Development of an Ultrasound-emitting Device for Performing Rapid Immunostaining Procedures

Although intraoperative rapid diagnosis is conventionally performed using hematoxylin–eosin (HE)-stained specimens, the use of additional special staining, together with immunostaining techniques, has been examined in recent years to improve diagnostic accuracy. In intraoperative rapid diagnosis, immunostaining should be completed within 7–10 min, because the pathologist is typically presented with an HE-stained specimen within the same time period. We hypothesized that ultrasound may enhance antigen–antibody reactions and reduce the number of immunostaining steps. To clarify the ability of ultrasound to support immunostaining, we first created an ultrasonic generator specifically for immunostaining. Next, we explored the optimal conditions for immunostaining of formalin-fixed specimens to examine the utility of the ultrasonic generator. Finally, we tried immunostaining with the ultrasonic generator using frozen specimens to simulate intraoperative rapid diagnosis. We report herein that ultrasound enables immunostaining of frozen specimens in ∼10 min. (J Histochem Cytochem 58:421–428, 2010)     

Contrasting effects of a cladoceran (Daphnia galeata) and a calanoid copepod (Eodiaptomus japonicus) on algal and microbial plankton in a Japanese lake, Lake Biwa

Macrozooplankton may affect algal and microbial plankton directly through grazing or predation and indirectly through nutrient regeneration. They may also affect potential prey positively by removing alternative predators. Here, we examined the effects of a cladoceran (Daphnia) and a calanoid copepod (Eodiaptomus) on algal and microbial plankton in a Japanese lake using in situ experiments in which we manipulated the nutrient supply and biomass of these macrozooplankton. The response of algal and microbial plankton to macrozooplankton was diverse and varied depending on the level of nutrient supply. Eodiaptomus seemed to feed mainly on large algae (>20 µm) and microzooplankton, while direct grazing by Daphnia on algae, bacteria, heterotrophic nanoflagellates (HNF), and microzooplankton (ciliates, heliozoa, and rotifers) was pronounced. Trophic linkages within these microbial plankton was also suggested; bacteria were grazed by HNF and these in turn were grazed by microzooplankton. When the nutrient supply was high, both HNF and microzooplankton were exposed to higher amounts of algae and lower bacterial abundance. Moreover, nutrient regeneration by daphnids and Eodiaptomus copepods seemed to differentially stimulate the growth of algae and bacteria. The results suggest that the relationship between macrozooplankton and microbial plankton cannot be fully understood without taking into consideration not only the feeding characteristics of the macrozooplankton, but also the food web structure, the subsidized algal resource, and nutrient regeneration from the macrozooplankton.     

Effect of phospholipids on the hydrolysis of cholesterol oleate liquid crystals by lysosomal acid lipase

Cholesterol oleate liquid crystals were prepared in vitro as a model of lipid droplets accumulated in the atheromatous aorta. The hydrolysis of cholesterol oleate crystals by lysosomal acid lipase was examined in the presence and absence of various phospholipids. Phosphatidylserine markedly stimulated the hydrolysis of cholesterol oleate liquid crystals (20 times the basal value); it increased the Vmax value about 15 times and decreased the Km value to 1/20 times the basal value. The polar head group and the acyl chains of phosphatidylserine were required for its stimulation of hydrolysis.     

Influence of salt stress on C4 photosynthesis in Miscanthus sinensis Anderss.

• Miscanthus sinensis Anderss. is a good candidate for C₄ bioenergy crop development for marginal lands. As one of the characteristics of marginal lands, salinization is a major limitation to agricultural production. The present work aimed to investigate the possible factors involved in the tolerance of M. sinensis C₄ photosynthesis to salinity stress.
• Seedlings of two accessions (salt‐tolerant ‘JM0119’ and salt‐sensitive ‘JM0099’) were subjected to 0 mm NaCl (control) or 250 mm NaCl (salt stress treatment) for 2 weeks. The chlorophyll content, parameters of photosynthesis and chlorophyll a fluorescence, activity of C₄ enzymes and expression of C₄ genes were measured.
• The results showed that photosynthesis rate, transpiration rate, chlorophyll content, PSII operating efficiency, coefficient of photochemical quenching, activity of phosphoenolpyruvate carboxylase (PEPC) and pyruvate, orthophosphate dikinase (PPDK) and gene expression of PEPC and PPDK under salinity were higher after long‐term salinity exposure in ‘JM0119’ than in ‘JM0099’, while activity of NADP‐malate dehydrogenase (NADP‐MDH) and NADP‐malic enzyme (NADP‐ME), together with expression of NADP‐MDH and NADP‐ME, were much higher in ‘JM0099’ than in ‘JM0119’.
• In conclusion, the increased photosynthetic capacity under long‐term salt stress in the salt‐tolerant relative to the salt‐sensitive M. sinensis accession was mainly associated with non‐stomatal factors, such as reduced chlorophyll loss, higher PSII operating efficiency, enhanced activity of PEPC and PPDK and relatively lower activity of NADP‐ME.

     

Experimental verification of the 'stability profile of mutant protein' (SPMP) data using mutant human lysozymes.

The stability profile of mutant protein (SPMP) (Ota,M., Kanaya,S. and Nishikawa,K., 1995, J. Mol. Biol., 248, 733-738) estimates the changes in conformational stability due to single amino acid substitutions using a pseudo-energy potential developed for evaluating structure-sequence compatibility in the structure prediction method, the 3D-1D compatibility evaluation. Nine mutant human lysozymes expected to significantly increase in stability from SPMP were constructed, in order to experimentally verify the reliability of SPMP. The thermodynamic parameters for denaturation and crystal structures of these mutant proteins were determined. One mutant protein was stabilized as expected, compared with the wild-type protein. However, the others were not stabilized even though the structural changes were subtle, indicating that SPMP overestimates the increase in stability or underestimates negative effects due to substitution. The stability changes in the other mutant human lysozymes previously reported were also analyzed by SPMP. The correlation of the stability changes between the experiment and prediction depended on the types of substitution: there were some correlations for proline mutants and cavity-creating mutants, but no correlation for mutants related to side-chain hydrogen bonds. The present results may indicate some additional factors that should be considered in the calculation of SPMP, suggesting that SPMP can be refined further.     

Involvement of receptor aggregation and reactive oxygen species in osmotic stress-induced Syk activation in B cells.

Syk has been shown to be activated by osmotic stress, however, the mechanisms involved are largely unknown. In this study, we demonstrated that cell shrinkage, rather than osmolarity, was responsible for osmotic stress-induced Syk activation. Osmotic stress-induced Syk activation depended partly upon aggregation of surface receptors. Moreover, intracellular reactive oxygen species were involved in mediating osmotic stress-induced Syk activation, with osmotic stress-induced Syk activation being inhibited by the pretreatment of cells with N-acetyl-cysteine and reduced glutathione. When cells were treated with the combination of sodium chloride and hydrogen peroxide, there was a synergistic activation of Syk. In conclusion, osmotic stress-induced Syk activation required suramin-inhibitable surface receptor aggregation and accumulation of intracellular reactive oxygen species.     

Mechanism of defective lysogenization by phage P1 in a lon-mutant of Escherichia coli K-12.

Phage P1 cannot lysogenize a lon- mutant of Escherichia coli K-12, which is defective in the regulation of cellular division cycle to result in snake formation (14). P1 mutants, called P1pla, can lysogenize the lon- host. These mutations have been classified into two complementation groups: one is cis-dominant; the other is trans-dominant. A temperature-sensitive lon- mutant was isolated, which exhibited the lon- phenotype at 42 C but not at 33 C. A temperature-shift experiment of the P1-lysogenic derivative of the lon- ts mutant showed lysis of the culture and induction of the phage production. It is proposed that P1 plasmid may be under a certain regulatory circuit of the division cycle of the host bacterium by indirectly regulating the production of P1 immune repressor, or alternatively by directly derepressing the functions of P1 prophage.