Prolongation of Oral Phase for Initial Swallow of Solid Food is Associated with Oral Diadochokinesis Deterioration in Nursing Home Residents in Japan: A Cross-Sectional Study.

Background: Prolongation of bolus forming complicates ingestion, in particular in older adults. Objectives: The purpose of this study is to examine which oral functions are associated with prolongation of the oral phase of forming a bolus until swallowing in older adults. Design: Cross-sectional study. Setting: three nursing homes in Kitakyushu, Japan from August 2017 to October 2018. Participants: 39 adults >60-years. Measurements: Number of functional teeth, chewing ability, swallowing ability, tongue and cheek pressure, saliva flow rate, oral diadochokinesis, global cognitive function, and body mass index, were examined. Time of oral phase until the first swallowing of solid food was measured as the outcome of the study using video, and audio recording of the swallowing sound by a throat microphone, with the cutoff point designated at 30 s. Based on the oral phase, participants were divided in two groups: normal and prolonged. Results: The 39 enrolled participants had a median age of 87 years, 17.3% were men, and 48.7% had prolonged oral phase. In the prolonged group, the swallowing ability, saliva flow rate, tongue and cheek pressure, and oral diadochokinesis were significantly lower than in the normal group. Binomial logistic regression analysis revealed that oral phase prolongation was associated with oral diadochokinesis (odds ratio 0.81, 95% confidence interval 0.67-0.98) after adjusting for potential covariates. Conclusion: Oral diadochokinesis deterioration is significantly associated with oral phase prolongation for initial swallowing of solid food in older adults.

Cloning and sequencing of the membrane-bound hydrogenase-encoding genes (hupS and hupL) from Pseudomonas hydrogenovora.

The membrane-bound hydrogenase (Hdg)-encoding structural genes were isolated from the hydrogen-utilizing bacterium Pseudomonas hydrogenovora (Ph). Nucleotide sequence analysis revealed two genes, hupS and hupL. The hupS gene encoded a 363-amino-acid (aa) polypeptide (40.4 kDa). The deduced aa sequence contained a putative 43-aa leader peptide sequence. The hupL gene started at 55 bp downstream from the stop codon of hupS and encoded a 622-aa polypeptide (69.3 kDa). The disruption of Ph hupS resulted in the loss of Hdg activity.

TH protein and mRNA in nigrostriatal dopaminergic neurons are down-regulated by continuous but not intermittent apomorphine.

Levels of tyrosine hydroxylase (TH) and TH mRNA were measured after administration of dopamine agonists for a long period of time to elucidate the long-term feedback inhibition of dopamine synthesis in nigrostriatal dopaminergic neurons. Continuous infusion, which desensitized presynaptic dopamine receptors, but not repeated administration, down-regulated TH and TH mRNA levels. This suggests levels of TH protein and mRNA are only feedback inhibited by the continuous stimulation of postsynaptic dopamine receptors.

Purification and properties of orotate phosphoribosyltransferases from Escherichia coli K-12, and its derivative purine-sensitive mutant.

Orotate phosphoribosyltransferase (OPT) was purified from both Escherichia coli K-12 strain and its derivative, a purine-sensitive mutant. The wild-type OPT had a molecular weight (M.W.) of 47,000 and was composed of two identical subunits (M.W. 23,500). The wild-type OPT showed maximum activity at pH 9.5, and no activity was seen in the absence of Mg2+ or Mn2+ ion. It also catalyzed a reverse reaction, namely orotidine-5'-monophosphate (OMP) pyrophosphorolysis. In this reverse reaction, tripolyphosphate, tetrapolyphosphate, and trimetaphosphate were also effective as pyrophosphate donors. The apparent Km values of the wild-type OPT were 30 microM for orotate and 40 microM for 5-phosphoribosyl 1-pyrophosphate (PRib-PP), and also 3.6 microM for OMP and 13 microM for PPi. On the other hand, the mutant OPT showed increased apparent Km values for all four substrates, 440 microM for orotate, 360 microM for PRib-PP, 33 microM for OMP, and 250 microM for PPi. The mutant OPT required a higher concentration of Mg2+ ion for maximum activity than the wild-type OPT. The nature of the purine-sensitive phenotype of the mutant is discussed from the standpoint of the reactivity of the mutant OPT, which has an increased Km value for PRib-PP (about 9-fold).

Enhanced formation of isoamyl alcohol in Zygosaccharomyces rouxii due to elimination of feedback inhibition of alpha-isopropylmalate synthase.

Mutants of the 'miso' yeast, Zygosaccharomyces rouxii, that produced a large amount of isoamyl alcohol, an important flavour in miso fermentation, were isolated from 5,5,5-trifluoro-DL-leucine-resistant mutants, an analogue of L-leucine. One of the mutants, M21-10, produced a three-fold higher level of isoamyl alcohol than the wild-type strain MY21 in miso fermentation. The activity of alpha-isopropylamalate synthase, one of the enzymes used for L-leucine synthesis, in the mutant M21-10 was not inhibited by the addition of L-leucine, a feedback inhibitor.

The hupC gene product is a component of the electron transport system for hydrogen oxidation in Pseudomonas hydrogenovora.

The hydrogenase gene cluster containing nine genes (hupSLCDFGHIJ) was identified by sequencing of an 8.8-kb DNA region from Pseudomonas hydrogenovora. To investigate the function of the hupC gene product, we isolated a hupC-null mutant (HID3) of P. hydrogenovora by introducing an in-frame deletion into the hupC. The mutant, HID3, could not grow autotrophically but retained half the level of hydrogenase activity of the wild-type strain. Results of the oxygen consumption test and Western blot analysis revealed that the hupC gene product is a b-type cytochrome but not involved in the hydrogenase maturation process.

Cloning and characterization of a new exo-cellulase gene, cel3, in Irpex lacteus.

A new cellulose-inducible gene (named cel3) was isolated from a strain of the white rot basidiomycete, Irpex lacteus MC-2. The cel3 open reading frame, containing two introns, encodes a polypeptide of 526 amino acids residues with a molecular mass of 55794 Da. Expression of the cel3 gene was induced by various insoluble celluloses and CM-cellulose. Transcription of cel3 was abolished when cells were cultivated in media containing the above cellulosic substrates, but added with glucose, fructose or lactose, while addition of glycerol or mannitol did not affect the cel3 mRNA level. The amino acid sequence of the catalytic domain of the Cel3 protein was homologous to that of fungal exo-type cellulases belonging to family 7 of the glycosyl hydrolases. A phylogenetic study showed that these exo-type cellulases can be clearly separated from family 7 endo-type cellulases.

Isolation of sake yeast mutants producing a high level of ethyl caproate and/or isoamyl acetate.

In the case of sake, ethyl caproate and isoamyl acetate are considered to be closely associated with flavor. Various mutant yeast strains producing a higher level of flavor compounds (ethyl caproate and/or isoamyl acetate) than the parent strain were isolated by ethyl methane sulfonate treatment followed by global selection. Two of the mutants obtained also showed a high malate productivity. These mutants would be promising for practical sake fermentation.

Effect of gene disruptions of the TCA cycle on production of succinic acid in Saccharomyces cerevisiae.

Succinate is the main taste component produced by yeasts during sake (Japanese rice wine) fermentation. The pathway leading to accumulation of succinate was examined in liquid culture in the presence of a high concentration (15%) of glucose under aerobic and anaerobic conditions using a series of Saccharomyces cerevisiae strains in which various genes that encode the expression of enzymes required in TCA cycle were disrupted. When cultured in YPD medium containing 15% glucose under aerobic conditions, the KGD1 (alpha-ketoglutarate dehydrogenase) gene disrupted mutant produced a lower level of succinate than the wild-type strain, while the SDH1 (succinate dehydrogenase) gene-disrupted mutant produced an increased level of succinate. On the other hand, the FUM1 (fumarase) gene disrupted mutant produced significantly higher levels of fumarate but did not form malate at all. These results indicate that succinate, fumarate and malate are mainly synthesized through the TCA cycle (oxidative direction) even in the presence of glucose at a concentration as high as 15%. When the growth condition was shifted from aerobic to anaerobic, the increased level of succinate in SDH1 disruptants was no longer observed, whereas the decreased level of succinate in the KGD1 diruptant was still observed. A double mutant of the two fumarate reductase isozyme genes (OSM1 and FRDS) showed a succinate productivity of 50% as compared to the parent when cells were incubated in glucose-buffered solution. These results indicate that succinate could be synthesized through two pathways, namely, alpha-ketoglutarate oxidation via the TCA cycle and fumarate reduction under anaerobic conditions.

Isolation and transcriptional analysis of a cellulase gene (cell) from the basidiomycete Irpex lacteus.

A gene (named cell) homologous to the cellobiohydrolase I gene (cbhl) of Trichoderma reesei was isolated and sequenced from the white rot basidiomycete Irpex lacteus MC-2. The cell open reading frame consists of 1551 bp, which is interrupted by two introns, encoding a polypeptide of 517 amino acid residues with a calculated molecular mass of 54,522 Da. The deduced amino acid sequence showed that CEL1 (the protein encoded by cell) has a modular structure consisting of a catalytic domain of 449 amino acids and a C-terminal cellulose-binding domain (CBD) of 36 amino acids separated by a proline-, serine-, threonine-rich linker region of 32 amino acids. The CEL1 catalytic domain is homologous with fungal cellobiohydrolases (CBHs) belonging to family 7 of the glycosyl hydrolases. The transcription of cell was induced in the presence of various cellulosic substrates and repressed by glucose. It was therefore concluded that the reported sequence represents the first cellulase gene isolated from the basidiomycete Irpex.

Isolation of sake yeast strains possessing various levels of succinate- and/or malate-producing abilities by gene disruption or mutation.

Succinate and malate are the main taste components produced by yeast during sake (Japanese alcohol beverage) fermentation. Sake yeast strains possessing various organic acid productivities were isolated by gene disruption. Sake fermented using the aconitase gene (ACO1) disruptant contained a two-fold higher concentration of malate and a two-fold lower concentration of succinate than that made using the wild-type strain K901. The fumarate reductase gene (OSM1) disruptant produced sake containing a 1.5-fold higher concentration of succinate as compared to the wild-type, whereas the alpha-ketoglutarate dehydrogenase gene (KGD1) and fumarase gene (FUMI) disruptants gave lower succinate concentrations. The Deltakgd1 disruptant exhibited lower succinate productivity in the earlier part of the sake fermentation, while the Deltafum1 disruptant showed lower succinate productivity later in the fermentation, indicating that succinate is mainly produced by an oxidative pathway of the TCA cycle in the early phase of sake fermentation and by a reductive pathway in the later phases. Sake yeasts with low succinate productivity and/or high malate productivity was bred by isolating mutants unable to assimilate glycerol as a carbon source. Low malate-producing yeasts were also obtained from phenyl succinate-resistant mutants. The mutation of one of these mutant strains with low succinate productivity was found to occur in the KGD1 gene. These strains possessing various succinate- and/or malate-producing abilities are promising for the production of sake with distinctive tastes.

Purification, characterization and gene analysis of exo-cellulase II (Ex-2) from the white rot basidiomycete Irpex lacteus.

A new exo-type cellulase, named exo-cellulase II (Ex-2), was purified from the crude enzyme preparation of Irpex lacteus. Ex-2 was very similar to the previously characterized exo-cellulase I (Ex-1) with respect to enzymatic features such as optimal pH, temperature, heat stability, and catalytic activity. However, Ex-2 exhibited greater pH stability than Ex-1. The molecular mass and carbohydrate content of Ex-2 (56,000, 4.0%) were different from those of Ex-1 (53,000, 2.0%). A cellulase gene (named cel2) encoding both Ex-2 and Ex-1 was isolated from an I. lacteus genomic library. The cel2 gene was found to consist of 1569 bp with an open reading frame encoding 523 amino acids, interrupted by two introns. The deduced amino acid sequences revealed that cel2 ORF has a modular structure consisting of a catalytic domain and a fungal-type cellulose-binding domain (CBD) separated by a serine-rich linker region. The catalytic domain was homologous to those of fungal cellobiohydrolases belonging to family 7 of the glycosyl hydrolases. Northern blot analysis showed that expression of the cel2 gene was induced by various cellulosic substrates and repressed by glucose, fructose, and lactose.

The bacterium Burkholderia gladioli strain CHB101 produces two different kinds of chitinases belonging to families 18 and 19 of the glycosyl hydrolases.

Two genes (chiA and chiB) coding for chitanases A and B (ChiA and ChiB) were isolated from the chitinolytic bacterium, Burkholderia gladioli strain CHB101. chiA contains an open reading frame that encodes a protein of 343 amino acids, whereas chiB encodes a protein of 307 amino acids. The deduced amino acid sequence of ChiA showed a high similarity to those of microbial chitinases belonging to family 18 of the glycosyl hydrolases, while ChiB showed significant sequence similarity to plant chitinases and Streptomyces spp. chitinases belonging to family 19.

Role of cellulose-binding domain of exocellulase I from white rot basidiomycete Irpex lacteus.

The core fragment (designated P-42), devoid of the cellulose-binding domain (CBD) in the C-terminus and prepared from Irpex lacteus exocellulase I (Ex-1), was isolated by limited proteolysis using papain. Both the hydrolytic activity and binding ability of the isolated P-42 toward insoluble cellulose were lower than those of the native Ex-1, whereas Ex-1 and P-42 showed similar hydrolytic activities toward soluble substrates. These results indicate that the CBD of I. lacteus Ex-1 is the important domain which could enhance hydrolytic activity and binding ability of the enzyme toward insoluble cellulose. In addition, the isolated P-42 was different from the native Ex-1 in terms of enzymatic properties such as pH and temperature stabilities. These differences in stability, with regard to pH and temperature, between P-42 and the native Ex-1 are probably due to the O-linked sugar chains existing in the linker region.

Expression of genes encoding membrane-bound hydrogenase in Pseudomonas hydrogenovora under autotrophic condition is dependent on two different promoters.

Expression of a membrane-bound hydrogenase of the hydrogen-utilizing bacterium Pseudomonas hydrogenovora was induced specifically in autotrophic condition. Dot blot and primer extension analysis showed that the transcription of the hydrogenase gene started from the region upstream of a hydrogenase structural gene, hupS, which contained three putative sigma54-type promoter sequences (Phup1, Phup2, and Phup3). The lacZ-fusion analysis of the hupS-upstream region combined with site-directed mutagenesis showed that Phup1 and Phup3 would be essential for a transcriptional initiation. It was also found that the region upstream of Phup sequences was concerned with regulation of transcription.

Chitosanase from the Plant Pathogenic Fungus, Fusarium solani f. sp. Phaseoli-Purification and Some Properties.

Among 162 strains of the genus Fusarium tested, 22 strains, mainly from F. solani and F. splendens, formed halos on chitosan-containing agar medium. Chitosanase secreted in the culture by the most effective producer, F. solani f. sp. phaseoli SUF386, was further investigated. N-Acetylglucosamine (GlcNAc) used as a carbon source was most effective for production of chitosanase. Chitosan, a substrate for chitosanase, inhibited cell growth completely and abolished production of chitosanase when used as a carbon source in the liquid medium. Chitosanase purified from the culture filtrate had a molecular mass of 36 kDa, and showed a maximum activity at pH 5.6 and 40°C. The enzyme catalyzed the hydrolysis of chitopentaose, chitosan (70% and 100% deacetylation), and glycolchitosan, but showed little activity toward chitobiose, chitotriose, chitotetraose, glycol chitin, and carboxymethyl cellulose. A rapid reduction in the viscosity of chitosan solutions suggested that the enzyme catalyzed an endo-type cleavage reaction.

Purine-mediated growth inhibition caused by a pyrE mutation in Escherichia coli K-12.

A purine-sensitive phenotype results from a previously described mutation in the structural gene (pyrE) for orotate phosphoribosyltransferase (OPT) in Escherichia coli K-12. OPT from both the mutant and the wild-type was partially inhibited by adenine and adenosine, although other purine derivatives were not effective for this inhibition. The Km values of the mutant OPT were 580 and 760 microM for orotate and 5'-phosphoribosyl-1'-pyrophosphate (PRib-PP), respectively, whereas the corresponding values for the wild-type OPT were 40 and 60 microM. The intracellular level of PRib-PP was decreased to less than 15% of the normal level when purine derivatives were added to exponentially growing cultures of both the parent and mutant strains. However, this decrease of the PRib-PP level was not found in strains derived from the mutant, in which the purine-sensitive phenotype was suppressed by a secondary mutation. The purine-sensitive phenotype was caused by retardation of the pyrimidine de novo pathway, when the intracellular level of PRib-PP was diminished by exogenously supplied purine derivatives.

Application of hybrid plasmids carrying glycolysis genes to ATP production by Escherichia coli.

The closely linked structural genes of phosphofructokinase (pfkA) and triosephosphate isomerase (tpi) of Escherichia coli were separately cloned onto plasmid pBR322. By gene dosage effects, transformed cells of E. coli C600 with these pBR322 hybrid plasmids showed 7- and 16-fold increases in the specific activities of phosphofructokinase and triosephosphate isomerase, respectively, over the specific activities in C600. Dried preparations of E. coli cells dosed with these genes showed appreciably high ATP-regenerating activity.

Effect of long term-administration with Gymnema sylvestre R. BR on plasma and liver lipid in rats.

Extract of Gymnema sylvestre leaves was administered to rats receiving either a high fat diet or normal fat diet for 10 weeks to investigate its influence on plasma and liver lipids and on visceral fat accumulation. In addition, its effect was compared with those of chitosan and the influence of combined use of these two substances was also evaluated. Within the high fat diet groups, the extract suppressed body weight gain and accumulation of liver lipids to the same extent as chitosan and the combined use. In addition, intraperitoneal fat and fat drop vacuoles on the epithelium of renal tubules, noted in the high fat diet group, were scattered by administration of the extract with the same results as for chitosan and combined use. Within the normal fat diet groups, plasma triglyceride levels decreased by administration of the extract, with similar results as chitosan and combined use. Concerning plasma total cholesterol, there was no decreasing effects with the extract, as found with chitosan and combined use. However, the effect of chitosan on plasma total cholesterol tended to be enhanced when used in combination with the extract. In addition, long-term administration of the extract did not show any influence on hematological and blood chemical parameters.