Micropipette Aspiration-Based Assessment of Single Channel Water Permeability.

Measurements of the unitary hydraulic conductivity of membrane channels, pf , may be hampered by difficulties in producing sufficient quantities of purified and reconstituted proteins. Low yield expression, the purely empiric choice of detergents, as well as protein aggregation and misfolding during reconstitution may result in an average of less than one reconstituted channel per large unilamellar vesicle. This limits their applicability for pf measurements, independent of whether light scattering or fluorescence quenching of encapsulated dyes is monitored. Here the micropipette aspiration technique is adopted because its superb sensitivity allows resolving pf values for one order of magnitude smaller protein densities in sphingomyelin and cholesterol rich giant unilamellar vesicles (GUVs). Protein density is derived from intensity fluctuations that fluorescently labeled channels in the aspirated GUV induce by diffusing through the diffraction limited spot. A perfusion system minimizes unstirred layers in the immediate membrane vicinity as demonstrated by the distribution of both encapsulated and extravesicular aqueous dyes. pf amounted to 2.4 ± 0.1 × 10-13 cm³ s-1 for aquaporin-1 that served as a test case. The new assay paves the way for directly monitoring the effect that interaction of aquaporins with other proteins or inhibitors may have on pf on a single sample.

Assessment of plasma biomarkers for their association with Multiple Sclerosis progression.

In a cross-sectional study involving 160 Multiple Sclerosis (MS) patients and 70 healthy controls we set out to determine the association of five blood biomarkers with MS risk and progression scores. High levels of Semaphorin3A (SEMA3A) in females, and low levels of prolactin and estradiol in males associated with MS risk. High MS disability correlated with higher SEMA3A levels in females. Our findings suggest the clinical applicability of SEMA3A, and prolactin as biomarkers for MS progression. However, these biomarkers had sex-specific associations with MS, and any therapeutic approaches utilizing them should take that into consideration.

Nonclinical safety assessment of recombinant human acid sphingomyelinase (rhASM) for the treatment of acid sphingomyelinase deficiency:the utility of animal models of disease in the toxicological evaluation of potential therapeutics.

Recombinant human acid sphingomyelinase (rhASM) is being developed as an enzyme replacement therapy for patients with acid sphingomyelinase deficiency (Niemann-Pick disease types A and B), which causes sphingomyelin to accumulate in lysosomes. In the acid sphingomyelinase knock-out (ASMKO) mouse, intravenously administered rhASM reduced tissue sphingomyelin levels in a dose-dependent manner. When rhASM was administered to normal rats, mice, and dogs, no toxicity was observed up to a dose of 30mg/kg. However, high doses of rhASM≥10mg/kg administered to ASMKO mice resulted in unexpected toxicity characterized by cardiovascular shock, hepatic inflammation, adrenal hemorrhage, elevations in ceramide and cytokines (especially IL-6, G-CSF, and keratinocyte chemoattractant [KC]), and death. The toxicity could be completely prevented by the administration of several low doses (3mg/kg) of rhASM prior to single or repeated high doses (≥20mg/kg). These results suggest that the observed toxicity involves the rapid breakdown of large amounts of sphingomyelin into ceramide and/or other toxic downstream metabolites, which are known signaling molecules with cardiovascular and pro-inflammatory effects. Our results suggest that the nonclinical safety assessment of novel therapeutics should include the use of specific animal models of disease whenever feasible.

Effect of acute exercise on the content of free sphinganine and sphingosine in different skeletal muscle types of the rat.

It has previously been shown that prolonged exercise of moderate intensity reduces the content of ceramide in each type of skeletal muscle. This was accompanied by a reduction in the activity of neutral, Mg++-dependent sphingomyelinase (the major enzyme responsible for ceramide formation from sphingomyelin) in the soleus and red gastrocnemius, but not in the white gastrocnemius (A. Dobrzyn and J. Górski, Am. J. Physiol.: Endorcinol. Metab. 282: E277 - E285, 2002). No other data on regulation of ceramide metabolism in contracting muscles are available. The aim of the present study was to examine the content of sphinganine (a key precursor of ceramide on the de novo synthesis route) and the content of sphingosine (the main product of ceramide catabolism) in different skeletal muscle types after two kinds of acute exercise. The experiments were carried out on 30 male Wistar rats, 250 - 280 g of body weight. The rats were divided equally into three groups: 1 - control, 2 - run until exhaustion (1200 m/h, +10 degree incline), 3 - a group in which the sciatic nerve was stimulated 10 min with tetanic pulses (60 pulses/min). Samples were taken of the soleus and of the red and white section of the gastrocnemius. These muscles are composed mostly of the slow-twitch oxidative, fast-twitch oxidative-glycolytic and fast-twitch glycolytic fibers, respectively. Lipids were extracted with chloroform/methanol. Sphinganine and sphingosine were quantified by high-performance liquid chromatography. At rest, the content of sphinganine in the soleus was higher than in the red gastrocnemius (p < 0.05), and in the latter, it was higher than in the white gastrocnemius (p < 0.01). Prolonged exercise increased the content of sphinganine approximately 6-fold in each muscle. The resting content of sphingosine in the soleus and in the red gastrocnemius was similar--higher than in the white gastrocnemius (p < 0.001 and p < 0.01, respectively). The content of sphingosine increased over 3-fold in the soleus and nearly 2-fold in the red and white sections of the gastrocnemius. Stimulation of the sciatic nerve increased the content of both compounds approximately 2-fold in each muscle. We conclude that acute exercise increases both de novo synthesis and catabolism of ceramide in skeletal muscles. Accumulation of sphingosine in contracting muscles may contribute to the development of fatigue.

Assessment of foetal lung maturity.

An important prerequisite for the management of high risk pregnancies is the accurate prediction of foetal lung maturity. A number of indices of foetal lung maturity based on the determination of surfactant constituents in the amniotic fluid have been proposed. Amniotic fluid contains phospholipids, including phosphatidylcholine (lecithin), sphingomyelin, phosphatidylinositol and phosphatidylglyerol (PG), some enzymes of the pathways of phospholipid synthesis, lamellar bodies, and lung specific apoproteins. The amount of these substances in amniotic fluid changes towards the end of gestation in a manner related to foetal lung maturity. Determination of the lecithin to sphingomyelin (L/S) ratio is by far the most widely used and accepted method. However, there is still controversy regarding the high incidence of false immature values, and the increased incidence of false mature values (from 1 to 15%) especially in pregnancies complicated by diabetes mellitus; an immature L/S ratio may predict respiratory distress syndrome (RDS) only in about 50% of cases. The incidence of false immature L/S ratio as well as other amniotic fluid tests depends upon patient variability, method employed, threshold taken for differentiating a normal from an abnormal condition, and on the fact that only few authors report their results in terms of sensitivity and specificity. Where laboratory facilities are minimal, it is advisable to perform the shake test or to measure the optical density of amniotic fluid. However, when these tests indicate immaturity, additional tests, such as determination of the L/S ratio or the lung profile (including PG), must be performed. The utilization of these tests is recommended for: 1) timing of delivery prior to elective caesarean section; 2) management of complicated pregnancies; and 3) recognizing indications for pharmacologic prevention of RDS in utero or at delivery.