Human induced pluripotent stem cells (BIONi010-C) generate tight cell monolayers with blood-brain barrier traits and functional expression of large neutral amino acid transporter 1 (SLC7A5).

The barrier properties of the brain capillary endothelium, the blood-brain barrier (BBB) restricts uptake of most small and all large molecule drug compounds to the CNS. There is a need for predictive human in vitro models of the BBB to enable studies of brain drug delivery. Here, we investigated whether human induced pluripotent stem cell (hiPSC) line (BIONi010-C) could be differentiated to brain capillary endothelial- like cells (BCEC) and evaluated their potential use in drug delivery studies. BIONi010-C hIPSCs were differentiated according to established protocols. BCEC monolayers displayed transendothelial electrical resistance (TEER) values of 5,829±354 Ω∙cm2, a Papp,mannitol of 1.09±0.15 ∙ 10-6 cm∙s-1 and a Papp,diazepam of 85.7 ± 5.9 ∙ 10-6 cm ∙s-1. The Pdiazepam/Pmannitol ratio of ~80, indicated a large dynamic passive permeability range. Monolayers maintained their integrity after medium exchange. Claudin-5, Occludin, Zonulae Occludens 1 and VE-Cadherin were expressed at the cell-cell contact zones. Efflux transporters were present at the mRNA level, but functional efflux of substrates was not detected. Transferrin-receptor (TFR), Low density lipoprotein receptor-related protein 1 (LRP1) and Basigin receptors were expressed at the mRNA-level. The presence and localization of TFR and LRP1 were verified at the protein level. A wide range of BBB-expressed solute carriers (SLC's) were detected at the mRNA level. The presence and localization of SLC transporters GLUT1 and LAT1 was verified at the protein level. Functional studies revealed transport of the LAT1 substrate [3H]-L-Leucine and the LRP1 substrate angiopep-2. In conclusion, we have demonstrated that BIONi010-C-derived BCEC monolayers exhibited, BBB properties including barrier tightness and integrity, a high dynamic range, expression of some of the BBB receptor and transporter expression, as well as functional transport of LAT1 and LRP1 substrates. This suggests that BIONi010-C-derived BCEC monolayers may be useful for studying the roles of LAT-1 and LRP1 in brain drug delivery.

Properties of the V-type ATPase from the excretory system of the usherhopper, Poekilocerus bufonius.

The bafilomycin A(1) and N-ethylmaleimide (NEM)-sensitive (V-type) ATPase was partially purified from the apical membrane-rich fractions of excretory system (Malpighian tubules and hind gut) of P. bufonius. Enzymatic activity was inhibited by bafilomycin A(1) (IC(50) = 1.3 nM) and NEM (IC(50) = 10.1 microM). The V-type ATPase activity is confined to the apical membrane fraction, while the activity of Na(+)/K(+) -ATPase forms the major part of the basal membrane fraction. The optimal pH required for maximal activity of V-type ATPase was pH 7.5. The effect of 30 mM of various salts on ATPase activity was investigated. NaCl and KCl caused increases of 175% and 184%, respectively. Other chloride salts also caused an increase in activity in the following ascending order: RbCl, LiCI, choline Cl, NaCI, KCl and tris-HCl. The activity of V-type ATPase was stimulated by a variety of different anions and cations, and HCO(3)(-) was found to be the most potent cationic activator of ATPase activity. The present results show that the properties of V-type ATPase of P. bufonius are similar to those reported for other insect tissues.

An enzyme-linked immunosorbent assay using Schistosoma mansoni purified egg antigen for the diagnosis of schistosomiasis in Saudi Arabia.

The enzyme-linked immunosorbent assay (ELISA) using purified Schistosoma mansoni egg antigen (CEF6) was compared with standard parasitological diagnostic tests (potassium hydroxide digestion of faeces and urine filtration) for diagnosis of schistosomiasis in humans in Saudi Arabia. Faecal, urine and finger-prick blood samples were collected from 2 groups of individuals of both sexes, aged 1-50 years, in 2 areas in the western region of Saudi Arabia: 983 in a schistosomiasis endemic area (Al-Hijaz highlands) and 192 in a non-endemic area (Jeddah). In the non-endemic area, almost 90% of the blood samples gave optical density readings at 492 nm (OD) < 0.25. The mean OD in the schistosome endemic area (0.31) was much higher than in the non-endemic area (0.14). The prevalence of S. mansoni infection by faecal examination in the endemic area was 10.2% and the specificity and sensitivity of the ELISA using a cut-off OD of 0.25 were 55% and 90%, respectively. In the endemic area, there was a positive correlation between egg intensity and OD value. No S. haematobium was detected. In the non-endemic area, the specificity was 90%. The main reasons for false positive results may have been inapparent or cured S. mansoni infection.

Lichen acids as uncouplers of oxidative phosphorylation of mouse-liver mitochondria.

Three lichen acids-namely, (+)usnic acid, vulpinic acid, and atranorin-were isolated from three lichen species (Usnea articulata, Letharia vulpina, and Parmelia tinctorum, respectively). The effects of these lichen products on mice-liver mitochondrial oxidative functions in various respiratory states and on oxidative phosphorylation were studied polarographically in vitro. The lichen acids exhibited characteristics of the 2,4-dinitrophenol (DNP), a classical uncoupler of oxidative phosphorylation. Thus, they released respiratory control and oligomycin inhibited respiration, hindered ATP synthesis, and enhanced Mg(+2)-ATPase activity. (+)Usnic acid at a concentration of 0.75 microM inhibited ADP/O ratio by 50%, caused maximal stimulation of both state-4 respiration (100%) and ATPase activity (300%). Atranorin was the only lichen acid with no significant effect on ATPase. The uncoupling effect was dose-dependent in all cases. The minimal concentrations required to cause complete uncoupling of oxidative phosphorylation were as follows: (+)usnic acid (1 microM), vulpinic acid, atranorin (5 microM) and DNP (50 microM). It was postulated that the three lichen acids induce uncoupling by acting on the inner mitochondrial membrane through their lipophilic properties and protonophoric activities.

Epidemiological studies on Schistosoma mansoni infection in the western region of Saudi Arabia using the potassium hydroxide stool digestion technique.

The potassium hydroxide stool digestion technique was used in 30 sites in the Western Region of Saudi Arabia (Hijaz highlands and Tihamah lowlands) for epidemiological studies of S. mansoni. The mean prevalence rate in 2792 individuals (1490 males and 1302 females) in Hijaz highlands (20 sites) was 6.09 (0.5-20.1%) and the geometric mean egg count of stool was 110.8 (63-285). The prevalence rate was greater in males (8.06%) than in females (3.79%). The peak prevalence in males was in the age group 11-15 years, and 31-50 years in females. In 10 sites in the Tihamah lowlands a total of 1771 persons were examined for schistosomiasis. Only one site was recorded with S. mansoni, at a prevalence rate of 0.05%; S. haematobium was recorded in two sites at prevalence rates of 0.55 and 1.13%. Praziquantel therapy (40 mg kg-1) of 170 S. mansoni cases showed a cure of 85-98% at 1-6 months after treatment and a considerable reduction in stool arithmetic mean egg count. Schistosoma haematobium was not recorded in the Hijaz highlands.

Different ouabain sensitivities of Na+/K(+)-ATPase from Poekilocerus bufonius tissues and a possible physiological cost.

1. The properties of Na+/K(+)-transporting ATPase in microsomal preparation from mid-gut of the grasshopper, Poekilocerus bufonius, were investigated and compared with the same enzyme from brain and excretory system. 2. Two components of ATPases activity are present in the three tissues studied. 3. The physiochemical properties of Na+/K(+)-transporting ATPase from mid-gut, brain and excretory system (hind-gut plus Malpighian tubules) are essentially the same. 4. The calculated values of PI50 were 2 (I50 = 1 x 10(-2) M), 3.7 (I50 = 2 x 10(-4) M) and 6.4 (I50 = 3.98 x 10(-7)) for Na+/K(+)-ATPase from mid-gut, excretory system and brain, respectively. The mid-gut contains the most ouabain-resistant Na+/K(+)-ATPase. 5. The results suggest that P. bufonius have developed some tolerance to toxic cardiac glycosides (CGS), but there is a possibility of autotoxicity as indicated by the presence of ouabain-sensitive ATPase from brain tissue. 6. It was concluded that the dissimilarities of Na+/K(+)-ATPases from different tissues of P. bufonius are probably due to tissue-dependent differences in ouabain sensitivity (or isoenzymes pattern) available in the same insect. 7. The atrophy of female flight muscle of P. bufonius suggests the possibility of physiological cost inflicted on insects consuming poisonous plants.