A rapid staining technique for the detection of the initiation of germination of bacterial spores

AIMS: We propose to apply the Wirtz-Conklin staining technique to evaluate spore germination. METHODS AND RESULTS: Spores at different stages of germination were stained with modified spore stain (Wirtz-Conklin) and evaluated for staining properties. Bacillus spores suspended in deionized water, which does not support germination, stained greenish-blue. Spores suspended in germination enhancers that did not form bacilli stained pink, indicating the initiation of germination. Spores suspended in culture media, which promotes bacterial outgrowth, formed bacilli and were also stained pink. CONCLUSIONS: Modified spore stain (Wirtz-Conklin) was found to be useful to detect the initiation of spore germination as early as 30 min following incubation in a germination environment. SIGNIFICANCE AND IMPACT OF THE STUDY: This simple staining procedure is useful in detecting the initiation of germination of bacterial spores.     

Sol-gel encapsulation of lactate dehydrogenase for optical sensing of L-lactate

Sol-gel encapsulation of lactate dehydrogenase and its cofactor can be employed as a disposable sensor for L-lactate. The sensor utilized the changes in absorbance or fluorescence from the reduced cofactor nicotinamide adenine dinucleotide (NADH) upon exposure to L-lactate. Although, problems such as diminished enzymatic activity and/or leaching of enzyme from the sol-gel matrix occurred, the sol-gel process is sufficiently mild to permit retention of enzymatic activity. The apparent activity of LDH in the sensor is at least 10% of that of the dissolved enzyme. The sensor has a linear dynamic range over the normal physiological L-lactate level and has a long-term storage stability of at least 3 weeks.     

Exposure to oxidized low-density lipoprotein reduces activable Ras protein in vascular endothelial cells

Oxidized low-density lipoprotein (ox-LDL) has been shown to alter the migratory and proliferative activities of the vascular endothelial cells (EC) in response to serum and growth factors. The mechanism underlying the antiproliferative effect of ox-LDL on vascular EC has not been fully elucidated. In this report, we show that exposure of vascular EC to ox-LDL results in a marked reduction of the membrane-associated Ras protein. Further study shows that in ox-LDL-treated EC, reduction of the membrane-associated Ras protein is correlated with a reduced amount of active Ras (Ras-guanosine triphosphate), indicating that the Ras signaling pathway is attenuated. The attenuation of the Ras signaling pathway in ox-LDL-treated EC may thus be responsible for the retarded response to the mitogenic stimulation of serum and growth factors.     

A salvianolic acid B-rich fraction of Salvia miltiorrhiza induces neointimal cell apoptosis in rabbit angioplasty model

Apoptosis has been suggested to participate in stabilizing cell number in restenosis. Salvia miltiorrhiza (SM) Bunge which is a Chinese herb widely used for the treatment of cardiovascular disorders contains a potent antioxidant, Salvianolic acid B. To determine whether the antioxidant affects vascular apoptosis, the present study examined the frequency of apoptotic cell death in atherosclerotic plaques and in restenotic lesions of cholesterol-fed rabbits. New Zealand White rabbits were treated with a normal diet (normal), a 2% cholesterol diet (HC), a 2% cholesterol diet and endothelial denudation (HC-ED), a 2% cholesterol diet with 5% water-soluble extract of SM (4.8 g/Kg B.W./day) and endothelial denudation (HC-ED-SM), or with a 2% cholesterol diet containing probucol (0.6 g/kg B.W./day) and endothelial denudation (HC-ED-probucol). Apoptosis and associated cell types were examined in serial paraffin sections by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and immunohistochemistry. The expression of p53, an apoptosis-related protein, was also examined. Apoptosis was mainly detected in the neointima of the three groups with endothelial denudation. The percentage of apoptotic cells in SM-treated group (68.5+/-5.9%) was significantly higher than that of normal (0%), HC (1.9+/-1.2%), HC-ED (46.1+/-5.4%), and probucol-treated (32.8+/-3.9%) groups. The SM treatment markedly reduced the thickness of the neointima which was mainly composed of smooth muscle cells with few macrophages. In accordance with the apoptotic cell counts, positive immunoreactivity for p53 was observed in restenotic lesions from HC-ED, SM-treated and probucol-treated groups but not in the intima of the other two groups. These results suggest that the treatment with salvianolic acid B-rich fraction of SM induces apoptosis in neointima which in turn may help prevent the neointimal thickening.     

Monoubiquitin carries a novel internalization signal that is appended to activated receptors

Ubiquitin modification of signal transducing receptors at the plasma membrane is necessary for rapid receptor internalization and downregulation. We have investigated whether ubiquitylation alters a receptor cytoplasmic tail to reveal a previously masked internalization signal, or whether ubiquitin itself carries an internalization signal. Using an alpha-factor receptor-ubiquitin chimeric protein, we demonstrate that monoubiquitin can mediate internalization of an activated receptor that lacks all cytoplasmic tail sequences. Furthermore, fusion of ubiquitin in-frame to the stable plasma membrane protein Pma1p stimulates endocytosis of this protein. Ubiquitin does not carry a functional tyrosine- or di-leucine-based internalization signal. Instead, the three-dimensional structure of the folded ubiquitin polypeptide carries an internalization signal that consists of two surface patches surrounding the critical residues Phe4 and Ile44. We conclude that ubiquitin functions as a novel regulated internalization signal that can be appended to a plasma membrane protein to trigger downregulation.     

Expression of SpC3, the sea urchin complement component,in response to lipopolysaccharide

The homologue of the vertebrate complement component C3 that is expressed in the coelomocytes of the purple sea urchin, Strongylocentrotus purpuratus, designated SpC3, was investigated for changes in response to immune challenge or injury. Immunoquiescent animals were used in this study because they have reduced or no detectable SpC3 in their coelomocytes or coelomic fluid (CF). Animals were injected with lipopolysaccharide (LPS) or sterile sea water (SSW, injury control). Changes in the amounts of SpC3 in coelomic fluid and in coelomocytes were then followed over time by Western blots and ELISA. Changes in mRNA from the SpC3 gene (Sp064) were also followed by RT-PCR. Although all animals responded to injury with increased levels of SpC3 in the coelomic fluid, those challenged with LPS had greater amounts of SpC3 in both CF and coelomocytes than those receiving SSW. In most of the animals receiving LPS, initial increases in SpC3 were observed within 1 h post-injection, while the earliest response in the animals receiving SSW was 6 h. The appearance of SpC3 in the coelomocytes was delayed compared to its appearance in CF, and was first detected several days after challenge. Changes in mRNA from the Sp064 gene paralleled the appearance of SpC3 in the coelomic fluid. Increases in the number of coelomocytes per milliliter of CF and in the percentage of coelomocytes that were SpC3+ also occurred after challenge with LPS or in response to injury, with a slightly greater increase in response to LPS. Although the changes in SpC3 were not as great as those identified previously for human C3 expressed in macrophages, the kinetics of the response are similar to that of acute-phase reactants in mammals.