Thermostabilization of protein A-luciferase fusion protein by single amino acid mutation

A gene expression plasmid, pMALU7, for coding a fusion protein between protein A (SpA) and mutated firefly luciferase (Luc), was constructed. The fused gene was expressed in Escherichia coli and the resulting protein (SpA-LucTS) was purified with affinity chromatography. By changing a single amino acid, from Glu to Lys at the position 354 in the luciferase moiety, the thermostability of luciferase was improved.     

Hyperproduction of a bifunctional hybrid protein, metapyrocatechase-protein A, by gene fusion.

A hybrid protein between metapyrocatechase and Staphylococcal protein A was produced by recombinant DNA techniques. A plasmid carrying the fusion gene that encodes the hybrid protein was constructed and expressed in E. coli. Over 70% of soluble proteins of the cell extracts was estimated to be the hybrid protein. This fusion protein is about 65,000. Both the IgG-binding activity of protein A and the metapyrocatechase activity were found in the hybrid protein. The optimum pH of metapyrocatechase in the fusion protein was at around 6.5 and Km was 1.3 X 10(-5) M. A simple immuno-enzymometric assay was developed for anti-BSA antibody using the fusion protein.     

Negative Phototaxis from Blue Light and the Role of Third Rhodopsinlike Pigment in Halobacterium Cutirubrum

Wild-type cells of Halobacterium cutirubrum show phototaxis. In negative phototaxis the cells are repelled by blue-near ultraviolet light, and in positive phototaxis the cells are attracted to green-red light. The extent of the responses are measured by monitoring the changes in the reversal frequency of the swimming direction of cells using a computer-linked automated method as described previously (Takahashi, T., and Y. Kobatake, 1982, Cell. Struct. Funct., 7:183-192). When the intensity of the background light (illumination for the observation) was dramatically reduced, the sensitivity of the cells to the repellent light decreased markedly. This result has been previously explained by Bogomolni and Spudich (1982, Proc. Natl. Acad. Sci. USA, 79:6250-6254), who proposed that the photoreceptor for negative phototaxis is the long-lifetime intermediate in the photocycle of slow-rhodospin. The behavioral response in the negative phototaxis is dependent upon the intensity of the actinic light and the background light. This agrees quantitatively with our model based on the aforementioned hypothesis.     

Effects and mechanisms of prolongevity induced by Lactobacillus gasseri SBT2055 in Caenorhabditis elegans

Lactic‐acid bacteria are widely recognized beneficial host associated groups of the microbiota of humans and animals. Some lactic‐acid bacteria have the ability to extend the lifespan of the model animals. The mechanisms behind the probiotic effects of bacteria are not entirely understood. Recently, we reported the benefit effects of Lactobacillus gasseriSBT2055 (LG2055) on animal and human health, such as preventing influenza A virus, and augmentation of IgA production. Therefore, it was preconceived that LG2055 has the beneficial effects on longevity and/or aging. We examined the effects of LG2055 on lifespan and aging of Caenorhabditis elegans and analyzed the mechanism of prolongevity. Our results demonstrated that LG2055 has the beneficial effects on longevity and anti‐aging of C. elegans. Feeding with LG2055 upregulated the expression of the skn‐1 gene and the target genes of SKN‐1, encoding the antioxidant proteins enhancing antioxidant defense responses. We found that feeding with LG2055 directly activated SKN‐1 activity via p38 MAPK pathway signaling. The oxidative stress response is elicited by mitochondrial dysfunction in aging, and we examined the influence of LG2055 feeding on the membrane potential of mitochondria. Here, the amounts of mitochondria were significantly increased by LG2055 feeding in comparison with the control. Our result suggests that feeding with LG2055 is effective to the extend lifespan in C. elegans by a strengthening of the resistance to oxidative stress and by stimulating the innate immune response signaling including p38MAPK signaling pathway and others.     

A theory of dynamic and steady responses in chemoreception

A theoretical model which can account for both the dynamic and steady responses is proposed based on the occupation theory. The reaction scheme used is;

Here, S and A are stimulus chemicals and receptor sites unbound, respectively. The binding of S to A leads to an active complex (SA)_active, which is successively transformed into an inactive complex (SA)_active. The response is assumed to be proportional to number of (SA)_active. When a stimulating solution is applied instantaneously at t = 0, the solution to the set of differential equations based on the above scheme is obtained as follows;

where p and C stand for the fraction of (SA)_active to the total number of receptor sites and stimulus concentration, respectively, and α_i, and ω_i (i = 1, 2) are numerical parameters depending on the rate constants and on C. The steady response is expressed as the third term in the above equation, which indicates that the response accords with the Beidler taste equation. Mathematical analysis of the above scheme shows that the dynamic response appears when k₁C > k_?2, and the calculated results for the dynamic response agree approximately with the Hill equation. The Hill coefficient lays within 1·00 and 0·79 and reaches unity with increasing $\text{k}{}_{\mathrm{?1}}\text{k}{}_2$, which implies that the dynamic response under this condition satisfies the Beidler taste equation. For the case of gradual application of stimuli, i.e. the experimental condition, the time course of p is simulated with use of an analogue computer rather than with a numerical solution to the above equation. The results indicate that the dynamic response diminishes with decreasing the application speed of stimulus solution. The present theory accounts consistently for various experimental data observed in the chemoreceptor systems.     

Marked enhancement of acyl-CoA synthetase activity and mRNA, paralleled to lipoprotein lipase mRNA, in adipose tissues of Zucker obese rats (fa/fa).

To clarify the role of acyl-CoA synthetase in development of obesity, the mRNA levels and activities were studied in Zucker fatty rats (fa/fa). In Zucker fatty rats compared with their lean littermates, marked enhancement of ACS were observed in adipose tissues. Obese/lean rats ratio of ACS activity and mRNA in abdominal subcutaneous fat (3.3- and 3.9-fold, respectively) were greater than in mesenteric fat (2.0- and 2.2-fold). The enhancement of ACS activity and mRNA in the liver of fatty rats (1.2- and 1.8-fold) were less than those in the adipose tissues. There were no enhancement of ACS activities and mRNA levels in heart tissue of the obese rats. LPL mRNA levels were also enhanced in adipose tissue of fatty rats and obese/lean ratio of LPL mRNA was also higher in abdominal subcutaneous fat than mesenteric fat (6.2- vs 3.1-fold). The larger obese/lean rats ratio of LPL and ACS parameters in abdominal subcutaneous fat than mesenteric fat may be related to the observation that the increase of subcutaneous fat weight was larger than that of mesenteric fat weight in fatty rats (21.1- vs 4.9-fold). Integrated enhancement of LPL and ACS gene expression in adipose tissue may play an important role in the development of obesity.     

Mitochondrial membrane potential estimated with the correction of probe binding

Lipophilic ions are widely used as the probe for estimation of the membrane potential. It is suggested that the correction of the probe binding to the membrane and/or intracellular constituents is a problem to be solved in order to evaluate the membrane potential accurately. Previously, we proposed a method for the correction of the probe binding (Demura, M., Kamo, N. and Kobatake, Y. (1985) Biochim. Biophys. Acta 820, 207-215). In this paper, the method was applied to the determination of the membrane potential of intact mitochondria. The probes used constitute a homologous series of (Phe)3-P+-(CH2)n-CH3 (n = 0-4) and tetraphenylphosphonium (TPP+). Binding of these probes to de-energized mitochondria followed the Langmuir isotherm. However, values of parameters determined at high (50-800 microM) and low (under 20 microM) probe concentrations were different, suggesting the existence at least two, high- and low-affinity, binding sites. With extrapolation to the 'state of no binding', the membrane potential of intact mitochondria was estimated to be -147 mV (interior-negative) when they were energized by 5 mM succinate in medium consisting of 125 mM KCl, 10 mM MgCl2, 5 mM phosphate, 0.4 mM EDTA and 50 mM Tris-HCl (pH 7.5) at 25 degrees C. Parameters appearing in the equation for the correction of probe binding were determined with the use of this value of the membrane potential. The validity of the equation and the value of the parameters were revealed by the fact that after the correction, all probes used gave approximately the same value under the same conditions. We expanded the method so as to include the langmuir adsorption isotherm. When the modified equation is used, the estimated membrane potentials were less dependent on a probe concentration less than 10 microM.     

Patterns in the distribution of intracellular ATP concentration in relation to coordination of amoeboid cell behavior in Physarum polycephalum

The Physarum plasmodium reacts tactically to external stimuli. The cell behavior of this giant amoeboid cell was studied by analysing intracellular ATP concentration. The two-dimensional (2D) spatial distribution of ATP depended on cell shape: a polar pattern for a unidirectionally migrating plasmodium, a bowl shape for a circular plasmodium, a hump shape for an oval plasmodium, or a wavy pattern for plasmodia stimulated with blue light or confined in a small chamber, etc. Local external stimulation brought about new patterns of ATP distribution. The ATP concentrations around the stimulated frontal region were reduced by about a half stimulation with KCl (repellent) or casamino acids (attractant). In both cases, migration was inhibited. Migration velocity increased almost linearly with increasing concentration of intracellular ATP above the threshold (about 20 micrograms/mg protein). Under anaerobic conditions or at low temperatures, the intracellular ATP oscillated slowly with a periodicity of about 30 min. Pattern formations in the intracellular ATP concentration and amoeboid coordination are discussed in terms of coupled chemical oscillators in a self-organizing system.     

Intracellular delivery of antibodies using TAT fusion protein A

Internalization of antibodies into mammalian cells is a useful method for analyzing and regulating cellular function. In this study, we developed a novel method for the delivery of antibodies into cells using the TAT-fused protein. This fusion protein consists of two functional domains, the protein transduction domain of HIV-1 TAT and the B domain of staphylococcal protein A (SpA), which has an ability to bind to the IgG. The TAT-SpA fusion protein was mixed with fluorescence-labeled rabbit IgG and added to cells. The internalization of antibody was analyzed using confocal microscopy and flow cytometry in living cells. As a result, fluorescence-labeled IgG with the TAT-SpA fusion protein was observed intracellularly. Flow cytometry results demonstrated time course and dose dependence relationships of antibody internalization. These results suggest that the TAT-SpA fusion protein can be a useful reagent for the delivery of antibody into cells.