Identification of the 23-kDa peptide derived from the precursor of Gly m Bd 28K, a major soybean allergen, as a new allergen

One of the major soybean allergens, Gly m Bd 28K, is suggested to be biosynthesized as a preproprotein form, which would be composed of a signal peptide, Gly m Bd 28K and the C-terminal peptide (the 23-kDa peptide). However, the 23-kDa peptide has never been characterized. In the present study, we prepared a monoclonal antibody (mAb) against a recombinant 23-kDa peptide expressed in Escherichia coli to detect the 23-kDa peptide in soybean. Several proteins were detected by immunoblotting with the mAb. All of the proteins were shown to have the identical N-terminal amino acid sequence, suggesting that the proteins correspond to the C-terminal part of the Gly m Bd 28K precursor. Furthermore, Gly m Bd 28K and the 23-kDa peptide were observed to come out at the 21st day after flowering and to locate in the crystalloid part of protein storage vacuoles in growing cotyledons. Some of the 23-kDa peptides were shown to be glycoproteins with an N-linked glycan moiety and exhibited the binding to IgE antibodies in the sera of patients sensitive to soybean. The binding of the peptides to IgE antibodies was suggested to be predominantly dependent on their glycan moiety. This study proves the occurrence of the 23-kDa peptide in soybean and that it is a new allergen.     

Oxidative stress-induced cell death of human oral neutrophils

Polymorphonuclear leukocytes (PMN) playcrucial roles in protecting hosts against invading microbes and in thepathogenesis of inflammatory tissue injury. Although PMN migrate intomucosal layers of digestive and respiratory tracts, only limitedinformation is available of their fate and function in situ. Wepreviously reported that, unlike circulating PMN (CPMN), PMN in theoral cavity spontaneously generate superoxide radical and nitric oxide (NO) in the absence of any stimuli. When cultured for 12 h under physiological conditions, oral PMN (OPMN) showed morphological changesthat are characteristic of those of apoptosis. Upon agarose gelelectrophoresis, nuclear DNA samples isolated from OPMN revealed ladder-like profiles characteristic of nucleosomal fragmentation. L-cysteine, reduced glutathione (GSH), and herbimycin A, aprotein tyrosine kinase inhibitor, suppressed the activation ofcaspase-3 and apoptosis of OPMN. Neither thiourea, superoxidedismutase (SOD), nor catalase inhibited the activation of caspase-3 and apoptosis. Moreover,N-acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), inhibitorfor caspase-3, inhibited the fragmentation of DNA. These resultssuggested that oxidative stress and/or tyrosine-kinase-dependent pathway(s) activated caspase-3 in OPMN, thereby inducing their apoptosis.

     

Silicon compatible with the height of human vertebral column

In the study on human vertebral composites, silicon was found to be related to the height of the vertebral column. The element ratio of silicon to calcium in the lumbar vertebra was twice that of the lowest one in the cervical vertebra. The element ratio gradually increased from approx 0.5 at the cervical vertebra to approx 1.0 at the lumbar one. Silicon is preserved catastropically much more in the lower height of the backbone in contrast with the calcium amount, and the ratio to sulfur appears to be unchanged. This suggests that silicon is compatible with the biokinetics of the vertebral bone structure of calcium.     

Cepharanthine, an anti-inflammatory drug, suppresses mitochondrial membrane permeability transition

Cepharanthine (CEP), a biscocrourine alkaloid, has been widely used in Japan for the treatment of several disorders. Furthermore, accumulated evidence shows that CEP protects against some cell death systems but not others. Recently, it was found that mitochondria play an important role in a mechanism of apoptosis involving membrane permeability transition (MPT). Although CEP stabilizes the mitochondrial membrane structure and protects some functions of mitochondria from damage, the mechanism of action of CEP on MPT remains obscure. In this study, therefore, we examined the effect of CEP on Ca2+- and Fe2+/ADP-induced MPT of isolated mitochondria. CEP inhibited Ca2+-induced swelling, depolarization, Cyt.c release, and the release of Ca2+ in a concentration dependent manner. CEP also inhibited Ca2+-induced generation of reactive oxygen species and Fe/ADP-induced swelling and lipid peroxidation. Furthermore, CEP suppressed Ca2+-induced thiol modification of adenine nucleotide transloase (ANT). These results suggested that CEP suppressed MPT by a decrease in affinity of cyclophilin D for ANT. From these results it was concluded that the suppression of MPT by CEP might be due to its inhibitory action on Ca2+ release and antioxidant activity and that CEP might suppress the mechanism of apoptotic cell death when directly interacted with mitochondria in cells.     

Cloning and expression of rapeseed procruciferin in Escherichia coli and crystallization of the purified recombinant protein

Two rapeseed cruciferin cDNAs (cru2/3a and cru2/3b) were cloned and sequenced. A comparison of their DNA and protein sequences with other cruciferins, indicated cru2/3b to be a novel clone and, among them, an inherent and highly conserved sequence of twelve amino acids was identified. Procruciferin 2/3a and 2/3b were expressed in Eschericha coli, and procruciferin 2/3a was obtained in a soluble form. The expressed procruciferin 2/3a has a trimeric structure and formed crystals although the quality was not good, suggesting that this expression system is useful for protein engineering of procruciferin 2/3a.     

Age-related changes of elements and relationships among elements in human tendons and ligaments

To elucidate compositional changes of the tendons and ligaments with aging, the authors investigated age-related changes of element contents in the insertion tendons of the biceps brachii muscle, central tendons of the diaphragma, Achilles’ tendons, posterior longitudinal ligaments (PLLs) of the cervical spine, ligamenta capitum femorum, and anterior cruciate ligaments. After ordinary dissections by medical students, the three tendons and three ligaments were resected and element contents were determined by inductively coupled plasma-atomic emission spectrometry. It was found that the elements, such as Ca, P, S, Mg, Na, Zn, and Fe, did not change significantly in the three tendons and two ligaments with aging, except for the PLLs where Ca and Mg increased significantly with aging and Fe decreased significantly with aging. With regard to the relationships among elements, the common finding that there were significant correlations between Ca and P contents and between Ca and Mg contents was obtained in the three ligaments. Likewise, the common finding that there was a significant correlation between Ca and Mg contents was obtained in the three tendons. Regarding the relationship between Ca and P contents, the three tendons were different from the three ligaments.     

Age-related changes of elements with their relationships in human cranial and spinal nerves

To elucidate compositional changes of peripheral nerves with aging, the authors investigated age-related changes of elements and their relationships in the optic, trigeminal, vagus, median, radial, ulnar, femoral, sciatic, tibial, and common peroneal nerves by inductively coupled plasma-atomic emission spectrometry. The subjects consisted of 10 men and 12 women, ranging in age from 65 to 91 yr. It was found that although accumulations of Ca and P occurred only in the trigeminal nerve at old age, it hardly occurred in the optic, vagus, median, radial, ulnar, femoral, sciatic, tibial, and common peroneal nerves at old age. The average contents of Ca and P were three and two times higher in the trigeminal nerve than in the other nine kinds of nerve, respectively. Likewise, the average content of Mg was a little higher in the trigeminal nerve compared with the other nerves. With regard to the relationships among elements, significant direct correlations were found among the contents of Ca, P, S, and Mg in most, but not all, 10 kinds of nerve. In the trigeminal nerve, a significant inverse correlation was found between the contents of S and the other elements, such as Ca, P, and Mg. Regarding the relationships between the contents of S and other elements, the nerves, except for the trigeminal nerve, differed from those found in the arteries previously reported.     

Accumulation of calcium in human common iliac artery, aortic valve, xiphoid process, costal cartilage, posterior longitudinal ligament, trigeminal nerve, and rib accompanied by increase of magnesium

To examine whether an accumulation of Ca in the tissues was accompanied by an increase of Mg, the authros investigated the relationships between Ca and Mg contents in the common iliac arteries, aortic valves, xiphoid processes, costal cartilages, posterior longitudinal ligaments, trigeminal nerves, and ribs by inductively coupled plasma-atomic emission spectrometry. After the ordinary dissections by medical students were finished, the common iliac arteries, aortic valves, xiphoid processes, bilateral the fourth costal cartilages, posterior longitudinal ligaments between the fourth and fifth cervical vertebrae, trigeminal nerves, and bilateral the sixth ribs were resected from the subjects and elements were determined. It was found that there were extremely significant direct correlations between Ca and Mg contents in all of the common iliac arteries, aortic valves, costal cartilages, posterior longitudinal ligaments, and trigeminal nerves, whereas there were significant direct correlations in both the xiphoid processes and ribs. As for the tissues containing Ca higher than 20 mg/g, the average mass ratios of Mg/Ca were similar among the seven tissues. As Ca increased in all of the common iliac arteries, aortic valves, xiphoid processes, costal cartilages, posterior longitudinal ligaments, trigeminal nerves, and ribs, Mg increased simultaneously in the seven tissues.     

Foreign gene products can be enhanced by introduction into low storage protein mutants

Transgenic rice expressing soybean glycinin in its endosperm was crossed with two types of low-glutelin mutants to determine how much storage the protein mutants can contribute to increases in glycinin accumulation. The glycinin level (102 microg/100 mg seed) in the parental transgenic line was enhanced to approximately 224-237 microg/100 mg seed within a genetic background deficient in glutelin (i.e. of low glutelins). The enrichment of this foreign gene product was compensated by a decrease in the expression of other endogenous prolamine and globulin storage proteins, resulting in an almost equivalent total amount of seed storage proteins. These results show that low storage protein mutants can provide potentially useful hosts for the expression of foreign genes, allowing a higher-level accumulation, because they can provide wider space for the accumulation of foreign gene products than in the normal host plant.