subclasses in either CD4(+) or CD8(+) T cells. In contrast to the unstimulated condition, the magnetic-field exposure reduced the viability of phytohemagglutinin (PHA)-activated T cells in both the CD4(+) and CD8(+) subclasses. In particular, the number of PHA-treated naive CD8(+) T cells (CD45RA(+)CD4(-)CD8(+)) was markedly decreased after the magnetic-field exposure, while PHA-treated memory CD8(+) cells (CD45RA(-)CD4(-)CD8(+)) were resistant to the exposure. The number of PHA-treated naive CD4(+) T cells (CD45RA(+)CD4(+)CD8(-)) and memory cells (CD45RA(-)CD4(+)CD8(-)) was markedly decreased to a similar degree. Thus the susceptibility of lymphocytes to the magnetic-field exposure differed among activated T-cell subtypes. The magnetic-field exposure significantly increased the death of PHA-stimulated lymphocytes by apoptosis. These results suggest that a strong static magnetic field has acute effects on immune cells during cell division, while the field exposure has a minimal effect on immune cells in a nondividing phase.     

Carbazole/dioxin-degrading car gene cluster is located on the chromosome of Pseudomonas stutzeri strain OM1 in a form different from the simple transposition of Tn4676

The carbazole-degrading (car) operon on the chromosome of Pseudomonas stutzeri strain OM1 showed >99% identity to that in the 72.8 kb catabolic transposon, Tn4676, on plasmid pCAR1. Southern hybridization using probes prepared from the pCAR1 sequence and sequencing analyses showed that the OM1 chromosome contained the 55 kb DNA region, almost all of which was a part of Tn4676, flanked by two copies of novel insertion sequence, ISPst3, and included the car gene.