Spindle pole body movement is affected by glucose and ammonium chloride in fission yeast.

The complexity of chromatin dynamics is orchestrated by several active processes. In fission yeast, the centromeres are clustered around the spindle pole body (SPB) and oscillate in a microtubule- and adenosine triphosphate (ATP)-dependent manner. However, whether and how SPB oscillation are affected by different environmental conditions remain poorly understood. In this study, we quantitated movements of the SPB component, which colocalizes with the centromere in fission yeast. We found that SPB movement was significantly reduced at low glucose concentrations. Movement of the SPB was also affected by the presence of ammonium chloride. Power spectral analysis revealed that periodic movement of the SPB is disrupted by low glucose concentrations. Measurement of ATP levels in living cells by quantitative single-cell imaging suggests that ATP levels are not the only determinant of SPB movement. Our results provide novel insight into how SPB movement is regulated by cellular energy status and additional factors such as the medium nutritional composition.

Handling unstable analytes: literature review and expert panel survey by the Japan Bioanalysis Forum Discussion Group.

Analyzing unstable small molecule drugs and metabolites in blood continues to be challenging for bioanalysis. Although scientific countermeasures such as immediate cooling, immediate freezing, addition of enzyme inhibitors, pH adjustment, dried blood spot or derivatization have been developed, selecting the best practices has become an issue in the pharmaceutical industry as the number of drugs with such problems is increasing, even for generic drugs. In this study, we conducted a comprehensive literature review and a questionnaire survey to determine a suitable practice for evaluating instability and implementing countermeasures. Three areas of focus, matrix selection, effect of hemolysis and selection of esterase inhibitors, are discussed.

Effect of sinusoidal leg cycling exercise period on brachial artery blood flow dynamics in humans.

PURPOSE: To quantify the dynamics of blood flow in brachial artery (BF-BA) in response to sinusoidal work rate (WR) leg cycling exercises of 2-, 4-, and 6-min periods and to examine their relationship with the forearm skin blood flow (SBF). METHODS: Seven healthy young male subjects performed upright leg ergometer exercise with a constant WR (mean sinusoidal WR) for 30 min followed by sinusoidal WR exercise of three different periods (number of repetitions): 2 min (7), 4 min (4), and 6 min (3). The WR fluctuated from 20 W to a peak WR corresponding to 60% peak oxygen uptake (VO2). We continuously measured pulmonary gas exchange, heart rate (HR), blood velocity and cross-sectional area of BA, and forearm SBF and sweating rate (SR). RESULTS: All variables were followed by the sinusoidal WR. The phases of the variables for gas exchange and central circulation, such as VO2 and HR with WR forcing were similar (e.g., phase shift (θ) in HR [°]: 2 min, 60 ± 7; 4 min, 45 ± 10; 6 min, 37 ± 8; mean ± SD) to previous study results, that is, a longer period showed a shorter θ and larger amplitude of responses. Contrarily, the BF-BA response showed anti-phase (approximately 180°) regardless of the period, whereas the θ of forearm SBF and SR were similar to gas exchange and central circulation. CONCLUSIONS: Inactive limb BF-BA during sinusoidal leg cycling exercise was out of phase relative to the regulation of O2-delivery to active muscles and thermoregulation. The response of BF-BA seems to not always reflect the response of forearm SBF in the downstream area.

Eliminating Lipopolysaccharide (LPS) Using Lactic Acid Bacteria (LAB) and a Fraction of its LPS-Elimination Protein.

Lactic acid bacteria (LAB) can improve human intraintestinal conditions. One reason is that ingestion of LAB prevents bacterial diarrhea. Furthermore, inflammation of human intestines can be caused by a lipopolysaccharide (LPS) component in the cell walls of gram-negative bacteria. This chapter describes a method of LPS elimination using lactic acid bacteria (LAB). First, the LPS concentration is assayed using an LPS assay kit with the limulus cascade reaction made by limulus amebocyte lysate. Some LABs, four bacillus strains and one coccus strain, have LPS-elimination activity. Particularly, the coccus strain Pediococcus pentosaceus eliminates LPS to 43%. The cells fractionate and eliminate four fractions: the extracellular fraction, cell membrane fraction, cytoplasm fraction, and cell wall fraction. Only the cell wall digesting fraction eliminates LPS to 45%. Results confirm that the LAB eliminates all LPS having O-antigen under a low-sugar medium condition at temperatures of 15-30 °C. This method can be used for assay of LPS elimination by LABs exactly and easily for the probiotics field.

Neutralization of Lipopolysaccharide by Heat Shock Protein in Pediococcus pentosaceus AK-23.

About 1000 species of bacteria are present in the human intestine. Some Gram-negative bacteria such as Escherichia coli or Salmonella spp. among intestinal bacteria have lipopolysaccharide (LPS), which might induce inflammation of human intestines. Actually, LPS, especially its lipid A constituent, is toxic. Small amounts of LPS in bacteria cause inflammation of mucosa and other tissues in humans. Such bacteria may be regulated by beneficial lactic acid bacteria to maintain human health. Many lactic acid bacteria show cancer prevention activity and anti-inflammatory activity in intestines. Recently, Pediococcus pentosaceus AK-23 was isolated from fermentative vegetable pickles for neutralization of LPS. For this study, a protein for LPS neutralization was purified partly from P. pentosaceus AK-23. For this study, a protein for LPS neutralization was purified partly from P. pentosaceus AK-23, by ultrafiltration using a 300 kDa membrane and a 100 kDa membrane after cell wall digestion by lysozyme. Gel running blue native electrophoresis revealed the existence of a 217 kDa protein. The band of the protein having the ability to bind LPS on the gel was analyzed for amino acid homology. As the result, it is revealed as part of a subunit of heat shock protein (HSP). Furthermore, it displayed LPS binding or hydrophobic motifs. The protein neutralized LPS to release fatty acid as myristic acid and glucose from polysaccharide. These findings suggest that HSP in P. pentosaceus AK-23 neutralizes LPS to decompose it compising fatty acid and polysaccharide.

Brachial artery blood flow dynamics during sinusoidal leg cycling exercise in humans.

To explore the control of the peripheral circulation of a nonworking upper limb during leg cycling exercise, blood flow (BF) dynamics in the brachial artery (BA) were determined using a sinusoidal work rate (WR) exercise. Ten healthy subjects performed upright leg cycling exercise at a constant WR for 30 min, followed by 16 min of sinusoidal WR consisting of 4-min periods of WR fluctuating between a minimum output of 20 W and a maximum output corresponding to ventilatory threshold (VT). Throughout the protocol, pulmonary gas exchange, heart rate (HR), mean arterial blood pressure (MAP), blood velocity (BV), and cross-sectional area of the BA, forearm skin BF (SBF), and sweating rate (SR) were measured. Each variable was fitted to a sinusoidal model with phase shift (θ) and amplitude (A). Nearly all variables closely fit a sinusoidal model. Variables relating to oxygen transport, such as VO2 and HR, followed the sinusoidal WR pattern with certain delays (θ: VO2; 51.4 ± 4.0°, HR; 41.8 ± 5.4°, mean ± SD). Conversely, BF response in the BA was approximately in antiphase (175.1 ± 28.9°) with a relatively large A, whereas the phase of forearm SBF was dissimilar (65.8 ± 35.9°). Thus, the change of BF through a conduit artery to the nonworking upper limb appears to be the reverse when WR fluctuates during sinusoidal leg exercise, and it appears unlikely that this could be ascribed exclusively to altering the downstream circulation to forearm skin.

Isolation of Endotoxin Eliminating Lactic Acid Bacteria and a Property of Endotoxin Eliminating Protein.

Recently, many scholars have reported lactic acid bacteria (LAB) functions, such as anticancer activity and anti-inflammatory activity for intestines. To decrease inflammatory substances such as endotoxins, LAB consumed safely with meals were isolated from food and food ingredients. First, LAB were isolated as 168 strains of bacillus LAB (49 strain) and coccus LAB (119 strains) from food ingredients and fermented foods such as rice, rice bran, malt, grains, miso soy paste, and some pickles. Their LAB (168 strains) were cultivated in medium containing endotoxin from Escherichia coli O18 LPS at 15 and 30 °C for 64 h to identify endotoxin-eliminating LAB. Consequently, the AK-23 strain was screened as an endotoxin-eliminating LAB strain. The strain decreased endotoxin in YP medium without sugar at 30 °C for 64 h until 9% of endotoxin. The strain was identified as Pediococcus pentosaceus according to morphological characteristics such as its cell shape, physiological characteristics related to its fermentation type, assimilation of sugars, pH tolerance, optimum growth temperature, and molecular biological characteristics as its homology to 16S rRNA. To investigate the location of the endotoxin-eliminating substance, 4 fractions were separated from AK-23 cells as extracellular, cell wall digestion, cytoplasm, and cell membrane fractions. The endotoxin-decreasing substance, located on a cell wall, was identified as a 217 kDa protein.