Quantitative Estimation of Urate Transport in Nephrons in Relation to Urinary Excretion Employing Benzbromarone-Loading Urate Clearance Tests in Cases of Hyperuricemia

Background: A four-component system for urate transport in nephrons has been proposed and widely investigated by various investigators studying the mechanisms underlying urinary urate excretion. However, quantitative determinations of urate transport have not been clearly elucidated yet. Methods: The equation Cua = {Ccr(1 – R1) + TSR}(1 – R2) was designed to approximate mathematically urate transport in nephrons, where R1 = urate reabsorption ratio; R2 = urate postsecretory reabsorption ratio; TSR = tubular secretion rate; Cua = urate clearance, and Ccr = creatinine clearance . To investigate relationships between the three unknown variables (R1, R2, and TSR), this equation was expressed as contour lines of one unknown on a graph of the other two unknowns. Points at regular intervals on each contour line for the equation were projected onto a coordinate axis and the high-density regions corresponding to high-density intervals of a coordinate were investigated for three graph types. For benzbromarone (BBR)-loading Cua tests, Cua was determined before and after oral administration of 100 mg of BBR and CuaBBR(∞) was calculated from the ratio of CuaBBR(100)/Cua. Results: Before BBR administration, points satisfying the equation on the contour line for R1 = 0.99 were highly dense in the region R2 = 0.87–0.92 on all three graphs, corresponding to a TSR of 40–60 ml/min in hyperuricemia cases (HU). After BBR administration, the dense region was shifted in the direction of reductions in both R1 and R2, but TSR was unchanged. Under the condition that R1 = 1 and R2 = 0, urate tubular secretion (UTS) was considered equivalent to calculated urinary urate excretion (Uex) in a model of intratubular urate flow with excess BBR; CuaBBR(∞) = TSR was deduced from the equation at R1 = 1 and R2 = 0. In addition, TSR of the point under the condition that R1 = 1 and R2 = 0 on the graph agreed with TSR for the dense region at excess BBR. TSR was thus considered approximately equivalent to CuaBBR(∞), which could be determined from a BBR-loading Cua test. Approximate values for urate glomerular filtration, urate reabsorption, UTS, urate postsecretory reabsorption (UR2), and Uex were calculated as 9,610; 9,510; 4,490; 4,150, and 440 µg/min for HU and 6,890; 6,820; 4,060; 3,610, and 520 µg/min for normal controls (NC), respectively. The most marked change in HU was the decrease in TSR (32.0%) compared to that in NC, but UTS did not decrease. Calculated intratubular urate contents were reduced more by higher UR2 in HU than in NC. This enhanced difference resulted in a 15.4% decrease in Uex for HU. Conclusion: Increased UR2 may represent the main cause of urate underexcretion in HU

Functional Characterization of YM928, a Novel Noncompetitive ␣-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Antagonist

ABSTRACT The ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor is thought to play an important role in the pathogenesis of several neurological disorders as well as normal brain function. The search for AMPA receptor antagonists as potential therapeutics is ongoing. Here, we describe the functional characterization of a novel noncompetitive AMPA receptor antagonist, 2-[N-(4-chlorophenyl)-N-methylamino]-4H-pyrido[3,2-e]-1,3-thiazin-4-one (YM928). This compound inhibited AMPA receptor-mediated toxicity in primary rat hippocampal cultures with an IC 50 of 2 M. Its manner of inhibition was noncompetitive as the agonist concentration was increased. YM928 blocked AMPA-induced intracellular calcium influx with an IC 50 of 3 M and antagonized AMPA-induced inward currents with an IC 50 of 1 M in cultured cells. YM928 displaced neither [ 3 H]AMPA binding nor other existing glutamate receptor-related ligand binding in rat brain membranes. In terms of in vivo activity, YM928 had an anticonvulsant effect in sound-induced seizures in DBA/2 mice 45 min after oral administration at 3 mg/kg. Thus, YM928 has potential as an oral therapeutic drug for various types of neurological disorders

Efficient depolymerization of polyethylene terephthalate (PET) and polyethylene furanoate by engineered PET hydrolase Cut190

The enzymatic recycling of polyethylene terephthalate (PET) can be a promising approach to tackle the problem of plastic waste. The thermostability and activity of PET-hydrolyzing enzymes are still insufficient for practical application. Pretreatment of PET waste is needed for bio-recycling. Here, we analyzed the degradation of PET films, packages, and bottles using the newly engineered cutinase Cut190. Using gel permeation chromatography and high-performance liquid chromatography, the degradation of PET films by the Cut190 variant was shown to proceed via a repeating two-step hydrolysis process; initial endo-type scission of a surface polymer chain, followed by exo-type hydrolysis to produce mono/bis(2-hydroxyethyl) terephthalate and terephthalate from the ends of fragmented polymer molecules. Amorphous PET powders were degraded more than twofold higher than amorphous PET film with the same weight. Moreover, homogenization of post-consumer PET products, such as packages and bottles, increased their degradability, indicating the importance of surface area for the enzymatic hydrolysis of PET. In addition, it was required to maintain an alkaline pH to enable continuous enzymatic hydrolysis, by increasing the buffer concentration (HEPES, pH 9.0) depending on the level of the acidic products formed. The cationic surfactant dodecyltrimethylammonium chloride promoted PET degradation via adsorption on the PET surface and binding to the anionic surface of the Cut190 variant. The Cut190 variant also hydrolyzed polyethylene furanoate. Using the best performing Cut190 variant (L136F/Q138A/S226P/R228S/D250C-E296C/Q123H/N202H/K305del/L306del/N307del) and amorphous PET powders, more than 90 mM degradation products were obtained in 3 days and approximately 80 mM in 1 day

Cellular analysis of SOD1 protein-aggregation propensity and toxicity: a case of ALS with slow progression harboring homozygous SOD1-D92G mutation

Mutations within Superoxide dismutase 1 (SOD1) cause amyotrophic lateral sclerosis (ALS), accounting for approximately 20% of familial cases. The pathological feature is a loss of motor neurons with enhanced formation of intracellular misfolded SOD1. Homozygous SOD1-D90A in familial ALS has been reported to show slow disease progression. Here, we reported a rare case of a slowly progressive ALS patient harboring a novel SOD1 homozygous mutation D92G (homD92G). The neuronal cell line overexpressing SOD1-D92G showed a lower ratio of the insoluble/soluble fraction of SOD1 with fine aggregates of the misfolded SOD1 and lower cellular toxicity than those overexpressing SOD1-G93A, a mutation that generally causes rapid disease progression. Next, we analyzed spinal motor neurons derived from induced pluripotent stem cells (iPSC) of a healthy control subject and ALS patients carrying SOD1-homD92G or heterozygous SOD1-L144FVX mutation. Lower levels of misfolded SOD1 and cell loss were observed in the motor neurons differentiated from patient-derived iPSCs carrying SOD1-homD92G than in those carrying SOD1-L144FVX. Taken together, SOD1-homD92G has a lower propensity to aggregate and induce cellular toxicity than SOD1-G93A or SOD1-L144FVX, and these cellular phenotypes could be associated with the clinical course of slowly progressive ALS

Associations in tumor infiltrating lymphocytes between clinicopathological factors and clinical outcomes in estrogen receptor-positive/human epidermal growth factor receptor type 2 negative breast cancer

The value of assessing tumor infiltrating lymphocytes (TILs) in estrogen receptor (ER) positive/human epidermal growth factor receptor type 2 (HER2) negative breast cancer has yet to be determined. In the present study, a total of 184 cases with early distant recurrence detected within 5 years following the primary operation, 134 with late distant recurrence diagnosed following 5 years or longer and 321 controls without recurrence for >10 years following starting the initial treatment for ER-positive/HER2 negative breast cancer, registered in 9 institutions, were analyzed. The distributions of TILs and their clinical relevance were investigated. TIL distributions did not differ significantly among the early, late and no recurrence groups, employing a 30% cut-off point as a dichotomous variable. In those who had received adjuvant chemotherapy as well as endocrine therapy, a trend toward higher TIL proportions was detected when the early recurrence group was compared with the no recurrence group employing the 30% cut-off point (P=0.064). The TIL distributions were significantly associated with nodal metastasis (P=0.004), ER status (P=0.045), progesterone receptor (PgR) status (P=0.002), tumor grade (P=0.021), and the Ki67 labeling index (LI) (P=0.002) in the no recurrence group and with the Ki67 LI in the recurrence groups (P=0.002 in early recurrence group, P=0.023 in late recurrence group). High TIL distributions also predicted shorter survival time following the detection of recurrence (P=0.026). However, these prognostic interactions were not significant in multivariate analysis (P=0.200). The present retrospective study demonstrated no significant interaction between TIL proportions and the timing of recurrence. However, higher TIL proportions were observed in breast cancer patients with aggressive biological phenotypes, which tended to be more responsive to chemotherapy. The clinical relevance of stromal TILs for identifying patients who would likely benefit from additional therapies merits further investigation in a larger patient population

Clinicopathological factors predicting early and late distant recurrence in estrogen receptor-positive, HER2-negative breast cancer

Background: Most studies analyzing prognostic factors for late relapse have been performed in postmenopausal women who received tamoxifen or aromatase inhibitors as adjuvant endocrine therapy for estrogen receptor (ER)-positive breast cancer. Methods: A total of 223 patients (108 premenopausal and 115 postmenopausal) with early distant recurrence and 149 patients (62 premenopausal and 87 postmenopausal) with late distant recurrence of ER-positive, HER2-negative breast cancer who were given their initial treatment between 2000 and 2004 were registered from nine institutions. For each late recurrence patient, approximately two matched control patients without relapse for more than ten years were selected. Clinicopathological factors and adjuvant therapies were compared among the three groups by menopausal status and age. Results: Factors predicting early recurrence in premenopausal women were large tumor size, high lymph node category and high tumor grade, whereas predictors for late recurrence were large tumor size and high lymph node category. In postmenopausal women under 60 years of age, factors predicting early recurrence were bilateral breast cancer, large tumor size, high lymph node category, low PgR expression and high Ki67 labeling index (LI), while predictors for late recurrence were large tumor size and high lymph node category. On the other hand, in postmenopausal women aged 60 years or older, factors predicting early recurrence were bilateral breast cancer, large tumor size, high lymph node category, high tumor grade, low ER expression and high Ki67 LI, whereas predictors for late recurrence were high lymph node category, low ER expression and short duration of adjuvant endocrine therapy. Conclusion: Predictors of early and late distant recurrence might differ according to menopausal status and age

Time-resolved serial femtosecond crystallography reveals early structural changes in channelrhodopsin

X線自由電子レーザーを用いて、光照射によるチャネルロドプシンの構造変化の過程を捉えることに成功. 京都大学プレスリリース. 2021-03-26.Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light . Light absorption causes retinal chromophore isomerization and subsequent protein conformational changes visualized as optically distinguished intermediates, coupled with channel opening and closing. However, the detailed molecular events underlying channel gating remain unknown. We performed time-resolved serial femtosecond crystallographic analyses of ChR by using an X-ray free electron laser, which revealed conformational changes following photoactivation. The isomerized retinal adopts a twisted conformation and shifts toward the putative internal proton donor residues, consequently inducing an outward shift of TM3, as well as a local deformation in TM7. These early conformational changes in the pore-forming helices should be the triggers that lead to opening of the ion conducting pore