Evidence for a structural mutation (347Ala to Thr) in a German family with 3-ketothiolase deficiency.

The molecular basis of 3-ketothiolase deficiency (3KTD) was examined in a 3KTD family. Immunochemical analyses showed that mitochondrial acetoacetyl-CoA thiolase (T2) biosynthesized in the patient's fibroblasts (GK06) was unstable and that the parents and brother were obligatory carriers of 3KTD. When sequencing the PCR-amplified patient's T2 cDNA, we noted a G to A replacement which caused 347Ala to Thr substitution of the mature T2 subunit. Transfection analysis revealed that this substitution resulted in an instability of the T2 protein. Analyses of the T2 cDNA and gene of the family indicated that the patient was a compound heterozygote; the allele that derived from the mother had a point mutation (347Ala to Thr) and the other allele from the father has a mutation which would abolish the T2 gene expression. This report is apparently the first definition of a mutant allele for 3KTD, at the gene level.     

Different effects of DNA adducts induced by carcinogenic and noncarcinogenic azo dyes on in vitro DNA synthesis

M13mp10 phage DNA modified with the carcinogen 3-methoxy-4-aminoazobenzene (3-MeO-AAB) or the noncarcinogen 2-methoxy-4-aminoazobenzene (2-MeO-AAB) was used as a template for E.coli DNA polymerase I. Analysis of the reaction products on DNA sequencing gels showed that with both types of compound the induced lesions blocked DNA synthesis, mainly at one base prior to guanine adducts, but that the inhibition by 3-MeO-AAB-adducts was substantially greater than that by 2-MeO-AAB-adducts. Thus different effects on DNA replication between 3-MeO-AAB- and 2-MeO-AAB-adducts might be a reflection of differences in their carcinogenic potency.     

Cloning and characterization of an amidase gene from Rhodococcus species N-774 and its expression in Escherichia coli

For investigation of an unknown open reading frame which is present upstream of the nitrile hydratase (NHase) gene from Rhodococcus sp. N-774, a longer DNA fragment covering the entire gene was cloned in Escherichia coli. Nucleotide sequencing and detailed subcloning experiments predicted a single open reading frame consisting of 521 amino acid residues of Mr 54,671. The amino acid sequence, especially its NH2-terminal portion, showed significant homology with those of indoleacetamide hydrolases from Pseudomonas savastanoi and Agrobacterium tumefaciens, and acetamidase from Aspergillus nidulans. The 521-amino acid coding region was therefore expressed by use of the E. coli lac promoter in E. coli, and was found to direct a considerable amidase activity. This amidase hydrolyzed propionamide efficiently, and also hydrolyzed, at a lower efficiency, acetamide, acrylamide and indoleacetamide. These data clearly show that the unknown open reading frame present upstream of the NHase coding region encodes an amidase. Because the TAG translational stop codon of the amidase is located only 75 base pairs apart from the ATG start codon of the alpha-subunit of NHase, these genes are probably translated in a polycistronic manner.     

A new method for quantifying keratinocyte differentiation using immunofluorescent staining of involucrin and cytofluorography

Involucrin is a precursor protein of detergent-insoluble cornified envelope and a marker of terminal differentiation of epidermal keratinocytes. To quantify differentiation of cultured human keratinocytes, the population of involucrin-positive cells was estimated by immunofluorescent staining using anti-involucrin antibody and flow cytometry. Normal human keratinocytes were cultured under three conditions for induction of differentiation: low Ca2+ concentration (0.1 mM Ca2+), high Ca2+ concentration (1.8 mM Ca2+), and high Ca2+ concentration with 10% fetal bovine serum (FBS). The relationship between fluorescence intensity and involucrin synthesis was confirmed by visual examination of sorted cells. The population of involucrin-positive cells increased from 7.2 to 18.1% by elevating Ca2+ concentration and to 37.0% by adding FBS. The extent of cornified envelope formation under the same culture conditions was consistent with the estimation of involucrin-positive cells. The cytofluorographic analysis of involucrin synthesis made it possible to determine the number of differentiated cells in a large number of samples precisely and reliably. Thus, it is a useful method for quantifying keratinocyte differentiation.     

A locus controlling the activity of UDP-galactose:GM2(NeuGc) galactosyltransferase in mouse liver is linked to the H-2 complex

Genetic polymorphism in the expression of the G_M1(NeuGc) ganglioside has been shown in the liver of inbred strains of mice. Through analysis of the gangliosides of H-2 congenic and recombinant strains, this polymorphism was demonstrated to be controlled by a locus mapped left outside of the H-2 complex on chromosome 17, and the locus was assumed to control the level of the activity of G_M1(NeuGc) synthetase, UDP-galactose:G_M2(NeuGc) galactosyltransferase (E.C.2.4.1.62) [Hashimotoet al., J Biochem (1983) 94:2049-54].In the present study we analyzed the genetic linkage between the activity of the galactosyltransferase and the H-2 haplotype. For this purpose, we selected two inbred strains of mice, WHT/Ht and BALB/c, because they have different levels of the transferase activity and show different H-2 haplotypes; the specific activity of the transferase obtained with BALB/c was one-eighth of that with WHT/Ht, and BALB/c expressed the la.7 antigen as one of the products encoded in their H-2^d complex, whereas WHT/Ht did not. To analyze the linkage between these two phenotypes, WHT/Ht were mated with BALB/c to obtain the F₁ mice, and the female F₁ mice were then backcrossed to WHT/Ht. It was found that one half of the backcross generation expressed the la.7 antigen derived from BALB/c and had a significantly lower specific activity of the transferase than that of WHT/Ht, while the other half did not express the la.7 antigen but had the same specific activity of the transferase as that obtained with WHT/Ht.These results suggest that the locus controlling the level of the transferase activity in mouse liver is linked to the H-2 complex on chromosome 17.Abbreviations NeuGc N-glycolylneuraminic acid The ganglioside nomenclature is based on the system of Svennerholm, J Neurochem (1963) 10:613-23. The sialic acid species present is shown in parentheses after the ganglioside abbreviation.     

Roles of Ca2+ on the inositol 1,4,5-trisphosphate-induced release of Ca2+ from saponin-permeabilized single cells of the porcine coronary artery

The release of Ca2+ from the intracellular store site, as induced by inositol 1,4,5-trisphosphate, was studied in relation to free Ca2+ concentrations or amounts of stored Ca2+ in smooth muscle cells. The maximal Ca2+ release induced by inositol 1,4,5-trisphosphate was observed when the amount of Ca2+ in the store site was about 50% of the maximal capacity of the Ca2+ storage, and when the extravesicular free Ca2+ concentration was less than 1.5 X 10(-6) M. The Ca2+ release induced by inositol 1,4,5-trisphosphate was accelerated by ATP and 5'-adenylylimidodiphosphate (AMPPNP), but not by ADP and AMP. This inositol 1,4,5-trisphosphate-induced Ca2+ release appeared to be specific for intracellular Ca2+ store sites (mainly sarcoplasmic reticulum), and this Ca2+ release was not apparent in the sarcolemmal fraction.     

Cytotoxic factor production by kupffer cells elicited with Lactobacillus casei and Corynebacterium parvum

Summary The ability of Kupffer cells, spleen macrophages, pulmonary macrophages, and peritoneal macrophages (PM) to produce cytotoxic factor (CTF) was investigated in vitro. The production of CTF by Kupffer cells elicited with Corynebacterium parvum (CP) or Lactobacillus casei YIT9018 (LC9018) was higher than that of spleen, pulmonary macrophages, or PM. In addition, oxygen radical (OR) production by Kupffer cells or PM was measured. The production of OR by Kupffer cells or PM was significantly augmented by i.v. or i.p. injection of LC9018 or CP. No significant correlation was observed between the increase in OR production by Kupffer cells or PM and CTF production by Kupffer cells or PM elicited with either organism. It was suggested that activated Kupffer cells may be one important source of CTF production in serum and that the CTF-producing macrophages may be different from the OR-producing macrophages.     

<Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> mechanosensitive channels: towards unpuzzling “glutamate efflux” for amino acid production

Corynebacterium glutamicum has been utilized for industrial amino acid production, especially for monosodium glutamate (MSG), the food-additive for the “UMAMI” category of taste sensation, which is one of the five human basic tastes. Glutamate export from these cells is facilitated by the opening of mechanosensitive channels in the cell membrane within the bacterial cell envelope following specific treatments, such as biotin limitation, addition of Tween 40 or penicillin. A long-unsolved puzzle still remains how and why C. glutamicum mechanosensitive channels are activated by these treatments to export glutamate. Unlike mechanosensitive channels in other bacteria, these channels are not simply osmotic safety valves that prevent these bacteria from bursting upon a hypo-osmotic shock. They also function as metabolic valves to continuously release glutamate as components of a pump-and-leak mechanism regulating the cellular turgor pressure. Recent studies have demonstrated that the opening of the mechanosensitive channel, MscCG, mainly facilitates the efflux of glutamate and not of other amino acids and that the “force-from-lipids” gating mechanism of channels also applies to the MscCG channel. The bacterial types of mechanosensitive channels are found in cell-walled organisms from bacteria to land plants, where their physiological functions have been specialized beyond their basic function in bacterial osmoregulation. In the case of the C. glutamicum MscCG channels, they have evolved to function as specialized glutamate exporters.     

Knockdown of AMPKα decreases ATM expression and increases radiosensitivity under hypoxia and nutrient starvation in an SV40-transformed human fibroblast cell line,LM217

Background

Presence of unperfused regions containing cells under hypoxia and nutrient starvation contributes to radioresistance in solid human tumors. It is well known that hypoxia causes cellular radioresistance, but little is known about the effects of nutrient starvation on radiosensitivity. We have reported that nutrient starvation induced decrease of mTORC1 activity and decrease of radiosensitivity in an SV40-transformed human fibroblast cell line, LM217, and that nutrient starvation induced increase of mTORC1 activity and increase of radiosensitivity in human liver cancer cell lines, HepG2 and HuH6 (Murata et al., BBRC 2015). Knockdown of mTOR using small interfering RNA (siRNA) for mTOR suppressed radiosensitivity under nutrient starvation alone in HepG2 cells, which suggests that mTORC1 pathway regulates radiosensitivity under nutrient starvation alone. In the present study, effects of hypoxia and nutrient starvation on radiosensitivity were investigated using the same cell lines.

Effect of a warm‐up sample on stabilizing the performance of untrained panelists in time–intensity evaluation

To improve the reliability of sensory evaluation, it is necessary to minimize response bias. In this study, we investigated the effect of a warm‐up sample on time–intensity evaluation of after‐flavor. Untrained panelists evaluated continuously perceived intensity of bitterness and retronasal aroma, using four types of samples (two brands of coffee beverages × two types of lids). Half of participants evaluated bitterness intensity in the first session and retronasal aroma intensity in the second session, whereas the other half evaluated them in the opposite order. Each session consisted of four trials, and we regarded the first trial sample of each session as a warm‐up. Results revealed that perceived intensity was significantly lower in the first trial than in the following trials, and that the time courses of the second, third, and fourth trials were similar except when participants evaluated bitterness intensity in the second session. In other words, the use of a warm‐up sample stabilized the performance of time–intensity evaluation of bitterness and retronasal aroma in the first session. Furthermore, no warm‐up sample was needed in the second session when participants evaluated bitterness intensity, but a warm‐up was needed when they evaluated retronasal aroma intensity.

Practical application

Based on the results of this study, we concluded that the use of a warm‐up sample stabilized performance in time–intensity evaluation of after‐flavor by untrained panelists. We proposed that in order to obtain reliable performance in time–intensity evaluation of after‐flavor, untrained panelists should be provided a training trial using warm‐up sample before staring the test trials.