Colorimetric cell proliferation assay for microorganisms in microtiter plate using water-soluble tetrazolium salts

A colorimetric method to assay cell proliferation of microorganisms in 96-well microtiter plates using water-soluble tetrazolium salts and electron mediators was developed. Combinations of 6 kinds of water-soluble tetrazolium salts and 27 kinds of electron mediators that considered the metabolic efficiency of microorganisms and the influence with medium components were investigated. 2-Methyl-1,4-naphthoquinone (NQ) was reduced most effectively by various species of microorganisms, and a combination of WST-8 as a water-soluble tetrazolium salt with 2-methyl-1,4-NQ repressed the increase in background due to medium components. In the presence of 2-methyl-1,4-NQ, WST-8 was reduced by microbial cells to formazan, which exhibited maximum absorbance at 460 nm. The proposed tetrazolium method could be applied to measure proliferations of various microbial cells including 3 kinds of yeast, 9 kinds of Gram-positive bacteria, and 10 kinds of Gram-negative bacteria. Linear relationships between the absorbance and viable microbial cell density were obtained in all microorganisms, suggesting that the absorbance change reflected the microbial cell proliferation.     

Specific cation effect on quenching reactions of excited tris(α,α-diimine)ruthenium(II) and tris(2,2-bipyridine)chromium(III) by tris(oxalato)- and tris(malnato)chromates(III) in aqueous solutions

Specific salt effects were studied on the quenching reaction of excited [Ru(N-N)₃]²⁺ (N-N=2,2^′-bipyridine (bpy), 1,10-phenanthrorine (phen)) and [Cr(bpy)₃]³⁺ by [Cr(ox)₃]³⁻ (ox=oxalate ion) and [Cr(mal)₃]³⁻ (mal=malonate ion) in aqueous solutions as a function of alkali metal ions which were added for adjustment of ionic strength. The value of k_q, quenching rate constants, and k₁, energy transfer rate constant in encounter complex, is changed by the cations as the order of Li⁺ > Na⁺ > K⁺ ≈ Rb⁺ ? Cs⁺, although diffusion rate constants are not changed by the co-existing cations. Among the quenching reactions investigated in this work, a ratio of k₁ values in the aqueous solutions whose ionic strength was adjusted with LiCl and KCl, k₁^LiCl/k₁^KCl, is larger for quenching systems of closely approached donor-acceptor pair than loosely bounded pair. These results indicate that co-existing alkali cation tunes the distance between donor and acceptor in encounter complex where energy transfer occurs.     

Vitamin C depletion increases superoxide generation in brains of SMP30/GNL knockout mice

Vitamin C (VC) has a strong antioxidant function evident as its ability to scavenge superoxide radicals in vitro. We verified that this property actually exists in vivo by using a real-time imaging system in which Lucigenin is the chemiluminescent probe for detecting superoxide in senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize VC in vivo. SMP30/GNL KO mice were given 1.5 g/L VC [VC(+)] for 2, 4, or 8 weeks or denied VC [VC(−)]. At 4 and 8 weeks, VC levels in brains from VC(−) KO mice were <6% of that in VC(+) KO mice. Accordingly, superoxide-dependent chemiluminescence levels determined by ischemia-reperfusion at the 4- and 8 weeks test intervals were 3.0-fold and 2.1-fold higher, respectively, in VC(−) KO mice than in VC(+) KO mice. However, total superoxide dismutase activity and protein levels were not altered. Thus, VC depletion specifically increased superoxide generation in a model of the living brain.     

Molecular chaperones involved in chloroplast protein import.

Transport of cytoplasmically synthesized precursor proteins into chloroplasts, like the protein transport systems of mitochondria and the endoplasmic reticulum, appears to require the action of molecular chaperones. These molecules are likely to be the sites of the ATP hydrolysis required for precursor proteins to bind to and be translocated across the two membranes of the chloroplast envelope. Over the past decade, several different chaperones have been identified, based mainly on their association with precursor proteins and/or components of the chloroplast import complex, as putative factors mediating chloroplast protein import. These factors include cytoplasmic, chloroplast envelope-associated and stromal members of the Hsp70 family of chaperones, as well as stromal Hsp100 and Hsp60 chaperones and a cytoplasmic 14-3-3 protein. While many of the findings regarding the action of chaperones during chloroplast protein import parallel those seen for mitochondrial and endoplasmic reticulum protein transport, the chloroplast import system also has unique aspects, including its hypothesized use of an Hsp100 chaperone to drive translocation into the organelle interior. Many questions concerning the specific functions of chaperones during protein import into chloroplasts still remain that future studies, both biochemical and genetic, will need to address.     

Diverse effects of ethanol on the pathway of inducible prostaglandin E2 production in macrophages.

The effects of ethanol on inducible prostaglandin production in RAW macrophages were investigated. Indomethacin (1 microM) or cycloheximide (1 microM) abolished prostaglandin E2 (PGE2) production induced by lipopolysaccharide (LPS, 1 microg/ml). Ethanol at concentrations from 100 mM to 600 mM concentration-dependently inhibited inducible PGE2 production, while ethanol only at higher concentrations (400 mM or more) showed cytotoxity to the cells. Cyclooxygenase-2 (COX-2) activity, estimated by transformation of exogenous arachidonic acid into PGE2, was not affected by ethanol (100-400 mM). LPS-induced expression of COX-2 mRNA was inhibited by ethanol (50-400 mM). On the other hand, protein expression of COX-2 by LPS was significantly increased by ethanol (100-400 mM). Ethanol alone at concentrations up to 600 mM did not induce expression of COX-2 protein. In a medium containing arachidonic acid (1 microM), ethanol at a low concentration (100 mM) did not significantly affect LPS-induced PGE2 production. These results suggest that ethanol shows diverse effects on the pathway of inducible PGE2 production in macrophages. Finally, ethanol may suppress utilization of arachidonic acid, resulting in reduction of inducible PGE2 production. Further study is needed to elucidate the mechanism of dissociation of ethanol effects on protein and mRNA expression.     

Power spectral analysis of heart rate variability for assessment of diurnal variation of autonomic nervous activity in guinea pigs.

We established characteristics of power spectral analysis of heart rate variability, and assessed the diurnal variations of autonomic nervous function in guinea pigs. For this purpose, an electrocardiogram (ECG) was recorded for 24 hr from conscious and unrestrained guinea pigs using a telemetry system. There were two major spectral components, at low frequency (LF) and high frequency (HF) bands, in the power spectrum of HR variability. On the basis of these data, we defined two frequency bands of interest: LF (0.07-0.7 Hz) and HF (0.7-3.0 Hz). The power of LF was higher than that of HF in the normal guinea pigs. Atropine significantly reduced power at HF. Propranolol also significantly reduced power at LF. Furthermore, the decrease in the parasympathetic mechanism produced by atropine was reflected in a slight increase in the LF/HF ratio. The LF/HF ratio appeared to follow the reductions of sympathetic activity produced by propranolol. Autonomic blockade studies indicated that the HF component reflected parasympathetic activity and the LF/HF ratio seemed to be a convenient index of autonomic balance. Nocturnal patterns, in which the values of heart rate in the dark phase (20:00-06:00) were higher than those in the light phase (06:00-20:00), were observed. However, the HF, LF and the LF/HF ratio showed no daily pattern. These results suggest that the autonomic nervous function in guinea pigs has no clear circadian rhythmicity. Therefore, this information may be useful for future studies concerning the autonomic nervous function in this species.     

The gene for alternative oxidase-2 (AOX2) from Arabidopsis thaliana consists of five exons unlike other AOX genes and is transcribed at an early stage during germination.

We investigated the expressions of genes for alternative oxidase (AOX1a, AOX1b, AOX1c and AOX2) and genes for cytochrome c oxidase (COX5b and COX6b) during germination of Arabidopsis thaliana, and examined oxygen uptakes of the alternative respiration and the cytochrome respiration in imbibed Arabidopsis seeds. A Northern blot analysis showed that AOX2 mRNA has already accumulated in dry seeds and subsequently decreased, whereas accumulation ofAOX1a mRNA was less abundant from 0 hours to 48 hours after imbibition and then increased. The increase of the capacity of the alternative pathway appeared to be dependent on the expressions of both AOX2 and AOX1a. On the other hand, steady-state mRNA levels of COX5b and COX6b were gradually increased during germination, and the capacity of the cytochrome pathway was correlated with the increase of expressions of the COX genes. Antimycin A, the respiratory inhibitor, strongly increased the expression of AOX1a but had no effect on the expression of AOX2. A 5'RACE analysis showed that AOX2 consists of five exons, which is different from the case of most AOX genes identified so far. Analysis of subcellular localization of AOX2 using green fluorescent protein indicated that the AOX2 protein is imported into the mitochondria.     

A Theoretical Model of Jigsaw-Puzzle Pattern Formation by Plant Leaf Epidermal Cells

Plant leaf epidermal cells exhibit a jigsaw puzzle–like pattern that is generated by interdigitation of the cell wall during leaf development. The contribution of two ROP GTPases, ROP2 and ROP6, to the cytoskeletal dynamics that regulate epidermal cell wall interdigitation has already been examined; however, how interactions between these molecules result in pattern formation remains to be elucidated. Here, we propose a simple interface equation model that incorporates both the cell wall remodeling activity of ROP GTPases and the diffusible signaling molecules by which they are regulated. This model successfully reproduces pattern formation observed in vivo, and explains the counterintuitive experimental results of decreased cellulose production and increased thickness. Our model also reproduces the dynamics of three-way cell wall junctions. Therefore, this model provides a possible mechanism for cell wall interdigitation formation in vivo.     

Immunohistochemical evidence for the existence of rat cytosolic sialidase in rat skeletal muscles

 Histochemical evidence is required to demonstrate the presence of biochemically defined cytosolic sialidase. To meet this requirement, we examined the immunohistochemical localization of the enzyme in rat skeletal muscles. Sections of chemically fixed tissues were incubated with a polyclonal antibody raised against a synthetic peptide which corresponded to a part of the enzyme protein. After incubation with the primary antibody, cryosections for fluorescence microscopy and resin sections for electron microscopy were incubated with a fluorochrome- and colloidal gold-labeled secondary antibody, respectively. Immunofluorescence was diffusely distributed in the muscle fibers and was also found in the perimysium and blood vessels. Many immunogold particles were scattered over the sarcoplasm, myofibrils, nucleoplasm, and matrix of mitochondria. The immunogold particles were also found in the equivalent compartments of axons, Schwann cells, and cells of endomysium and blood vessels. The specificity of the primary antibody was elucidated by immunoblotting and an immunoprecipitation test. These findings clearly indicate that this type of sialidase is essentially located in the cytosolic compartment. Consequently, the name, cytosolic sialidase, will be appropriate for this enzyme. Additionally it is indicated that this enzyme is also present in cells other than skeletal muscle fibers. Accepted: 29 January 1997