Simultaneous determination of estriol and estriol 3-sulfate in serum by column-switching semi-micro high-performance liquid chromatography with ultraviolet and electrochemical detection

A column-switching HPLC with semi-microcolumn enabled us a direct and simultaneous analysis of estriol (E3) and estriol 3-sulfate (E3 S) in human serum in combination with ultraviolet (for E3 S) and electrochemical (for E3) detectors. The mobile phases (phosphate buffer pH 7.0) contained 5 mM tetra-n-butylammonium ion (TBA) as a counter ion for E3 S. Serum samples were diluted with 200 mM phosphate buffer (pH 7.0) containing 100 mM TBA, then injected to the pre-column. After serum proteins had flowed out from the pre-column, E3 and E3 S were transferred to the enrichment column. Subsequently the analytes were eluted to the analytical column. Detection limits of E3 and E3 S in human serum were 2.5 ng/ml and 295 ng/ml. Serum E3 and E3 S levels (mean±SD) of umbilical artery from 18 full-term healthy neonates were 33±23 ng/ml and 1.26±0.69 μg/ml, respectively.     

MMP-7 cleaves amyloid β fragment peptides and copper ion inhibits the degradation

The extracellular deposition of amyloid β (Aβ) is known to be the fundamental cause of Alzheimer’s disease (AD). Aβ1-42, generated by β-secretases from the amyloid precursor protein (APP), is the main component of neuritic plaque, and the aggregation of this protein is shown to be dependent to an extent on metal ions such as copper and zinc. However, the mechanism by which Cu²⁺ affects the physicochemical properties of Aβ1-42 or the central nervous system is still under debate. A recent series of studies have demonstrated that both the soluble-type matrix metalloproteinases (MMP-2 and MMP-9) and the membrane-type matrix metalloproteinase (MT1-MMP) are capable of degrading Aβ peptides. MMP-7, one of the soluble-type matrix metalloproteinases, is expressed in hippocampal tissue; however, less information is available concerning the pathophysiological roles of this enzyme in the process and/or progress of Alzheimer’s disease. In this study, we examined the degradation activity of MMP-7 against various Aβ1-42’s fragment peptides and the effect of Cu²⁺. Although Aβ22-40 was degraded by MMP-7 regardless of Cu²⁺, Cu²⁺ inhibited the degradation of Aβ1-19, Aβ11-20, and Aβ11-29 by MMP-7. These results indicate that MMP-7 is capable of degrading Aβ1-42, and that Aβ1-42 acquired resistance against MMP-7 cleavage through Cu²⁺-binding and structure changes. Our results demonstrate that MMP-7 may play an important role in the defensive mechanism against the aggregation of Aβ1-42, which gives rise to the pathology of AD.     

Constant light disrupts the circadian but not the circatidal rhythm in mangrove crickets

Mangrove crickets have a circatidal activity rhythm (~12.6 h cycles) with a circadian modulation under constant darkness (DD), whereby activity levels are higher during subjective night low tides than subjective day low tides. This study explored the locomotor activity rhythm of mangrove crickets under constant light (LL). Under LL, the crickets also exhibited a clear circatidal activity rhythm with a free-running period of 12.6 ± 0.26 h (mean ± SD, n = 6), which was not significantly different from that observed under DD. In contrast, activity levels were almost the same between subjective day and night, unlike those under DD, which were greater during subjective night. The loss of circadian modulation under LL may be explained by the suspension of the circadian clock in these conditions. These results strongly suggest that the circatidal activity rhythm is driven by its own clock system, distinct from the circadian clock.     

Aflatoxin M<Subscript>1</Subscript> in human breast milk in southeastern Turkey

This study was performed to determine aflatoxin M₁ (AFM₁) in human breast milk samples collected in ?anl?urfa, located in Southeastern region of Turkey, and to investigate a possible correlation between AFM₁ occurrence (frequency and levels) and sampling seasons. Human breast milk samples collected in December 2014 and in June 2015 from a total of 74 nursing women, both outpatient and inpatient volunteers in hospitals located in ?anl?urfa, Turkey, were analyzed using competitive enzyme-linked immunosorbent assay (ELISA) for the presence of AFM₁. AFM₁ was detected in 66 (89.2%) out of 74 samples at an average concentration of 19.0 ± 13.0 ng/l (min.-max., 9.6–80 ng/l). There was a statistically significant difference between December and June concerning AFM₁ levels (p < 0.05). Further detailed studies will be needed to determine the main sources of aflatoxins in food, to establish protection strategies against maternal and infant exposure to these mycotoxins.     

Genetic characterization of an avian H4N6 influenza virus isolated from the Izumi plain,Japan

An influenza A virus of H4N6 subtype was isolated from the Izumi plain, Japan, in 2013. Genetic analyses revealed that two viral genes (M and NS gene segments) of this isolate were genetically distinct from those of the H4N6 virus isolated from the same place in 2012. Furthermore, three viral genes (PB2, PB1 and M gene segments) of this isolate share high similarity with those of the North American isolates of 2014. These results suggest a high frequency of genetic reassortment of avian influenza viruses in Asian waterfowl and intercontinental movements of avian influenza viruses via migratory waterfowl.

     

Muscular outputs during dynamic bench press under stable versus unstable conditions

Previous studies have suggested that resistance training exercise under unstable conditions decreases the isometric force output, yet little is known about its influence on muscular outputs during dynamic movement. The objective of this study was to investigate the effect of an unstable condition on power, force, and velocity outputs during the bench press. Twenty male collegiate athletes (mean age, 21.3 +/- 1.5 years; mean height, 167.7 +/- 7.7 cm; mean weight, 75.9 +/- 17.5 kg) participated in this study. Each subject attempted 3 sets of single bench presses with 50% of 1 repetition maximum (1RM) under a stable condition with a flat bench and an unstable condition with a Swiss ball. Acceleration data were obtained with an accelerometer attached to the center of a barbell shaft, and peak outputs of power, force, and velocity were computed. Although significant loss of the peak outputs was found under the unstable condition (p < 0.017), their reduction rates remained relatively low, approximately 6% for force and 10% for power and velocity outputs, compared with previous findings. Such small reduction rates of muscular outputs may not compromise the training effect. Prospective studies are necessary to confirm whether the resistance training under an unstable condition permits the improvement of dynamic performance and trunk stability.     

Prolactin induces MFG-E8 production in macrophages via transcription factor C/EBPβ-dependent pathway

The lactogenic hormone prolactin (PRL) regulates milk protein gene expression in mammary glands. To maintain homeostatic balance in the body, milk fat globule epidermal growth factor 8 (MFG-E8) is vital for phagocytic clearance of apoptotic cells. We investigated the effects of PRL on MFG-E8 expression in macrophages by evaluating its promoter function. Macrophages were stimulated with PRL, and the expression of MFG-E8 was determined using real-time PCR and Western blotting. The role of MFG-E8 on phagocytosis of apoptotic cells in PRL-treated macrophages was assessed using microscopy, while the response of PRL to MFG-E8 expression was evaluated using luciferase assay. Following treatment with PRL, significant up-regulations of the PRL receptor and MFG-E8 were observed in macrophages, though PRL-treated macrophages more efficiently engulfed apoptotic cells. The results of MFG-E8 promoter analysis showed considerable up-regulation of promoter activity in macrophages following PRL treatment and results from mutation analysis of the MFG-E8 promoter suggested that the C/EBPβ binding site was responsible for PRL-induced activation of the MFG-E8 promoter. C/EBPβ activity was found to be up-regulated in PRL-treated cells as revealed by an electrophoretic mobility shift assay (EMSA). In conclusion, PRL is a potent inducer of MFG-E8 expression in macrophages, while its effect is mediated by the presence of a responsive element in the MFG-E8 promoter.     

Pan-Vertebrate Toll-Like Receptors During Evolution

Human toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs) to raise innate immune responses. The human TLR family was discovered because of its sequence similarity to fruit fly (Drosophila) Toll, which is involved in an anti-fungal response. In this review, we focus on the origin of the vertebrate TLR family highlighted through functional and phylogenetic analyses of TLRs in non-mammalian vertebrates. Recent extensive genome projects revealed that teleosts contain almost all subsets of TLRs that correspond to human TLRs (TLR1, 2, 3, 4, 5, 7, 8, and 9), whereas the urochordate Ciona intestinalis contains only a few TLR genes. Therefore, mammals likely obtained almost all TLR family members at the beginning of vertebrate evolution. This premise is further supported by several functional analyses of non-mammalian TLRs. We have summarized several teleost TLRs with unique properties distinct from mammalian TLRs to outline their specific roles. According to Takifugu rubripes genome project, the puffer fish possesses fish-specific TLR21 and 22. Surprisingly, phylogenetic analyses indicate that TLR21 and 22 emerged during an early period of vertebrate evolution in parallel with other TLRs and that the mammalian ancestor lost TLR21 and 22 during evolution. Our laboratory recently revealed that TLR22 recognizes double-strand RNA and induces interferon production through the TICAM-1 adaptor, as in TLR3, but unlike TLR3, TLR22 localizes to the cell surface. Therefore, differential expression of TLR3 and TLR22, rather than simple redundancy of RNA sensors, may explain the effective protection of fish from RNA virus infection in the water. In this review, we summarize the similarities and differences of the TLR family in various vertebrates and introduce these unique TLRs for a possible application to the field of clinical practices for cancer or virus infection.     

4-(Anilino)pyrrole-2-carboxamides: Novel non-steroidal/non-anilide type androgen antagonists effective upon human prostate tumor LNCaP cells with mutated nuclear androgen receptor

Various 4-(anilino)pyrrole-2-carboxamides were designed and synthesized as novel androgen receptor (AR) antagonists without steroidal or anilide structure, based on our strategy for developing full antagonists of nuclear receptors. Introduction of a bulky N-alkyl group, such as a cyclohexylmethyl or benzyl group, increased the binding affinity for wild-type AR and the potency for growth inhibition of androgen-dependent SC-3 cells. Among the compounds obtained, N-[4-[(benzyl)(4-nitrophenyl)amino]-1-methylpyrrole-2-carbonyl]pyrrolidine (22) is as potent an AR antagonist as the typical anilide-type AR antagonists hydroxyflutamide and bicalutamide. Further, compound 22 had potent binding affinity for T877A mutated AR, and dose-dependently inhibited the testosterone-induced production of prostate-specific antigen in LNCaP cells bearing T877A AR.