Cryoprotectant permeability of aquaporin-3 expressed in Xenopus oocytes

It has been shown that aquaporin-3, a water channel, is expressed in mouse embryos. This type of aquaporin transports not only water but also neutral solutes, including cell-permeating cryoprotectants. Therefore, the expression of this channel may have significant influence on the survival of cryopreserved embryos. However, permeability coefficients of aquaporin-3 to cryoprotectants have not been determined except for glycerol. In addition, permeability coefficients under concentration gradients are important for developing and improving cryopreservation protocols. In this study, we examined the permeability of aquaporin-3 to various cryoprotectants using Xenopus oocytes. The permeability of aquaporin-3 to cryoprotectants was measured by the volume change of aquaporin-3 cRNA-injected oocytes in modified Barth's solution containing either 10% glycerol, 8% ethylene glycol, 10% propylene glycol, 1.5 M acetamide, or 9.5% DMSO (1.51-1.83 Osm/kg) at 25 degrees C. Permeability coefficients of aquaporin-3 for ethylene glycol and propylene glycol were 33.50 and 31.45 x 10(-3) cm/min, respectively, which were as high as the value for glycerol (36.13 x 10(-3) cm/min). These values were much higher than those for water-injected control oocytes (0.04-0.11 x 10(-3) cm/min). On the other hand, the coefficients for acetamide and DMSO were not well determined because the volume data were poorly fitted by the two parameter model, possibly because of membrane damage. To avoid this, the permeability for these cryoprotectants was measured under a low concentration gradient by suspending oocytes in aqueous solutions containing low concentrations of acetamide or DMSO dissolved in water (0.20 Osm/kg). The coefficient for acetamide (24.60 x 10(-3) cm/min) was as high as the coefficients for glycerol, ethylene glycol, and propylene glycol, and was significantly higher than the value for control (6.50 x 10(-3) cm/min). The value for DMSO (6.33 x 10(-3) cm/min) was relatively low, although higher than the value for control (0.79 x 10(-3) cm/min). This is the first reported observation of DMSO transport by aquaporin-3.     

Correlation between unirradiated cell TP53 protein levels and radiosensitivity in MOLT-4 cells.

MOLT-4 cells undergo apoptosis after X irradiation. A radiosensitive variant, MOLT-4N1, and a radioresistant variant, MOLT-4N2, have been studied with respect to their radiosensitivity and its relationship to the levels of TP53 protein (formerly known as p53). X irradiation induces apoptosis in both cell lines with the following difference: The induction of apoptosis in MOLT-4N2 cells occurred later than in MOLT-4N1 cells as determined by the morphological changes and DNA fragmentation. The levels of cell death measured by the dye exclusion test coincided with the levels of apoptosis in both cell lines, suggesting that radiation-induced cell killing is determined by the induction of apoptosis. Unirradiated MOLT-4N1 cells contained a significantly higher intracellular level of TP53 protein and a much higher level of TP53 mRNA compared to MOLT-4N2 cells. X irradiation led to an accumulation of TP53 protein in both cell lines that was greater in MOLT-4N1 cells. This accumulation of TP53 protein preceded changes in DNA degradation and ladder formation and in nuclear morphology. These results strongly suggest that the radiosensitivity of MOLT-4 cells correlates well with the unirradiated control levels of TP53 mRNA and TP53 protein, and that the quantitative levels of TP53 protein must reach a threshold for the cells to undergo apoptosis.     

Inhibitory effects of metachromins L–Q and its related analogs against receptor tyrosine kinases EGFR and HER2

Metachromins are a series of sesquiterpenoid quinones isolated from Okinawan marine sponges. Inhibitory effects of metachromins L–Q (1–6), sesquiterpenoid quinones with an amino acid residue, and their related analogs (7–18) prepared from metachromins A (19) and C (20) against receptor tyrosine kinases EGFR and HER2 were investigated. Two analogs 11 and 12 showed relatively stronger inhibitory activity against EGFR, while metachromins L–Q (1-6) and seven analogs (8, 10, 11, and 15–18) showed inhibitory activities against HER2.     

Effects of lactoferrin and lactoperoxidase‐containing food on the oral microbiota of older individuals

The oral microbiota influences health and disease states. Some gram‐negative anaerobic bacteria play important roles in tissue destruction associated with periodontal disease. Lactoferrin (LF) and lactoperoxidase (LPO) are antimicrobial proteins found in saliva; however, their influence on the whole oral microbiota currently remains unknown. In this randomized, double‐blinded, placebo‐controlled study, the effects of long‐term ingestion of LF and LPO‐containing tablets on the microbiota of supragingival plaque and tongue coating were assessed. Forty‐six older individuals ingested placebo or test tablets after every meal for 8 weeks. The relative abundance of bacterial species was assessed by 16S rRNA gene high‐throughput sequencing. Most of the bacterial species in supragingival plaque and tongue coating that exhibited significant decreases in the test group were gram‐negative bacteria, including periodontal pathogens. Decreases in the total relative abundance of gram‐negative organisms in supragingival plaque and tongue coating correlated with improvements in assessed variables related to oral health, such as oral malodor and plaque accumulation. Furthermore, there was significantly less microbiota diversity in supragingival plaque at 8 weeks in the test group than in the placebo group and low microbiota diversity correlated with improvements in assessed variables related to oral health. These results suggest that LF and LPO‐containing tablets promote a shift from a highly diverse and gram‐negative‐dominated to a gram‐positive‐dominated community in the microbiota of supragingival plaque and tongue coating. This microbial shift may contribute to improvements in oral health, including oral malodor and state of the gingiva.

     

The different effects of recombinant human tumor necrosis factor on rat fibrosarcoma sublines

Summary The antitumor effect of recombinant human tumor necrosis factor (rH-TNF) on two clones of rat fibrosarcoma with different metastatic potential to lymph nodes was examined. The colony formation of clone A, which has high metastatic potential, was completely inhibited by continuous exposure to rH-TNF at 50 U/ml. In contrast, colony formation of clone G, which has low metastatic potential, was not inhibited by high concentrations of rH-TNF (10,000 U/ml). The inhibitory effect of rH-TNF on colony formation by clone A was also observed with a 1-h exposure to rH-TNF. This effect was time and concentration dependent, as determined by the colony assay, ³H-thymidine uptake assay, and ⁵¹Cr-release assay. ³H-thymidine and ³H-uridine uptake per cell of clone A exposed to rH-TNF was not decreased. This suggests that the mechanisms of the antitumor effect of rH-TNF were not due to inhibition of DNA and RNA synthesis of tumor cells. In vivo growth and lymph node metastases of clone A inoculated i.p. to Donryu strain rats were completely suppressed by 14 consecutive i.p. injections of 10⁵ or 10⁶ U/kg per day of rH-TNF. On the other hand the growth of clone G was not influenced by rH-TNF administration.     

Seasonal changes in the spermatogenic epithelium of adult Japanese macaques (Macaca fuscata fuscata)

A histological study was undertaken to clarify seasonal changes in the spermatogenic epithelium of Japanese macaques. Testicular tissue samples were excised by biopsies from five adult laboratory-maintained males in mating and non-mating seasons. The samples were fixed with Bouin's solution, embedded in paraffin, and stained with PAS and hematoxylin. Microscopic observations on cross-sections of seminiferous tubules revealed that the seminiferous epithelium in the mating season was thicker than in the non-mating season. PAS-stained granules were found in some of the dark A-type spermatogonia, which significantly increased in the non-mating season. Spermatids of the steps preceding the appearance of the acrosomic cap in stages I to III were observed significantly more often than those in the step coinciding with the formation of the acrosomic cap in stage IV. In stage I, the ratio of mature spermatids or spermatozoa to immature spermatids in the mating season was higher than that in the non-mating season. These findings suggest that spermiogenesis, as well as spermatocytogenesis, is inhibited in the non-mating season.     

Effect of host shoot clipping on carbon and nitrogen sources for arbuscular mycorrhizal fungi

The effect of clipping of the host-plant shoot on the sources of carbon and nitrogen for the arbuscular mycorrhizal (AM) fungus Gigaspora margarita was determined by measuring ¹³C in spores and hyphae in cocultures of C₃ and C₄ plants and by differential ¹⁵N labeling. C₃ and C₄ plants, which have different δ¹³C values, were grown in the same container separated by a series of hyphal compartments. The C₃ and C₄ plants were applied with ¹⁴N- and ¹⁵N-urea, respectively. After clipping of the C₃ shoots, spore δ ¹³C gradually approached that of the C₄ roots. Hyphal δ ¹³C paralleled that of spores. Spore % ¹⁵N was similar to that of mineral N in the C₄ plant compartment. Thus C in G. margarita coming from the clipped plants decreased with time. This demonstrates that C in AM fungi comes from living plants, whilst the N in spores comes mostly from the soil. Accepted: 28 November 2000     

Potato and rabbit muscle phosphorylases: comparative studies on the structure,function and regulation of regulatory and nonregulatory enzymes

Summary Phosphorylases (EC 2.4.1.1) from potato and rabbit muscle are similar in many of their structural and kinetic properties, despite differences in regulation of their enzyme activity. Rabbit muscle phosphorylase is subject to both allosteric and covalent controls, while potato phosphorylase is an active species without any regulatory mechanism. Both phosphorylases are composed of subunits of approximately 100 000 molecular weight, and contain a firmly bound pyridoxal 5-phosphate. Their actions follow a rapid equilibrium random Bi Bi mechanism. From the sequence comparison between the two phosphorylases, high homologies of widely distributed regions have been found, suggesting that they may have evolved from the same ancestral protein. By contrast, the sequences of the N-terminal region are remarkably different from each other. Since this region of the muscle enzyme forms the phosphorylatable and AMP-binding sites as well as the subunit-subunit contact region, these results provide the structural basis for the difference in the regulatory properties between potato and rabbit muscle phosphorylases. Judged from CD spectra, the surface structures of the potato enzyme might be significantly different from that of the muscle enzyme. Indeed, the subunit-subunit interaction in the potato enzyme is tighter than that in the muscle enzyme, and the susceptibility of the two enzymes toward modification reagents and proteolytic enzymes are different. Despite these differences, the structural and functional features of the cofactor, pyridoxal phosphate, site are surprisingly well conserved in these phosphorylases. X-ray crystallographic studies on rabbit muscle phosphorylase have shown that glucose-1-phosphate and orthophosphate bind to a common region close to the 5-phosphate of the cofactor. The muscle enzyme has a glycogen storage site for binding of the enzyme to saccharide substrate, which is located away from the cofactor site. We have obtained, in our reconstitution studies, evidence for binding of saccharide directly to the cofactor site of potato phosphorylase. This difference in the topography of the functional sites explains the previously known different specificities for saccharide substrates in the two phosphorylases. Based on a combination of these and other studies, it is now clear that the 5-phosphate group of pyridoxal phosphate plays a direct role in the catalysis of this enzyme. Information now available on the reaction mechanism of phosphorylase is briefly described.