NTS Region Research Articles

Exploring the neural mechanisms underlying achalasia: A study of functional connectivity and regional brain activity

Background and AimsThe pathophysiology of achalasia, which involves central nuclei abnormalities, remains unknown. We investigated the resting-state functional MRI (rs-fMRI) features of patients with achalasia. MethodsWe applied resting-state functional MRI (rs-fMRI) to investigate the brain features in patients with achalasia (n = 27), compared to healthy controls (n = 29). Focusing on three regions of interest (ROIs): the dorsal motor nucleus of the vagus (DMV), the nucleus ambiguus (NA), and the nucleus of the solitary tract (NTS), we analyzed variations in resting-state functional connectivity (rs-FC), fractional amplitude of low-frequency fluctuations (fALFF), and regional homogeneity (ReHo). ResultsAchalasia patients demonstrated stronger functional connectivity between the NA and the right precentral gyrus, left postcentral gyrus, and left insula. No significant changes were found in the DMV or NTS. The fMRI analysis showed higher rs-FC values for NA-DMV and NA-NTS connections in achalasia patients. Achalasia patients exhibited decreased fALFF values in the NA, DMV, and NTS regions, as well as increased ReHo values in the NA and DMV regions. A positive correlation was observed between fALFF values in all six ROIs and the width of the barium meal. The NTS fALFF value and NA ReHo value displayed a positive correlation with integrated relaxation pressure (IRP), while the ReHo value in the right precentral gyrus showed an inverse correlation with the height of the barium meal. ConclusionsAbnormal rs-FC and regional brain activity was found in patients with achalasia. Our study provides new insights into the pathophysiology of achalasia and highlights the potential of rs-fMRI in improving the diagnosis and treatment of this condition.

A brainstem circuit for nausea suppression.

SUMMARYNausea is a discomforting sensation of gut malaise that remains a major clinical challenge. Several visceral poisons induce nausea through the area postrema, a sensory circumventricular organ that detects blood-borne factors. Here, we use genetic approaches based on an area postrema cell atlas to reveal inhibitory neurons that counteract nausea-associated poison responses. The gut hormone glucose insulinotropic peptide (GIP) activates area postrema inhibitory neurons that project locally and elicit inhibitory currents in nausea-promoting excitatory neurons through γ-aminobutyric acid (GABA) receptors. Moreover, GIP blocks behavioral responses to poisons in wild-type mice, with protection eliminated by targeted area postrema neuron ablation. These findings provide insights into the basic organization of nausea-associated brainstem circuits and reveal that area postrema inhibitory neurons are an effective pharmacological target for nausea intervention.

Altered GABAergic neurotransmission at the nucleus tractus solitarii following Hindlimb Unloading

Hindlimb Unloading (HU) in rats induces dysautonomia and cardiovascular deconditioning (CVD) similar to that seen in bedridden individuals. The autonomic alterations in CVD and HU are related to plasticity within the central nervous system. The nucleus tractus solitarii (nTS) is a major integrative brainstem region for autonomic modulation and processing of cardiovascular reflexes. GABA and glutamate are the main inhibitory and excitatory neurotransmitters within this nucleus. HU increases nTS glutamatergic neurotransmission yet decreases neuronal excitability. In this study, we investigated the effects of HU on the GABAergic neurotransmission in the nTS. We hypothesized that HU increases GABA synaptic modulation of nTS neurons, which contributes to impaired autonomic and cardiovascular control. HU was induced by placing stainless wire rings in the tail of male Sprague‐Dawley rats (3 weeks old), and after one week of recovery suspending rats at an angle of 30–35° for two weeks with free access to food and water. Control animals were maintained in normal postural conditions. After HU or the control period, coronal brainstem slices were generated and whole‐cell patch clamp was performed to evaluate GABAergic neurotransmission in the nTS region. The magnitude of GABA release was evaluated via an intensity‐based GABA sensing fluorescence reporter (iGABASnFR). Block of GABAA receptors (Gabazine, 20µM and Bicuculline, 20µM) depolarized membrane potential in HU but not in Control, suggesting greater tonic GABA inhibition in HU. In response to GABA interneuron stimulation the evoked IPSC amplitude and area, as well as iGABASnFR fluorescence, was greater in HU than Control. HU also elevated the frequency of spontaneous miniature IPSCs but not their amplitude. Puffer application of GABA (100µM, 15ms) produced similar postsynaptic current responses in nTS neurons of HU and Control. These results indicate that HU increases GABAergic signaling in the nTS likely via augmented release of GABA from presynaptic terminals. This synaptic plasticity may be a component of the mechanisms underlying the autonomic and cardiovascular alterations following CVD.

Characterization and use of Tetrahymena thermophila artificial chromosome 2 (TtAC2) constructed by biomimetic of macronuclear rDNA minichromosome

Efficient expression vectors for unicellular ciliate eukaryotic Tetrahymena thermophila are still needed in recombinant biology and biotechnology applications. Previously, the construction of the T. thermophila Macronuclear Artificial Chromosome 1 (TtAC1) vector revealed additional needs for structural improvements such as better in vivo stability and maintenance as a recombinant protein expression platform. In this study, we designed an efficiently maintained artificial chromosome by biomimetic of the native macronuclear rDNA minichromosome. TtAC2 was constructed by sequential cloning of subtelomeric 3′NTS region (1.8 kb), an antibiotic resistance gene cassette (2 kb neo4), a gene expression cassette (2 kb TtsfGFP), rDNA coding regions plus a dominant C3 origin sequence (10.3 kb), and telomeres (2.4 kb) in a pUC19 backbone plasmid (2.6 kb). The 21 kb TtAC2 was characterized using fluorescence microscopy, qPCR, western blot and Southern blot after its transformation to vegetative T. thermophila CU428.2 strain, which has a recessive B origin allele. All experimental data show that circular or linear forms of novel TtAC2 were maintained as free replicons in T. thermophila macronucleus with or without antibiotic treatment. Notably, TtAC2 carrying strains expressed a TtsfGFP marker protein, demonstrating the efficacy and functionality of the protein expression platform. We show that TtAC2 is functionally maintained for more than two months, and can be efficiently used in recombinant DNA, and protein production applications.

Calcium Imaging Analysis of Cellular Responses to Hypercapnia and Hypoxia in the NTS of Newborn Rat Brainstem Preparation.

It is supposed that the nucleus of the solitary tract (NTS) in the dorsal medulla includes gas sensor cells responsive to hypercapnia or hypoxia in the central nervous system. In the present study, we analyzed cellular responses to hypercapnia and hypoxia in the NTS region of newborn rat in vitro preparation. The brainstem and spinal cord were isolated from newborn rat (P0-P4) and were transversely cut at the level of the rostral area postrema. To detect cellular responses, calcium indicator Oregon Green was pressure-injected into the NTS just beneath the cut surface of either the caudal or rostral block of the medulla, and the preparation was superfused with artificial cerebrospinal fluid (25–26°C). We examined cellular responses initially to hypercapnic stimulation (to 8% CO2 from 2% CO2) and then to hypoxic stimulation (to 0% O2 from 95% O2 at 5% CO2). We tested these responses in standard solution and in two different synapse blockade solutions: (1) cocktail blockers solution including bicuculline, strychnine, NBQX and MK-801 or (2) TTX solution. At the end of the experiments, the superfusate potassium concentration was lowered to 0.2 from 3 mM to classify recorded cells into neurons and astrocytes. Excitation of cells was detected as changes of fluorescence intensity with a confocal calcium imaging system. In the synaptic blockade solutions (cocktail or TTX solution), 7.6 and 8% of the NTS cells responded to hypercapnic and hypoxic stimulation, respectively, and approximately 2% of them responded to both stimulations. Some of these cells responded to low K+, and they were classified into astrocytes comprising 43% hypercapnia-sensitive cells, 56% hypoxia-sensitive cells and 54% of both stimulation-sensitive cells. Of note, 49% of the putative astrocytes identified by low K+ stimulation were sensitive to hypercapnia, hypoxia or both. In the presence of a glia preferential blocker, 5 mM fluoroacetate (plus 0.5 μM TTX), the percentage of hypoxia-sensitive cells was significantly reduced compared to those of all other conditions. This is the first study to reveal that the NTS includes hypercapnia and hypoxia dual-sensitive cells. These results suggest that astrocytes in the NTS region could act as a central gas sensor.

Distribution and difference of APOBEC-induced mutations in the TpCpW context of HBV DNA between HCC and non-HCC.

Hepatitis B virus (HBV) DNA is vulnerable to editing by human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminases. However, the distribution of APOBEC-induced mutations on HBV DNA is not well characterized. To this end, we obtained the HBV DNA sequence of HBV-infected individuals with and without hepatocellular carcinoma (HCC and non-HCC groups, respectively) from NCBI database and calculated the rapo values of APOBEC-induced TpCpW→TpKpW mutation prevalence in HBV DNA. The results showed that the APOBEC-induced mutations were mainly distributed in the minus strand of non-HCC-derived HBV DNA (rapo = 2.04), while the mutation on the plus-strand was weaker (rapo = 0.99). There were high APOBEC-induced mutation regions in the minus strand of HBV DNA 1 to 1000 nucleotides (nts) region and in the plus-strand of HBV DNA 1000 to 1500 nts region; the mutations in the 1 to 1000 nts region were mainly TpCpW→TpTpW mutation types (total T/G: 111/18) and a number of these were missense mutations (missense/synonymous: 35/94 in P gene, 17/15 in S gene, and 5/10 in X gene). The difference between minus to plus-strand rapo of HCC-derived HBV DNA (1.96) was greater than that of the non-HCC group (1.05). The minus-strand rapo of HCC-derived HBV DNA regions 1000 to1500nts and 1500 to 2000 nts (rapo = 4.2 and 4.2) was also higher than that of the same regions of non-HCC-derived HBV DNA (rapo = 1.2 and 1.1). Finally, the ratio of minus to plus-strand rapo was used to distinguish HCC-derived HBV DNA from non-HCC-derived HBV DNA. This study unraveled the distribution characteristics of APOBEC-induced mutations on double strands of HBV DNA from HCC and non-HCC samples. Our findings would help understand the mechanism of APOBECs on HBV DNA and may provide important insights for the screening of HCC.

Molecular organization of the 5S rDNA gene type II in elasmobranchs

ABSTRACTThe 5S rDNA gene is a non-coding RNA that can be found in 2 copies (type I and type II) in bony and cartilaginous fish. Previous studies have pointed out that type II gene is a paralog derived from type I. We analyzed the molecular organization of 5S rDNA type II in elasmobranchs. Although the structure of the 5S rDNA is supposed to be highly conserved, our results show that the secondary structure in this group possesses some variability and is different than the consensus secondary structure. One of these differences in Selachii is an internal loop at nucleotides 7 and 112. These mutations observed in the transcribed region suggest an independent origin of the gene among Batoids and Selachii. All promoters were highly conserved with the exception of BoxA, possibly due to its affinity to polymerase III. This latter enzyme recognizes a dT4 sequence as stop signal, however in Rajiformes this signal was doubled in length to dT8. This could be an adaptation toward a higher efficiency in the termination process. Our results suggest that there is no TATA box in elasmobranchs in the NTS region. We also provide some evidence suggesting that the complexity of the microsatellites present in the NTS region play an important role in the 5S rRNA gene since it is significantly correlated with the length of the NTS.

Phylogeny of Cucurbitaceae species in Korea based on 5S rDNA non-transcribed spacer

The 5S rDNA coding region and its spacer have been successfully utilized in phylogenetic studies of plants. However, it has not been utilized in the phylogenetic analysis of Cucurbitaceae. Here, we obtained the 5S rDNA sequences of 12 Cucurbitaceae species by direct PCR or cloning. The 5S rDNA sequences ranged from 275 to 359 bp, and the coding regions of all species were 120 bp long, except for that of Cucurbita, which was 119 bp. Some genus-specific SNPs were observed in the coding regions of Cucurbita, Lagenaria, Melothria, and Tricosanthes. The GC content of the coding regions was generally higher than that of the NTS regions, and the difference in GC content between the coding and NTS regions varied among species, with Gynostemma pentaphyllum having the greatest difference of 20.3. The phylogenetic trees generated using maximum parsimony and maximum likelihood were congruent and well supported by the recently published classification of Cucurbitaceae. These results demonstrated the utility of the 5S rDNA sequence in inferring phylogenetic relationships among 12 Cucurbitaceae species, and its utility could be extended by using a greater number of species in future studies.

Phylogenetic Analysis of Common Millet (Panicum miliaceum L.) using NTS of 5S Ribosomal DNA

Twenty two common millet (Panicum miliaceum L.) varieties collected from Korea, China and Russia were investigated for their phylogenetic relationship using 5S ribosomal DNA sequences with a hope to provide the basic information on their exact origin. Sequences of 5S rDNA were isolated by PCR. The primers, 5s-rRNA1 and 5s-rRNA2, were designed to isolate the complete NTS. Genomic DNA amplification produced two fragments with different length, 900 bp and 400 bp fragments, confirming the presence of two types of 5S rDNA repeats that differed from each other in the length of the NTS region. Amplified DNAs of 400 bp fragment were subcloned and used for further investigation. The obtained NTS sequences ranged from 200 to 300 bp and homology of sequences among plant materials was much higher than long repeat. CLUSTALW multiple aligment of 5S rDNA sequences from 22 different common millets revealed the clear difference by their origin. And critically different areas with insert or deletion were also confirmed. Those sequence difference seems to be used for discrimination of cultivars from different origin and use as molecular markers for origin identification. In phylogenic tree construction, the clear classification was shown where the genotypes from China and Russia is positioned together and stay away from domestic genotypes.

Systemic administration of leptin potentiates the response of neurons in the nucleus of the solitary tract to chemoreceptor activation in the rat

Leptin microinjections into the nucleus of the solitary tract (NTS) have been shown to elicit sympathoexcitatory responses, and potentiate the cardiovascular responses to activation of the chemoreflex. In this study, experiments were done in Sprague–Dawley rats initially to provide a detailed mapping within the NTS complex of cells containing immunoreactivity to the long form of the leptin receptor (Ob-Rb). In a second series, this NTS region containing Ob-Rb immunoreactive cells was explored for single units antidromically activated by stimulation of pressor sites in the rostral ventrolateral medulla (RVLM). These antidromically identified neurons were then tested for their response to intra-carotid injections of leptin (50–100ng/0.1ml), and to activation of peripheral chemoreceptors following an injection of potassium cyanide (KCN) (80μg/0.1ml) into the carotid artery. Cells containing Ob-Rb-like immunoreactivity were found predominantly in the caudal NTS: within the medial, commissural and gelatinous (sub-postremal area) subnuclei of the NTS complex. Of 73 single units tested in these NTS regions, 48 were antidromically activated by stimulation of RVLM pressor sites and 25 of these single units responded with an increase in discharge rate after intra-carotid injections of leptin. In addition, 17 of these leptin responsive neurons were excited by the intra-carotid injections of KCN (80μg/0.1ml). Furthermore, the excitatory response of these single units to KCN was potentiated (59–83%) immediately following the leptin injection. These data indicate that leptin responsive neurons in NTS mediate chemoreceptor afferent information to pressor sites in the RVLM, and suggest that leptin may act as a facilitator on neuronal circuits within the NTS that potentiates the sympathoexcitatory responses elicited during the reflex activation of arterial chemoreceptors.

Acute reductions in blood flow restricted to the dorsomedial medulla induce a pressor response in rats

The brainstem nucleus of the solitary tract (nucleus tractus solitarii, NTS) is a pivotal region for regulating the set-point of arterial pressure, the mechanisms of which are not fully understood. Based on evidence that the NTS exhibits O2-sensing mechanisms, we examined whether a localized disturbance of blood supply, resulting in hypoxia in the NTS, would lead to an acute increase in arterial pressure. Male Wistar rats were used. Cardiovascular parameters were measured before and after specific branches of superficial dorsal medullary veins were occluded; we assumed these were drainage vessels from the NTS and would produce stagnant hypoxia. Hypoxyprobe-1, a marker for detecting cellular hypoxia in the post-mortem tissue, was used to reveal whether vessel occlusion induced hypoxia within the NTS. Following vessel occlusion, blood flow in the dorsal surface of the medulla oblongata including the NTS region showed an approximately 60% decrease and was associated with hypoxia in neurons located predominantly in the caudal part of the NTS as revealed using hypoxyprobe-1. Arterial pressure increased and this response was pronounced significantly in both magnitude and duration when baroreceptor reflex afferents were sectioned. These results suggest that localized hypoxia in the NTS increases arterial pressure. We suggest this represents a protective mechanism whereby the elevated systemic pressure is a compensatory mechanism to enhance cerebral perfusion. Whether this physiological mechanism has any relevance to neurogenic hypertension is discussed.

Variation in Genotype and Higher Virulence of a Strain of Sporothrix schenckii Causing Disseminated Cutaneous Sporotrichosis

Sporotrichosis is usually a localized, lymphocutaneous disease, but its disseminated type was rarely reported. The main objective of this study was to identify specific DNA sequence variation and virulence of a strain of Sporothrix schenckii isolated from the lesion of disseminated cutaneous sporotrichosis. We confirmed this strain to be S. schenckii by(®) tubulin and chitin synthase gene sequence analysis in addition to the routine mycological and partial ITS and NTS sequencing. We found a 10-bp deletion in the ribosomal NTS region of this strain, in reference to the sequence of control strains isolated from fixed cutaneous sporotrichosis. After inoculated into immunosuppressed mice, this strain caused more extensive system involvement and showed stronger virulence than the control strain isolated from a fixed cutaneous sporotrichosis. Our study thus suggests that different clinical manifestation of sporotrichosis may be associated with variation in genotype and virulence of the strain, independent of effects due to the immune status of the host.

Simultaneous POMC gene transfer to hypothalamus and brainstem increases physical activity, lipolysis and reduces adult-onset obesity

Pro-opiomelanocortin (POMC) neurons are identified in two brain sites, the arcuate nucleus of the hypothalamus and nucleus of the solitary tract (NTS) in brainstem. Earlier pharmacological and POMC gene transfer studies demonstrate that melanocortin activation in either site alone improves insulin sensitivity and reduces obesity. The present study, for the first time, investigated the long-term efficacy of POMC gene transfer concurrently into both sites in the regulation of energy metabolism in aged F344xBN rats bearing adult-onset obesity. Pair feeding was included to reveal food-independent POMC impact on energy expenditure. We introduced adeno-associated virus encoding either POMC or green fluorescence protein to the two brain areas in 22-month-old rats, then recorded food intake and body weight, assessed oxygen consumption, serum leptin, insulin and glucose, tested voluntary wheel running, analysed POMC expression, and examined fat metabolism in brown and white adipose tissues. POMC mRNA was significantly increased in both the hypothalamus and NTS region at termination. Relative to pair feeding, POMC caused sustained weight reduction and additional fat loss, lowered fasting insulin and glucose, and augmented white fat hormone-sensitive lipase activity and brown fat uncoupling protein 1 level. By wheel running assessment, the POMC animals ran twice the distance as the Control or pair-fed rats. Thus, the dual-site POMC treatment ameliorated adult-onset obesity effectively, involving a moderate hypophagia lasting ∼60 days, enhanced lipolysis and thermogenesis, and increased physical activity in the form of voluntary wheel running. The latter finding provides a clue for countering age-related decline in physical activity.

Pro-opiomelanocortin gene transfer to the nucleus of the solitary track but not arcuate nucleus ameliorates chronic diet-induced obesity

Short-term pharmacological melanocortin activation deters diet-induced obesity (DIO) effectively in rodents. However, whether central pro-opiomelanocortin (POMC) gene transfer targeted to the hypothalamus or hindbrain nucleus of the solitary track (NTS) can combat chronic dietary obesity has not been investigated. Four-weeks-old Sprague–Dawley rats were fed a high fat diet for 5 months, and then injected with either the POMC or control vector into the hypothalamus or NTS, and body weight and food intake recorded for 68 days. Insulin sensitivity, glucose metabolism and adrenal indicators of central sympathetic activation were measured, and voluntary wheel running (WR) assessed. Whereas the NTS POMC-treatment decreased cumulative food consumption and caused a sustained weight reduction over 68 days, the hypothalamic POMC-treatment did not alter cumulative food intake and produced weight loss only in the first 25 days. At death, only the NTS-POMC rats had a significant decrease in fat mass. They also displayed enhanced glucose tolerance, lowered fasting insulin and increased QUICK value, and elevated adrenal indicators of central sympathetic activation. Moreover, the NTS-POMC animals exhibited a near 20% increase in distance ran relative to the respective controls, but the ARC-POMC rats did not. In conclusion, POMC gene transfer to the NTS caused modest anorexia, persistent weight loss, improved insulin sensitivity, and increased propensity for WR in DIO rats. These metabolic improvements may involve stimulation of energy expenditure via centrally regulated sympathetic outflow. The similar POMC treatment in the hypothalamus had minimal long-term physiological or metabolic impact. Thus, melanocortin activation in the brainstem NTS region effectively ameliorates chronic dietary obesity whilst that in the hypothalamus fails to do so.

Independent chromosomal localization of two different size 5S rDNA of Allium victorialis var. platyphyllum by sequential fluorescence in situ hybridization in accordance with sequence polymorphism

5S rRNA gene repeat units in a species are usually organized as either one relatively close size with numbers of intraspecific variations in NTS region or two different sizes with completely different sequence in NTS. Allium victorialis var. platyphyllum revealed two different size products of approximately 0.39 kb and 0.51 kb with highly conserved coding region of 120 bp. However, an extra sequences of approximately 120 bp between at 324 and 443 bp in long NTS region revealed, besides the remaining sequences of two NTS regions of short and long size were highly conserved giving the identity of 94.9%. To identify whether two different size 5S rDNA are occupied by a mixed state as random repeat or an independent group by each size in a particular locus, two rounds of FISH was sequentially performed using two probes of independent different size 5S rDNA and additional probe of only extra sequences of 120 bp in long NTS. Due to the highly conserved coding regions of both 5S rDNA, two different size 5S rDNA were detected in 3 loci in short arm of chromosome 6, however, extra sequences of long NTS was shown only in one locus within detected 5S rDNA from all examined chromosomes and interphase cells. This independent localization of two different size 5S rDNA suggests that 5S rDNA may be organized as a tandem repeat with random positions in a molecular level, but of cytogenetic view in chromosomes and interphase cells, they are organized as an independent group in a significant loci consisting of own size by the patterns of nucleotide variations.

Hyperoxia decreases brain derived neurotrophic factor (BDNF) production in the carotid body and brainstem and catecholaminergic neurons in nucleus tractus solitarii (nTS).

BDNF in the carotid body (CB) has trophic effects on the development of primary sensory afferents. Hyperoxic exposure (HYP) during early postnatal development causes hypoplasia of the CB, reduces sensory neurons, and ablates hypoxic chemosensitivity. We hypothesized that HYP would directly affect BDNF production in the CB and brainstem (BS) and tyrosine hydroxylase (TH) expression in the nTS.MethodsTime‐dated SD rats were exposed to HYP (60% O2) or (21% O2) for 48h prior to delivery and for 7 or 14 days after delivery and then returned to 21% O2 until postnatal day 9, 16 or 22 when the CB and BS were harvested and processed for gene (qRT‐PCR) and protein expression.ResultsHYP exposure for 7days reduced BDNF mRNA levels by 80% and 50% in the CB and BS, respectively (p=0.002) along with a 90% reduction in protein expression (p=0.04) in both tissues. HYP also up‐regulated p75 receptor gene expression in BS (1.6‐fold, p=0.002) without significant change in TrkB receptor. BS BDNF mRNA levels decreased from day 9 to 16 (p=0.04) but was not different between HYP and control animals at day 16. At day 22, there was a reduction in TH‐ir+ neurons and in TH protein levels (15%) in the nTS region of animals exposed to HYP, while no change in TH protein levels was observed in the locus coeruleus.ConclusionThe reduction in TH+ neurons in the nTS in hyperoxic exposed animals may be linked to reduced BDNF production in the CB.Supported by NIH R01HL080725

Biological diversity of Saccharomyces yeasts of spontaneously fermenting wines in four wine regions: Comparative genotypic and phenotypic analysis

Combination of molecular genetic analysis (karyotyping, PCR-RFLP of MET2, the ITS1-ITS2 region and the NTS region) and physiological examination (melibiose and mannitol utilization, sugar-, ethanol- and copper tolerance, killer activity, fermentation vigor and production of metabolites) of yeasts isolated from spontaneously fermenting wines in four wine regions revealed very high diversity in the Saccharomyces cerevisiae populations. Practically each S. cerevisiae isolate showed a unique pattern of properties. Although the strains originating from the same wine were quite similar in certain traits, they showed diversity in other properties. These results indicate that alcoholic fermentation in grape wines is performed by highly diverse yeast consortia rather than by one or two dominating strains. The less frequent Saccharomyces uvarum strains were less diverse, showed lower karyotype variability, were Mel +, Man +, more sensitive to 60% sugar, and ethanol or copper in the medium. They produced less acetic acid and fermented better at 14 °C than most of the S. cerevisiae isolates, but certain S. cerevisiae strains showed comparably high fermentation rates at this temperature, indicating that it is not a general rule that S. uvarum ferments better than S. cerevisiae at low temperatures. The segregation of certain traits (melibiose utilization, mannitol utilization and copper resistance) in both species indicates that the genomes can easily change during vegetative propagation. The higher diversity among the S. cerevisiae isolates suggests that the S. cerevisiae genome may be more flexible than the S. uvarum genome and may allow more efficient adaptation to the continuously changing environment in the fermenting wine.

長期間の差しもとを可能とする小仕込み試験による焼酎製造技術の開発と酵母の安定性

In traditional Shochu making, the fermented mash of the first–stage is generally utilized as the seed of the next first–stage fermentation, which is called Sashimoto. However, with the repetition of Sashimoto, the activity of yeast cells decreases and therefore generally results in bacterial contamination. In this study, a method for activating yeast cells was studied to develop a Shochu making technology by the repetition of Sashimoto for a long–term process. In addition, the yeast stability during the repetition of Sashimoto was studied. Though the trehalose concentration and the activity of yeast cells increased twice after the first Sashimoto, they continued to decrease with the repetition of Sashimoto. However, the yeast cells were successfully activated by shaking the first–stage fermented mash for 10h and the activity of yeast cells remained high during the entire test period. As a result, the ethanol concentration of the second–stage fermented mash improved to more than 18%(v/v). Comparing the flavor compounds of Shochu produced by the repetition of Sashimoto with those of the conventional method, no obvious difference was found in the concentrations of the low–, middle– and high–boiling flavor compounds, nor in the concentrations of isoamyl acetate and β–phenethyl acetate, which are desired flavor compounds. The yeast strain seemed to be stable during the repetition of Sashimoto, according to the results of the nucleotide sequence analysis of NTS regions.

Fluorescent indication that nitric oxide formation in NTS neurons is modulated by glutamate and GABA

Nitric oxide (NO) in NTS plays an important role in regulating autonomic function to the cardiovascular system. Using the fluorescent dye DAF-2 DA, we evaluated the NO concentration in NTS. Brainstem slices of rats were loaded with DAF-2 DA, washed, fixed in paraformaldehyde and examined under fluorescent light. In different experimental groups, NTS slices were pre-incubated with 1 mM l-NAME (a non-selective NOS inhibitor), 1 mM d-NAME (an inactive enantiomere of l-NAME), 1 mM kynurenic acid (a non-selective ionotropic receptors antagonist) or 20 μM bicuculline (a selective GABA A receptors antagonist) before and during DAF-2 DA loading. Images were acquired using a confocal microscope and the intensity of fluorescence was quantified in three antero-posterior NTS regions. In addition, slices previously loaded with DAF-2 DA were incubated with NeuN or GFAP antibody. A semi-quantitative analysis of the fluorescence intensity showed that the basal NO concentration was similar in all antero-posterior aspects of the NTS (rostral intermediate, 15.5 ± 0.8 AU; caudal intermediate, 13.2 ± 1.4 AU; caudal commissural, 13.8 ± 1.4 AU, n = 10). In addition, the inhibition of NOS and the antagonism of glutamatergic receptors decreased the NO fluorescence in the NTS. On the other hand, d-NAME did not affect the NO fluorescence and the antagonism of GABA A receptors increased the NO fluorescence in the NTS. It is important to note that the fluorescence for NO was detected mainly in neurons. These data show that the fluorescence observed after NTS loading with DAF-2 DA is a result of NO present in the NTS and support the concept that NTS neurons have basal NO production which is modulated by l-glutamate and GABA.

Molecular organization of 5S rDNA in bitterlings (Cyprinidae)

Molecular organization and nucleotide sequences of the 5S rRNA gene and NTS were investigated in freshwater fish, bitterlings (Acheilognathinae), including 10 species/subspecies of four genera, Acheilognathus, Pseudoperilampus, Rhodeus, and Tanakia, to understand the evolutionary trait of 5S rDNA arrays. Southern hybridization analysis revealed a general trend with tandem repeats of 5S rDNA in all the examined bitterlings. Sequence analysis demonstrated a conserved 120 bp sequence of the 5S rRNA gene and a short NTS of 56-67 bp with two distinct portions, a conserved (5'-flanking portion; at positions -1 to -38) and a variable part (3'-flanking portion), in 6 of 10 species/subspecies examined. The conserved NTS region was most likely an external promoter so far observed in various vertebrates, whereas the variable NTS region could be divided into two types due to its nucleotide polymorphisms. Molecular phylogeny using the 5S rRNA gene and NTS sequences suggested the occurrence of 5S rDNA duplication before speciation and a concerted evolution for the gene and conserved NTS regions, but a birth-and-death process to maintain the variable NTS region. Thus, the 5S rDNA in the examined bitterlings might have evolved under a mixed process of evolution.