Growth hormone resistance induced by amino acid deprivation in fao cells is independent of FGF21.

Adequate dietary intake of amino acids is imperative for normal animal growth. Our previous work using rat hepatocarcinoma Fao cells demonstrated that growth hormone (GH) resistance, coupled with a concurrent reduction in insulin-like growth factor 1 (Igf1) mRNA levels, may underlie the growth retardation associated with a low-protein diet (LPD). In this study, we investigated whether FGF21 contributes to liver GH resistance in Fao rat hepatoma cells under amino acid deprivation conditions. Mice subjected to an LPD exhibited growth retardation, compromised GH signaling in the liver, and decreased blood IGF-1 levels compared with those on a control diet. To assess the potential involvement of fibroblast growth factor (FGF) 21, produced in response to amino acid deficiency, in the development of GH resistance, we examined GH signaling and Igf1 mRNA levels in Fao cells cultured in amino acid-deprived medium. Despite the inhibition of Fgf21 expression by the integrated stress response inhibitor, an inhibitor of the eukaryotic initiation factor 2-activating transcription factor 4 pathway, GH resistance persisted in response to amino acid deprivation. Additionally, the introduction of FGF21 into the control medium did not impair either GH signaling or GH-induced Igf1 transcription. These data suggest that, in Fao cells, amino acid deprivation induces GH resistance independently of FGF21 activity. By shedding light on the mechanisms behind growth retardation-associated GH resistance linked to amino acid deficiencies, our findings provide valuable insights for clinicians in formulating effective treatment strategies for individuals facing these challenges.

Ecdysone and gene expressions for chromatin remodeling, histone modification, and Broad Complex in relation to pupal commitment in Bombyx mori.

In the present study, we tried to clarify when and how pupal commitment (PT) better to use PC occurs and what is involved in the PT of Bombyx mori. To clarify this, we examined the responsiveness of a wing disc to ecdysone, referring to metamorphosis-related BR-C, development-related Myc and Wnt, and chromatin remodeling-related genes at around the predicted PT stage of the Bombyx wing disc. Wing disc responsiveness to juvenile hormone (JH) and ecdysone was examined using Methoprene and 20-hydroxyecdysone (20E) in vitro. The body weight of B. mori increased after the last larval ecdysis, peaked at Day 5 of the fifth larval instar (D5L5), and then decreased. The responsiveness of the wing disc to JH decreased after the last larval ecdysis up to D3L5. Bmbr-c (the Broad Complex of B. mori) showed enhanced expression in D4L5 wing discs with 20E treatment. Some chromatin remodeler and histone modifier genes (Bmsnr1, Bmutx, and Bmtip60) showed upregulation after being cultured with 20E in D4L5 wing discs. A low concentration of 20E is suggested to induce responsiveness to 20E in D4L5 wing discs. Bmbr-c, Bmsnr1, Bmutx, and Bmtip60 were upregulated after being cultured with a low concentration of 20E in D4L5 wing discs. The expression of Bmmyc and Bmwnt1 did not show a change after being cultured with or without 20E in D4L5 wing discs, while enhanced expression was observed with 20E in D5L5 wing discs. From the present results, we concluded that PT of the wing disc of B. mori occurred beginning on D4L5 with the secretion of low concentrations of ecdysteroids. Bmsnr1, Bmutx, Bmtip60, and BR-C are also involved.

[Method Improvement for Quantitative Analysis of Chlorophyll Degradation Compounds, Including Pheophorbide, in Chlorella Products].

An official analytical method for chlorophyll degradation compounds, including pheophorbide, in chlorella products, is described in notification Kanshoku No. 99 (May 8, 1981). However, this method has several operational issues, such as the formation of emulsion during liquid-liquid partitioning. Additionally, impurities present in the reagents (sodium sulfate decahydrate or anhydrous sodium sulfate) used to prepare saturated sodium sulfate solution can degrade pheophorbide and other related compounds, resulting in a significant decrease in analytical values. In this study, we thoroughly examined each step of the official method to enhance the operability and develop an alternative method that eliminates the need for saturated sodium sulfate solution. The developed method was evaluated for pheophorbide a and pyropheophorbide a at 100 mg%. Satisfactory analytical performance was achieved with trueness of 100% for pheophorbide a and 90% for pyropheophorbide a, and relative standard deviations of intra- and inter-day precision below 5% for both compounds. The proposed method is considered suitable for regulatory analysis of chlorophyll degradation compounds and would be useful for quality control of chlorella products.

Identification of cytotoxic T cells and their T cell receptor sequences targeting COVID-19 using MHC class I-binding peptides.

Since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) was first reported in China in December 2019, various variants have been identified in different areas of the world such as United Kingdom (alpha), South Africa (beta and omicron), Brazil (gamma), and India (delta). Some of SARS-CoV-2 variants, each of which is characterized by a unique mutation(s) in spike protein, are concerned due to their high infectivity and the capability to escape from neutralizing antibodies elicited by vaccinations. To identify peptide epitopes that are derived from SARS-CoV-2 viral proteins and possibly induce CD8+ T cell immunity, we investigated SARS-CoV-2-derived peptides that are likely to bind to major histocompatibility complex (MHC) class I molecules. We identified a total of 15 peptides that bind to human leukocyte antigen (HLA)-A*24:02, HLA-A*02:01, or HLA-A*02:06, and possibly induce cytotoxic T lymphocytes (CTLs); thirteen of them corresponded to ORF1ab polyprotein, one peptide to spike protein and the remaining one to membrane glycoprotein. CD8+ T cells that recognize these peptides were detected in peripheral blood samples in three individuals recovered from COVID-19 as well as non-infected individuals. Since most of these peptides are commonly conserved among other coronaviruses including SARS-CoV and/or MERS-CoV, these might be useful to maintain T cell responses to coronaviruses that are pandemic at present and will become the future threat. We could define pairs of TRA and TRB sequences of nine CTL clones that recognize SARS-CoV-2-derived peptides. We might use these SARS-CoV-2-derived peptide-reactive TCR sequences for investigating the history of SARS-CoV-2 infection.

Chronic HDAC6 Activation Induces Atrial Fibrillation Through Atrial Electrical and Structural Remodeling in Transgenic Mice.

Atrial fibrillation (AF) is a relatively common complication of hypertension. Chronic hypertension induces cardiac HDAC6 catalytic activity. However, whether HDAC6 activation contributes to hypertension-induced AF is still uncertain. We examined whether chronic cardiac HDAC6 activation-induced atrial remodeling, leading to AF induction.The HDAC6 constitutively active transgenic (TG) (HDAC6 active TG) mouse overexpressing the active HDAC6 protein, specifically in cardiomyocytes, was created to examine the effects of chronic HDAC6 activation on atrial electrical and structural remodeling and AF induction in HDAC6 active TG and non-transgenic (NTG) mice. Left atrial burst pacing (S1S1 = 30 msec) for 15-30 sec significantly increased the frequency of sustained AF in HDAC6 active-TG mice compared with NTG mice. Left steady-state atrial pacing (S1S1 = 80 msec) decreased the atrial conduction velocity in isolated HDAC6 active TG compared with NTG mouse atria. The atrial size was similar between HDAC6 active TG and NTG mice. In contrast, atrial interstitial fibrosis increased in HDAC6 active TG compared with that of NTG mouse atria. While protein expression levels of both CX40 and CX43 were similar between HDAC6 active TG and NTG mouse atria, a heterogeneous distribution of CX40 and CX43 occurred in HDAC6 active-TG mouse atria but not in NTG mouse atria. Gene expression of interleukin 6 increased in HDAC6 active TG compared with NTG mouse atria.Chronic cardiac HDAC6 activation induced atrial electrical and structural remodeling, and sustained AF. Hypertension-induced cardiac HDAC6 catalytic activity may play important roles in the development of AF.

Pretreatment with KGA-2727, a selective SGLT1 inhibitor, is protective against myocardial infarction-induced ventricular remodeling and heart failure in mice.

Recent studies demonstrated that sodium-glucose co-transporter 1 (SGLT1) is associated with human ischemic cardiomyopathy. However, whether SGLT1 blockade is effective against ischemic cardiomyopathy is still uncertain. We examined the effects of KGA-2727, a selective SGLT1 inhibitor, on myocardial infarction (MI)-induced ischemic cardiomyopathy. To create MI, left anterior descending coronary artery (LAD) ligation with or without KGA-2727 administration was performed in C57BL/6J mice. Four weeks after the operation, all mice were investigated. Left ventricular fractional shortening (LVFS) was reduced and KGA-2727 significantly improved it in LAD-ligated MI mice. The cardiomyocyte diameter, and ANP, BNP, ß-MHC, and IL-18 gene expressions significantly increased in LAD-ligated mouse left ventricles compared with those of sham-operated mouse left ventricles, and KGA-2727 inhibited increases in them. Myocardial fibrosis and upregulation of CTGF and MMP-3 gene expressions in the left ventricle were increased in LAD-ligated mice compared with sham-operated mice, and KGA-2727 decreased them in the LAD-ligated left ventricles. SGLT1 protein expression level was significantly higher in LAD-ligated compared with sham-operated mouse ventricles regardless of KGA-2727 treatment. These results suggest that KGA-2727 pretreatment protects against MI-induced left ventricular remodeling through SGLT1 blockade and that it may become a new pharmacological therapy for ischemia-induced cardiomyopathy.

8-HEPE-Concentrated Materials from Pacific Krill Improve Plasma Cholesterol Levels and Hepatic Steatosis in High Cholesterol Diet-Fed Low-Density Lipoprotein (LDL) Receptor-Deficient Mice.

Eicosapentaenoic acid (EPA), one of the N-3 polyunsaturated fatty acids (n-3 PUFAs), is a major active ingredient of fish that contributes to improve dyslipidemia. Recently, we demonstrated that 8-hydroxyeicosapentaenoic acid (8-HEPE) had a more positive effect on metabolic syndrome than EPA, and that 8-HEPE induced peroxisome proliferator-activated receptor (PPAR)α activation in the liver. We investigated the effects of 8-HEPE-concentrated materials from Pacific krill on dyslipidemia and hepatic steatosis in low-density lipoprotein (LDL) receptor-deficient (LDLR-KO) mice. Eight-week-old male LDLR-KO mice were fed a Western diet (0.15% cholesterol, WD), WD supplemented with 8-HEPE-concentrated materials from Pacific krill (8-HEPE included; WD +8-HEPE), or a standard diet (SD) for eighteen weeks, respectively. Murine J774.1 macrophages were incubated in the absence or presence of 8-HEPE (50 µM) or EPA (50 µM). 8-HEPE-concentrated materials significantly increased the plasma high-density lipoprotein (HDL)-cholesterol level, and decreased the plasma LDL-cholesterol and hepatic triglyceride levels in WD-fed LDLR-KO mice. Moreover, the rate of Oil Red O-positive staining was higher in the liver of WD-fed LDLR-KO mice than in that of 8-HEPE + WD-fed LDLR-KO mice. 8-HEPE but not EPA significantly increased gene expression levels of ABCA1, CD36, and interleukin 6 (IL-6) in murine J774.1 macrophages compared with those in the control. These results suggest that 8-HEPE-concentrated materials improve dyslipidemia and hepatic steatosis increasing ABCA1, CD36, and IL-6 gene expressions in macrophages.

TRAb elevations occurred even in the third trimester; a case of a mother of a child with neonatal thyroid dysfunction, who received radioactive iodine therapy for Graves' disease.

Activity of Graves' disease (GD) is known to improve during gestation, as values of thyrotropin (TSH) receptor antibody (TRAb) also improve. However, the risk of neonatal hyperthyroidism increases when maternal TRAb values are high in the second to third trimester. A 29-year-old woman who had undergone radioactive iodine (RAI) therapy for GD 10 years earlier visited our hospital at 17 weeks of gestation, showing subclinical hypothyroidism and a positive TRAb value of 2.6 IU/L (reference range, <2.0 IU/L). Thyroid hormone replacement therapy was commenced and thyroid function normalized within 4 weeks, although TRAb was elevated at the time (3.8 IU/L). Prenatal check-up showed normal growth development and no irregularities. At 29 weeks of gestation, serum TRAb was extremely elevated, up to 16.8 IU/L. Since the risk of neonatal hyperthyroidism was of great concern, delivery was planned at an advanced-care medical center. At 38 weeks 5 days of gestation, she delivered a female neonate without any complications, although blood testing of the neonate showed subclinical hyperthyroidism with positive TRAb and TSH receptor stimulating antibody (TSAb). According to the American Thyroid Association guidelines, the TRAb value should be checked in the third trimester if mothers show a TRAb elevation between the initial visit after pregnancy and 18-22 weeks of gestation. However, if the mother has a history of RAI therapy for GD, regardless of thyroid function during gestation, the possibility of TRAb values elevating over time even years after the definitive therapy must be considered.

An educational intervention improved knowledge of dietary supplements in college students.

BACKGROUND: We have previously reported on the prevalence of dietary supplements among college students; it was deduced that their intake of supplements increased according to their grade (i.e., 13.1% in the first grade to 20.5% in the sixth grade). We also reported that some students had experienced adverse events in Japan due to their intake of these supplements. However, awareness of dietary supplements among college students remains limited, even among pharmaceutical students. Being appropriately educated about them is important for pharmaceutical students, both for themselves as well as for their future careers as pharmacists. METHODS: We conducted a lecture-based educational intervention about dietary supplements on 328 college students in Japan-184 from pharmaceutical science and 144 from environmental science or food and life science disciplines. The purpose of this study was to evaluate the effects of an educational intervention on college students' understanding of dietary supplements. The intervention involved a lecture that covered the quality of dietary supplements, how they differed from drugs, and a summary of their adverse events. The lecture was evaluated using a 14-question questionnaire. We then compared the pre- and post-intervention responses to the same questionnaire using a Wilcoxon signed-rank test. The questions were assessed using a Likert scale that ranged from "strongly agree" to "strongly disagree"; the latter being the preferred answer. RESULTS: Before the intervention had taken place, the students' understanding of dietary supplements was shown to be deficient. Conversely, post-intervention, their knowledge levels had significantly improved, especially concerning agreement on whether "Dietary supplements are safe because they are just food items". Pre-intervention, 2.7% strongly agreed and 37.5% agreed; post-intervention, 1.2% strongly agreed and 15.6% agreed. On whether "Dietary supplements made from natural ingredients or herbs are safe", at the pre-intervention stage 2.8% strongly agreed and 44.0% agreed and post-intervention, 2.2% strongly agreed and 16.9% agreed. On whether "Dietary supplements made from food items are safe", 4.0% strongly agreed and 43.6% agreed pre-intervention and 0.9% strongly agreed and 16.6% agreed post-intervention. Despite there being a greater number of pharmaceutical students who had a correct understanding of dietary supplements before the intervention, these students still showed improvement after the lecture. CONCLUSION: An intervention in the form of a single educational lecture has the capacity to improve college students' understanding of dietary supplements. It is important for pharmacists to be appropriately educated about dietary supplements when they consult with patients. We will evaluate the long-term effects of the intervention on the alumni (pharmacists) in a subsequent study.

A Microbial Platform Based on Conducting Polymers for Evaluating Metabolic Activity.

Bacterial cells possessing a certain zeta potential are immobilized by electrochemical deposition within conducting polymers such as poly(3,4-ethylenedioxythiophene) (PEDOT) and polypyrrole (PPy). These conducting polymers serve as a biocompatible matrix for trapping bacteria on an indium-tin-oxide (ITO)-coated glass substrate. The biological functions of bacteria were not affected by the chemical structure and electrical conductivity of the matrix. The viability of the bacteria on the ITO glass was monitored by dark-field microscopy. The cell density of Escherichia coli increased logarithmically during incubation in nutrient broth medium, leading to definitive formation of a biofilm on PPy. The facultative E. coli anaerobe sustains metabolism under aerobic and anaerobic conditions, but proliferates more extensively in the presence of oxygen. The conducting PPy film also facilitates electrochemical evaluation of the respiratory activity of bacterial cells and establishes that facultative anaerobic and aerobic bacteria exhibit similar respiratory activities under aerobic conditions.

IL-1ß Plays an Important Role in Pressure Overload-Induced Atrial Fibrillation in Mice.

Hypertension is one risk for atrial fibrillation (AF) and induces cardiac inflammation. Recent evidence indicates that pressure overload-induced ventricular structural remodeling is associated with the activation of nucleotide binding-oligomerization domain (NOD)-like receptor P3 (NLRP3) inflammasomes, including an apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC). We hypothesized that NLRP3 inflammasomes are an initial sensor for danger signals in pressure overload-induced atrial remodeling, leading to AF. Transverse aortic constriction (TAC) or a sham procedure was performed in mice deficient for ASC-/- and interleukin-1ß (IL-1ß-/-). One week after the procedure, electrical left atrial burst pacing from the esophagus was performed for 30 s to induce AF. IL-1ß, monocyte chemotactic protein 1 (MCP-1), connective tissue growth factor (CTGF), and collagen 1 gene expression were also examined. The electrical burst pacing induced AF in TAC-operated wild-type (WT) (p < 0.001) and ASC-/- (p < 0.05) mice, compared to no AF in the sham-operated WT and ASC-/- mice, respectively. In contrast, the number of mice in which sustained AF was induced was similar between TAC-operated IL-1ß-/- and sham-operated IL-1ß-/- mice (p > 0.05). The expression of all genes tested was increased in TAC-operated WT and ASC-/- mice compared with sham-operated WT and ASC-/- mouse atria, respectively. CTGF and collagen 1, but not MCP-1, gene expressions were increased in TAC-operated IL-1ß-/- mouse atria compared with sham-operated WT and IL-1ß-/- mouse atria. In contrast, the IL-1ß gene was not detected in either TAC-operated or sham-operated IL-1ß-/- mouse atria. These results suggest that an IL-1ß activation pathway, different from NLRP3 inflammasomes, plays an important role in pressure overload-induced sustained AF.

Scanning RNA-Seq and RAD-Seq approach to develop SNP markers closely linked to MALE STERILITY 1 (MS1) in Cryptomeria japonica D. Don.

Cryptomeria japonica is a major forestry tree species in Japan. Male sterility of the species is caused by a recessive gene, which shows dysfunction of pollen development and results in no dispersed pollen. Because the pollen of C. japonica induces pollinosis, breeding of pollen-free C. japonica is desired. In this study, single nucleotide polymorphism (SNP) markers located at 1.78 and 0.58 cM to a male sterility locus (MS1) were identified from an analysis of RNA-Seq and RAD-Seq, respectively. SNPs closely linked to MS1 were first scanned by a method similar to MutMap, where a type of index was calculated to measure the strength of the linkage between a marker sequence and MS1. Linkage analysis of selected SNP markers confirmed a higher efficiency of the current method to construct a partial map around MS1. Allele-specific PCR primer pair for the most closely linked SNP with MS1 was developed as a codominant marker, and visualization of the PCR products on an agarose gel enabled rapid screening of male sterile C. japonica. The allele-specific primers developed in this study would be useful for establishing the selection of male sterile C. japonica.

Chronic Pressure Overload Induces Cardiac Hypertrophy and Fibrosis via Increases in SGLT1 and IL-18 Gene Expression in Mice.

Increased gene expression levels of sodium-glucose cotransporter 1 (SGLT1) are associated with hypertrophic and ischemic cardiomyopathy. However, it remains unclear whether chronic pressure overload increases SGLT1 expression, which in turn induces hypertrophic cardiomyopathy. We hypothesized that pressure overload could increase SGLT1 gene expression, leading to the development of hypertrophic cardiomyopathy.To create pressure overload-induced cardiomyopathy, transverse aortic constriction (TAC) was performed in SGLT1-deficient (SGLT1-/-) and wild-type (WT) mice. Six weeks after surgery, all mice were investigated. We observed a reduction of left ventricular fractional shortening and left ventricular dilatation in TAC-operated WT but not in TAC-operated SGLT1-/- mice. SGLT1, interleukin 18, connective tissue growth factor, and collagen type 1 gene expression levels were increased in TAC-operated WT mouse hearts compared with that of sham-operated WT mouse hearts. Moreover, heart/body weight ratio and ventricular interstitial fibrosis were increased in TAC-operated WT mice compared with that of sham-operated WT mice. Interestingly, these factors did not increase in TAC-operated SGLT1-/- mice compared with that of sham-operated WT and SGLT1-/- mice. Phenylephrine, an adrenergic α1 receptor agonist, caused cardiomyocyte hypertrophy in neonatal WT mouse hearts to a significantly larger extent than in neonatal SGLT1-/- mouse hearts.In conclusion, the results indicate that chronic pressure overload increases SGLT1 and IL-18 gene expressions, leading to the development of hypertrophic cardiomyopathy. These results make SGLT1 a potential candidate for the therapeutic target for hypertension-induced cardiomyopathy.

A comparison between the human sense of smell and neural activity in the olfactory bulb of rats.

Generally, odor qualities are evaluated via sensory tests in which predefined criteria are assessed by panelists and stochastically analyzed to reduce human inconsistencies. Because this method requires multiple, well-trained human subjects, a more convenient approach is required to enable predictions of odor qualities. In this article, we propose an approach involving linking internal states of the olfactory system with perceptual characteristics. In the study, the glomerular responses of rats were taken to represent internal olfactory system states. Similarities between the glomerular responses of rats were quantified by correlations between glomerular activity patterns, overlap rate of strongly activated part across glomerular activity patterns, and the similarity between histograms of the strength of activity. These indices were then compared with perceptual similarities measured from human subjects in sensory tests. The results of experiments involving 22 odorants showed medium strength correlations between each index and perceptual similarity. In addition, when the 3 indices were combined using their Euclidean distance, we observed middle to high correlations (r = 0.65-0.79) to human perceptual similarity. We also report the results of our use of a machine learning technique to classify the odorants into a similar and dissimilar category. Although the correct rate of classification varied from 33.3% to 92.9%, these results support the feasibility of linking the glomerular responses of rats to human perception.

Cloning and expression of a gene encoding a novel thermostable thiocyanate-degrading enzyme from a mesophilic alphaproteobacteria strain THI201.

Strain THI201, a member of the alphaproteobacteria, is a novel thiocyanate (SCN(-))-degrading bacterium isolated from lake water enriched with potassium thiocyanate (KSCN). This bacterium carries the enzyme thiocyanate hydrolase (SCNase) that hydrolyses thiocyanate to carbonyl sulfide and ammonia. Characterization of both native and recombinant SCNase revealed properties different from known SCNases regarding subunit structure and thermostability: SCNase of strain THI201 was composed of a single protein and thermostable. We cloned and sequenced the corresponding gene and determined a protein of 457 amino acids of molecular mass 50 267 Da. Presence of a twin-arginine (Tat) signal sequence of 32 amino acids was found upstream of SCNase. The deduced amino acid sequence of SCNase showed 83% identity to that of a putative uncharacterized protein of Thiobacillus denitrificans ATCC 25259, but no significant identity to those of three subunits of SCNase from Thiobacillus thioparus strain THI115. The specific activities of native and recombinant enzyme were 0.32 and 4-15 µmol min(-1) (mg protein)(-1), respectively. The maximum activity of SCNase was found in the temperature range 30-70 °C. The thiocyanate-hydrolysing activity in both enzymes was decreased by freeze-thawing, although 25-100% of the activity of recombinant protein could be retrieved by treating the enzyme at 60 °C for 15 min. Furthermore, both native and recombinant enzymes retained the activity after pre-treatment of the protein solution at temperatures up to 70 °C.

Agonist-induced receptor internalization in Chinese hamster ovary cells stably co-expressing ß(1)- and ß(2)-adrenergic receptors.

ß(1)- and ß(2)-Adrenergic receptors (ß(1)-AR and ß(2)-AR) are co-expressed in numerous tissues, for example, heart and bladder. They play a very important role in the responses of a variety of organs to sympathetic nerve stimulation. Recent studies suggest that many G protein-coupled receptors, such as ß(1)-AR, ß(2)-AR, µ opioid receptor and δ opioid receptor, can form homo- and heterooligomers. Previous studies demonstrated that the ß(1)-AR and ß(2)-AR formed dimers in living HEK 293 cells. The aim of the present study is to investigate whether such heterooligomerization affect the agonist-induced receptor internalization in the CHO-K1 cells stably co-expressing ß(1)-AR and ß(2)-AR. Using co-immunoprecipitation, we confirmed that ß(1)-AR and ß(2)-AR formed heterooligomers in the CHO-K1 cells. In cells co-expressing ß(1)-AR and ß(2)-AR, 30% of ß(1)-AR was internalized by isoproterenol, whereas only 20% of ß(1)-AR was internalized in cells expressing the ß(1)-AR alone. Heterooligomerization did not affect the ratio of internalized ß(2)-AR. Salmeterol, a specific ß(2)-AR agonist, broke ß(1)-AR/ß(2)-AR heterooligomers, and induced ß(2)-AR-specific internalization in cells co-expressing ß(1)-AR and ß(2)-AR. The present study demonstrated that heterooligomerization between ß(1)-AR and ß(2)-AR accelerates the isoproterenol-promoted internalization of the ß(1)-AR, and that salmeterol induces ß(2)-AR-specific internalization in Chinese hamster ovary (CHO) cells stably co-expressing ß(1)-AR and ß(2)-AR.

The construction of a high-density linkage map for identifying SNP markers that are tightly linked to a nuclear-recessive major gene for male sterility in Cryptomeria japonica D. Don.

BACKGROUND: High-density linkage maps facilitate the mapping of target genes and the construction of partial linkage maps around target loci to develop markers for marker-assisted selection (MAS). MAS is quite challenging in conifers because of their large, complex, and poorly-characterized genomes. Our goal was to construct a high-density linkage map to facilitate the identification of markers that are tightly linked to a major recessive male-sterile gene (ms1) for MAS in C. japonica, a species that is important in Japanese afforestation but which causes serious social pollinosis problems. RESULTS: We constructed a high-density saturated genetic linkage map for C. japonica using expressed sequence-derived co-dominant single nucleotide polymorphism (SNP) markers, most of which were genotyped using the GoldenGate genotyping assay. A total of 1261 markers were assigned to 11 linkage groups with an observed map length of 1405.2 cM and a mean distance between two adjacent markers of 1.1 cM; the number of linkage groups matched the basic chromosome number in C. japonica. Using this map, we located ms1 on the 9th linkage group and constructed a partial linkage map around the ms1 locus. This enabled us to identify a marker (hrmSNP970_sf) that is closely linked to the ms1 gene, being separated from it by only 0.5 cM. CONCLUSIONS: Using the high-density map, we located the ms1 gene on the 9th linkage group and constructed a partial linkage map around the ms1 locus. The map distance between the ms1 gene and the tightly linked marker was only 0.5 cM. The identification of markers that are tightly linked to the ms1 gene will facilitate the early selection of male-sterile trees, which should expedite C. japonica breeding programs aimed at alleviating pollinosis problems without harming productivity.