Frequent SLC12A3 mutations in Chinese Gitelman syndrome patients: structure and function disorder

Purposes: This study was conducted to identify the frequent mutations from reported Chinese Gitelman syndrome (GS) patients, to predict three-dimensional structure change of human Na-Cl co-transporter (hNCC), and to test the activity of these mutations and some novel mutations in vitro and in vivo. Methods: SLC12A3 gene mutations in Chinese GS patients previously reported in the PubMed, CNKI and Wanfang database were summarized. Predicted configurations of wild type (WT) and mutant proteins were achieved using the I-TASSER workplace. Six missense mutations (T60M, L215F, D486N, N534K, Q617R and R928C) were generated by site-directed mutagenesis. 22Na+ uptake experiment was carried out in the Xenopus laevis oocyte expression system. 35 GS patients and 20 healthy volunteers underwent the thiazide test. Results: T60M, T163M，D486N, R913Q, R928C and R959 frameshift were frequent SLC12A3 gene mutations (mutated frequency >3%) in 310 Chinese GS families. The protein’s three-dimensional structure was predicted to be altered in all mutations. Compared with WT hNCC, the thiazide-sensitive 22Na+ uptake was significantly diminished for all 6 mutations: T60M 22±9.2%, R928C 29±12%, L215F 38±14%, N534K 41±15.5%, Q617R 63±22.1% and D486N 77±20.4%. In thiazide test, the net increase in chloride fractional excretion in 20 healthy controls was significantly higher than GS patients with or without T60M or D486N mutations. Conclusions: Frequent mutations (T60M, D486N, R928C) and novel mutations (L215F, N534K and Q617R) lead to protein structure alternation and protein dysfunction verified by 22Na+ uptake experiment in vitro and thiazide test on patients.

Preliminary Morphological Study and Food Uptake Experiment for Transmission of Gregarine Infection from Infected Bivalves, Anadara cornea (Reeve, 1844) to the Mud Crab Genus Scylla as an Intermediate Host.

Objective: Research about gregarine become important due to the problem reported by this parasite especially in commercial bivalve i.e. Oyster. Diagnose of these parasites are important to secure the aquaculture industry in the future. Due to the advanced technologies nowadays, this research regarding to these parasites are become relevant to be study. The objective of this study is to determine the occurrence of gregarine parasites in wild mud crab and the food uptake transmission of gregarine infection from infected bivalves, Anadara cornea (Reeve, 1844) to the mud crab genus Scylla. Result: Preliminary study show that high prevalence of infection was reported in the Hairy Cockle (Anadara cornea) from Setiu Lagoon, Malaysia. From the analysis, the infection intensity was high and each phagocyte (Pha) contain maximum of 15 oocyst (Oc). Each oocyst has a single cell wall, longitudinal shape and contained sporozoite (Sz). Parasitophorous Vacuole (Pv) cover by membrane wall. There is no transmission of parasites reported in this experiment.

Cell-to-Medium Concentration Ratio Overshoot in the Uptake of Statins by Human Hepatocytes in Suspension, but Not in Monolayer: Kinetic Analysis Suggesting a Partial Loss of Functional OATP1Bs

Suspended human hepatocytes (SHH) have long been used in assessing hepatic drug uptake, while plated human hepatocytes in short-term monolayer culture (PHH) have gained use in recent years. This study aimed to cross-evaluate SHH and PHH in measuring the hepatic uptake mediated by organic anion transporting polypeptide 1Bs (OATP1Bs). We compared the time courses of cell-to-medium (C/M) concentration ratios and initial uptake clearance values of the OATP1B substrates (pitavastatin, rosuvastatin, cerivastatin, pravastatin, dehydropravastatin, and SC-62807) between SHH and PHH. For all compounds except cerivastatin, the C/M ratios in SHH displayed an apparent overshoot (an initial increase followed by a decrease) during the 180-min uptake experiment, but not in PHH. Based on the literature evidence suggesting the possible internalization of OATP1Bs in primary hepatocytes, separate experiments measured the drug uptake after varying lengths of pre-incubation in the drug-free medium. The initial uptake clearances of pitavastatin and rosuvastatin declined in SHH beyond an apparent threshold time of 20-min drug-free pre-incubation, but not in PHH. Kinetic modeling quantitatively captured the decline in the active uptake clearance in SHH, and more than half of the active uptake clearances of pitavastatin and rosuvastatin were prone to loss during the 180-min uptake experiment. These results suggested a partial, time-delayed loss of the functional OATP1Bs in SHH upon prolonged incubation. Our results indicate that PHH is more appropriate for experiments where a prolonged incubation is required, such as estimation of unbound hepatocyte-to-medium concentration ratio (Kp,uu) at the steady-state.

Transparency, ultraviolet transmittance, and miscibility of poly(lactic acid)/poly(methyl methacrylate) blends

Poly(lactic acid)/poly(methyl methacrylate) (PLA/PMMA) blends were prepared by melt compounding technique. The miscibility of PLA/PMMA blends at various blending ratios (i.e. 80/20, 60/40, 40/60, and 20/80) was investigated using a dynamic mechanical analyzer (DMA) and solvent uptake experiment. The solvent uptake experiment was conducted to estimate the interaction of PLA/PMMA blends based on the calculation of Flory–Huggins interaction parameter ( χ12). DMA results revealed that only single glass transition temperature ( Tg) existed along the PLA/PMMA blends. Smallest χ12 (i.e. −0.03) was found on the PLA/PMMA20 blend, suggesting interaction between PLA and PMMA at this composition. The incorporation of PMMA slightly improved the UV protection properties of the PLA/PMMA blend, while maintaining their optical transparency.

PENGARUH TINGKAT KEASAMAN PADA KARAKTERISTIK ZEOLIT ALAM SEBAGAI ADSORBEN AMMONIUM HIDROKSIDA (NH4OH)

Natural zeolite is a mineral that are widely available in Indonesia. Among many other benefits, the natural zeolite can be used as an adsorbent to reduce water pollutant caused by ammonia compounds and combination of derivatives. This research aimed to study the effect of different acids on zeolite characterizatics as adsorbent of ammonium hydroxide (NH4OH). The effect of acids addition on natural zeolite was studied by varying the concentration of H2SO4(0,5 N; 3 N and 5 N) and concentration of H3PO4 (0,5 N; 3 N and 5 N). In this study, zeolite surface was observed by Fourier Transform Infrared (FTIR). Results were showed that the amount of NH4OH adsorbed on H- zeolite has a maximum (23,6 mg/gram zeolit) at 5 N H2SO4. From the NH4OH uptake experiment, it was noted that zeolite obtained from H-Zeolite-H2SO4 has higher NH4OH adsorption capacity than that of zeolite obtained from H-Zeolite-H3PO4. The adsorption capacity of H-Zeolite-H2SO4, H-Zeolite-H3PO4, are 23,6 mg/g and 12,9 mg/g, respectively. FTIR analysis was showed that NH4OH adsorbed on the surface of H- zeolite on Brønsted acid peak.

Transport of Kynurenic Acid by Rat Organic Anion Transporters rOAT1 and rOAT3: Species Difference between Human and Rat in OAT1

A tryptophan catabolite, kynurenic acid, is involved in schizophrenia and uremia; there is little information on the mechanism of its disposition. Recently, our laboratory showed that kynurenic acid is a good substrate of human organic anion transporters hOAT1 and hOAT3. In this study, we performed uptake experiment using Xenopus laevis oocytes to characterize the transport of kynurenic acid by rat homologs of the transporters, rOAT1, and rOAT3. These transporters stimulated the uptake of kynurenic acid into oocytes, and transport by rOAT3 was marked. The Km values of the transport were estimated to be 8.46 μM for rOAT1 and 4.81 μM for rOAT3, and these values are comparable to their human homologs. The transport activity of kynurenic acid by rOAT1 was about one quarter of that of p-aminohippurate, although they were at the similar levels in hOAT1. A comparative experiment with hOAT1 was added in this study, showing that uptake amounts of kynurenic acid by hOAT1-expressing oocytes were 4 times greater than rOAT1-expressing oocytes. rOAT3 transported kynurenic acid as efficiently as estrone sulfate; this phenomenon was also observed in hOAT3. In conclusion, transport of kynurenic acid by rOAT1 and rOAT3 was shown. The characteristics of rOAT3 were similar to hOAT3, but low transport activity of kynurenic acid by rOAT1 was exhibited compared with hOAT1.

Re-plumbing in a Mediterranean sponge

Observations are reported for Dysidea avara sponges where once functioning oscula (outlets) are converted through internal re-plumbing into functioning oversized ostia (OSO; inlets). Flow tank studies employed high-speed photography and particle tracking of laser-illuminated 0.5–6.0 μm diameter glass beads to trace particles streaming into OSO. A fluorescein dye/glass bead uptake experiment showed that an oversized ostium was connected through internal structures to the lone osculum. Beginning 30 s after uptake and continuing over a 20 min period, dye streamed from the osculum, but no beads emerged. Scanning electron microscopy revealed that beads were deposited only on the inhalant side of particle filtering choanocyte chambers and not on the exhalant side, suggesting that internal re-plumbing had occurred. Functioning OSO were also found on freshly collected specimens in the field, making it highly unlikely that formation of OSO was only an artefact of sponges being held in a laboratory tank.