Development of preparative and analytical methods of the hop bitter acid oxide fraction and chemical properties of its components

The bitter acids in hops (Humulus lupulus L.) and beer, such as α-, β-, and iso-α-acids, are known to affect beer quality and display various physiological effects. However, these compounds readily oxidize, and the effect of the oxides on the properties of beer or their potential health benefits are not well understood. In this study, we developed a simple preparative method for the bitter acid oxide fraction derived from hops and designated the constituents as matured hop bitter acids (MHBA). HPLC-PDA-ESI/HRMS and MS² revealed that MHBA are primarily composed of α-acid-derived oxides, which possess a common β-tricarbonyl moiety in their structures similar to α-, β-, and iso-α-acids. We also developed a quantitative analytical method of whole MHBA by HPLC, which showed high precision and reproducibility. Using our newly developed method, the concentration of whole MHBA in several commercial beers was evaluated. Our results will promote the study of bitter acid oxides.     

Sema6B,Sema6C,and Sema6D Expression and Function during Mammalian Retinal Development

In the vertebrate retina, the formation of neural circuits within discrete laminae is critical for the establishment of retinal visual function. Precise formation of retinal circuits requires the coordinated actions of adhesive and repulsive molecules, including repulsive transmembrane semaphorins (Sema6A, Sema5A, and Sema5B). These semaphorins signal through different Plexin A (PlexA) receptors, thereby regulating distinct aspects of retinal circuit assembly. Here, we investigate the physiological roles of three Class 6 transmembrane semaphorins (Sema6B, Sema6C, and Sema6D), previously identified as PlexA receptor ligands in non-retinal tissues, in mammalian retinal development. We performed expression analysis and also phenotypic analyses of mice that carry null mutations in each of genes encoding these proteins using a broad range of inner and outer retinal markers. We find that these Class 6 semaphorins are uniquely expressed throughout postnatal retinal development in specific domains and cell types of the developing retina. However, we do not observe defects in stereotypical lamina-specific neurite stratification of retinal neuron subtypes in Sema6B^−/− or Sema6C^−/−; Sema6D^−/− retinas. These findings indicate these Class 6 transmembrane semaphorins are unlikely to serve as major PlexA receptor ligands for the assembly of murine retinal circuit laminar organization.     

Human Leukocyte Antigen and Systemic Sclerosis in Japanese: The Sign of the Four Independent Protective Alleles,DRB1*13:02, DRB1*14:06, DQB1*03:01, and DPB1*02:01

ObjectiveSeveral studies on associations between human leukocyte antigen (HLA) allele frequencies and susceptibility to systemic sclerosis (SSc) have been reported. Anti-centromere antibodies (ACA) and anti-topoisomerase I antibodies (ATA) are found in SSc patients. Here, we sought to identify HLA alleles associated with SSc in Japanese, and explored their associations with SSc phenotypes including the presence of autoantibodies.MethodsAssociations of HLA-DRB1, DQB1, and DPB1 were analyzed in 463 Japanese SSc patients and 413 controls.ResultsWe found that DRB1*13:02 (P = 0.0011, Pc = 0.0319, odds ratio [OR] 0.46, 95% confidence interval [CI] 0.29–0.73), DRB1*14:06 (P = 6.60X10^-5, Pc = 0.0020, OR 0.05, 95%CI 0.01–0.41), DQB1*03:01 (P = 0.0009, Pc = 0.0150, OR 0.56, 95%CI 0.40–0.79), and DPB1*02:01 (P = 5.16X10^-6, Pc = 8.77X10^-5, OR 0.52, 95%CI 0.39–0.69) were protectively associated with SSc. In addition, these four alleles seemed to be independently associated with the protection against the susceptibility of SSc. On the other hand, we could not find predisposing alleles for overall SSc. With respect to SSc subsets, a tendency for these four alleles to be protectively associated was observed. However, there was a significant association between DRB1*01:01, DRB1*10:01, DQB1*05:01, and DPB1*04:02 and the susceptibility to SSc with ACA. On the other hand, the presence of DRB1*15:02, DQB1*06:01, DPB1*03:01, and DPB1*09:01 was associated with SSc with ATA.ConclusionThus, the present study has identified protective associations of the four HLA class II alleles with overall Japanese SSc and predisposing associations of HLA class II alleles with Japanese SSc subsets.     

An enzyme combination assay for serum sphingomyelin: Improved specificity through avoiding the interference with lysophosphatidylcholine

Serum sphingomyelin (SM) has predictive value in the development of atherosclerosis. Furthermore, SM plays important roles in cell membrane structure, signal transduction pathways, and lipid raft formation. A convenient enzymatic method for SM is available for routine laboratory practice, but the enzyme specificity is not sufficient because of nonspecific reactions with lysophosphatidylcholine (LPC). Based on the differential specificity of selected enzymes toward choline-containing phospholipids, a two-step assay for measuring SM was constructed and its performance was evaluated using sera from healthy individuals on a Hitachi 7170 autoanalyzer. Results from this assay were highly correlated with theoretical serum SM concentrations estimated by subtracting phosphatidylcholine (PC) and LPC concentrations from that of total phospholipids determined using previously established methods. There was a good correlation between the results of SM assayed by the proposed method and the existing enzymatic method in sera from healthy individuals. Moreover, the proposed method was superior to the existing method in preventing nonspecific reactions with LPC present in sera. The proposed method does not require any pretreatment, uses 2.5 μl of serum samples, and requires only 10 min on an autoanalyzer. This high-throughput method can measure serum SM with sufficient specificity for clinical purposes and is applicable in routine laboratory practice.     

Phosphorylated TandeMBP: A unique protein substrate for protein phosphatase assay

To analyze a variety of protein phosphatases, we developed phosphorylated TandeMBP (P-TandeMBP), in which two different mouse myelin basic protein isoforms were fused in tandem, as a protein phosphatase substrate. P-TandeMBP was prepared efficiently in four steps: (1) phosphorylation of TandeMBP by a protein kinase mixture (Ca²⁺/calmodulin-dependent protein kinase Iδ, casein kinase 1δ, and extracellular signal-regulated kinase 2); (2) precipitation of both P-TandeMBP and protein kinases to remove ATP, Pi, and ADP; (3) acid extraction of P-TandeMBP with HCl to remove protein kinases; and (4) neutralization of the solution that contains P-TandeMBP with Tris. In combination with the malachite green assay, P-TandeMBP can be used to detect protein phosphatase activity without using radioactive materials. Moreover, P-TandeMBP served as an efficient substrate for PPM family phosphatases (PPM1A, PPM1B, PPM1D, PPM1F, PPM1G, PPM1H, PPM1K, and PPM1M) and PPP family phosphatase PP5. Various phosphatase activities were also detected with high sensitivity in gel filtration fractions from mouse brain using P-TandeMBP. These results indicate that P-TandeMBP might be a powerful tool for the detection of protein phosphatase activities.     

Minority species influences microbiota formation: the role of Bifidobacterium with extracellular glycosidases in bifidus flora formation in breastfed infant guts

The human body houses a variety of microbial ecosystems, such as the microbiotas on the skin, in the oral cavity and in the digestive tract. The gut microbiota is one such ecosystem that contains trillions of bacteria, and it is well established that it can significantly influence host health and diseases. With the advancement in bioinformatics tools, numerous comparative studies based on 16S ribosomal RNA (rRNA) gene sequences, metabolomics, pathological and epidemical analyses have revealed the correlative relationship between the abundance of certain taxa and disease states or amount of certain causative bioactive compounds. However, the 16S rRNA-based taxonomic analyses using next-generation sequencing (NGS) technology essentially detect only the majority species. Although the entire gut microbiome consists of 10¹³ microbial cells, NGS read counts are given in multiples of 10⁶, making it difficult to determine the diversity of the entire microbiota. Some recent studies have reported instances where certain minority species play a critical role in creating locally stable conditions for other species by stabilizing the fundamental microbiota, despite their low abundance. These minority species act as ‘keystone species’, which is a species whose effect on the community is disproportionately large compared to its relative abundance. One of the attributes of keystone species within the gut microbiota is its extensive enzymatic capacity for substrates that are rare or difficult to degrade for other species, such as dietary fibres or host-derived complex glycans, like human milk oligosaccharides (HMOs). In this paper, we propose that more emphasis should be placed on minority taxa and their possible role as keystone species in gut microbiota studies by referring to our recent studies on HMO-mediated microbiota formation in the infant gut.     

Structural basis of the catalytic reaction mechanism of novel 1,2-alpha-L-fucosidase from Bifidobacterium bifidum

1,2-alpha-L-fucosidase (AfcA), which hydrolyzes the glycosidic linkage of Fucalpha1-2Gal via an inverting mechanism, was recently isolated from Bifidobacterium bifidum and classified as the first member of the novel glycoside hydrolase family 95. To better understand the molecular mechanism of this enzyme, we determined the x-ray crystal structures of the AfcA catalytic (Fuc) domain in unliganded and complexed forms with deoxyfuconojirimycin (inhibitor), 2'-fucosyllactose (substrate), and L-fucose and lactose (products) at 1.12-2.10 A resolution. The AfcA Fuc domain is composed of four regions, an N-terminal beta region, a helical linker, an (alpha/alpha)6 helical barrel domain, and a C-terminal beta region, and this arrangement is similar to bacterial phosphorylases. In the complex structures, the ligands were buried in the central cavity of the helical barrel domain. Structural analyses in combination with mutational experiments revealed that the highly conserved Glu566 probably acts as a general acid catalyst. However, no carboxylic acid residue is found at the appropriate position for a general base catalyst. Instead, a water molecule stabilized by Asn423 in the substrate-bound complex is suitably located to perform a nucleophilic attack on the C1 atom of L-fucose moiety in 2'-fucosyllactose, and its location is nearly identical near the O1 atom of beta-L-fucose in the products-bound complex. Based on these data, we propose and discuss a novel catalytic reaction mechanism of AfcA.     

Effects of aerobic exercise on metabolic syndrome improvement in response to weight reduction

Objective: The objective was to test effects of aerobic exercise training on metabolic syndrome (MetSyn) improvement in response to weight reduction. Research Methods and Procedures: A total of 459 overweight and obese women (age, 49 ± 9 years; BMI, 28 ± 3 kg/m²) were recruited for a baseline examination to test the relationship between cardiorespiratory fitness and metabolic syndrome prevalence; among these, 67 subjects with MetSyn were treated with 14‐week weight‐loss programs, which included low‐calorie diet and aerobic exercise. The MetSyn was defined according to the Examination Committee of Criteria for “Metabolic Syndrome” in Japan. Maximal oxygen uptake (V?o _2max) during a maximal cycling test was measured as an index of cardiorespiratory fitness at baseline and after the intervention. Results: In the baseline examination, age‐ and BMI‐adjusted odds ratios for MetSyn prevalence in the low, middle, and upper thirds of V?o _2max were 1.0 (referent), 0.50 (95% confidence interval, 0.26 to 0.95), and 0.39 (95% confidence interval, 0.14 to 0.96), respectively (linear trend, p = 0.02). The adjusted odds ratios for MetSyn improvement in the two interventions with diet alone and diet plus exercise were 1.0 and 3.68 (95% confidence interval, 1.02 to 17.6; p = 0.04), respectively. Discussion: These results suggest that adding aerobic exercise training to a dietary weight‐reduction program further improves MetSyn (adjusted odds ratio, 3.68) in obese women, compared with diet alone. Further studies on an association between V?o _2max change and MetSyn improvement are needed.