Plasma membrane block to sperm entry occurs in mouse eggs upon parthenogenetic activation

The ability of parthenogenetically activated mouse eggs to establish a plasma membrane (PM) block to sperm penetration was studied. Zona-free eggs preloaded with Hoechst 33342 were activated by exposure to ethanol or OAG (1-oleoyl-2-acetyl-sn-glycerol) and inseminated after different periods. Eggs challenged with sperm at 30- or 60-min postactivation displayed a fertilization frequency significantly lower than that of control eggs. Conversely, when insemination was carried out at 120-min postactivation, the proportion of fertilized eggs was equivalent to that observed in the control group. Moreover, we report that when the eggs were induced to resume meiosis without any notable loss of CGs (egg exposure to OAG at 100 μM external Ca²⁺ or to heat shock), a normal ability to be penetrated was recorded at 30-min postactivation. Similar behaviour was exhibited by eggs that underwent a CG exocytosis close to that triggered by sperm in absence of nuclear activation (microinjection of inositol 1,4,5-trisphosphate into the egg at 1 μM cytosolic concentration). Present data support the conclusion that parthenogenetically activated mouse eggs are capable of a transitory PM block response that requires both CG exocytosis and meiosis resumption to occur. © 1994 Wiley-Liss, Inc.     

Angiopoietin/Tie2 Dysbalance Is Associated with Acute Kidney Injury after Cardiac Surgery Assisted by Cardiopulmonary Bypass

Introduction

The pathophysiology of acute kidney injury (AKI) after cardiac surgery is not completely understood. Recent evidence suggests a pivotal role for the endothelium in AKI. In experimental models of AKI, the endothelial specific receptor Tie2 with its ligands Angiopoietin (Ang) 1 and Ang2 are deranged. This study investigates their status after cardiac surgery, and a possible relation between angiopoietins and AKI.

Methods

From a cohort of 541 patients that underwent cardiac surgery, blood and urine was collected at 5 predefined time points. From this cohort we identified 21 patients who had at least 50% post-operative serum creatinine increase (AKI). We constructed a control group (n = 21) using propensity matching. Systemic levels of Ang1, Ang2, and sTie2 were measured in plasma and the AKI markers albumin, kidney injury molecule-1 (KIM-1) and N-acetyl-beta-D-glucosaminidase (NAG) were measured in the urine.

Results

Ang2 plasma levels increased over time in AKI (from 4.2 to 11.6 ng/ml) and control patients (from 3.0 to 6.7 ng/ml). Ang2 levels increased 1.7-fold more in patients who developed AKI after cardiac surgery compared to matched control patients. Plasma levels of sTie2 decreased 1.6-fold and Ang1 decreased 3-fold over time in both groups, but were not different between AKI and controls (Ang1 P = 0.583 and sTie2 P = 0.679). Moreover, we found a positive correlation between plasma levels of Ang2 and urinary levels of NAG.

Conclusions

The endothelial Ang/Tie2 system is in dysbalance in patients that develop AKI after cardiac surgery compared to matched control patients.     

Targeting Rapamycin to Podocytes Using a Vascular Cell Adhesion Molecule-1 (VCAM-1)-Harnessed SAINT-Based Lipid Carrier System

Together with mesangial cells, glomerular endothelial cells and the basement membrane, podocytes constitute the glomerular filtration barrier (GFB) of the kidney. Podocytes play a pivotal role in the progression of various kidney-related diseases such as glomerular sclerosis and glomerulonephritis that finally lead to chronic end-stage renal disease. During podocytopathies, the slit-diaphragm connecting the adjacent podocytes are detached leading to severe loss of proteins in the urine. The pathophysiology of podocytopathies makes podocytes a potential and challenging target for nanomedicine development, though there is a lack of known molecular targets for cell selective drug delivery. To identify VCAM-1 as a cell-surface receptor that is suitable for binding and internalization of nanomedicine carrier systems by podocytes, we investigated its expression in the immortalized podocyte cell lines AB8/13 and MPC-5, and in primary podocytes. Gene and protein expression analyses revealed that VCAM-1 expression is increased by podocytes upon TNFα-activation for up to 24 h. This was paralleled by anti-VCAM-1 antibody binding to the TNFα-activated cells, which can be employed as a ligand to facilitate the uptake of nanocarriers under inflammatory conditions. Hence, we next explored the possibilities of using VCAM-1 as a cell-surface receptor to deliver the potent immunosuppressant rapamycin to TNFα-activated podocytes using the lipid-based nanocarrier system Saint-O-Somes. Anti-VCAM-1-rapamycin-SAINT-O-Somes more effectively inhibited the cell migration of AB8/13 cells than free rapamycin and non-targeted rapamycin-SAINT-O-Somes indicating the potential of VCAM-1 targeted drug delivery to podocytes.     

Cardiac effects of postconditioning depend critically on the duration of index ischemia

Postconditioning (POC) is known as the phenomenon whereby brief intermittent ischemia applied at the onset of reperfusion following index ischemia limits myocardial infarct size. Whereas there is evidence that the algorithm of the POC stimulus is an important determinant of the protective efficacy, the importance of the duration of index ischemia on the outcome of the effects of POC has received little attention. Pentobarbital sodium-anesthetized Wistar rats were therefore subjected to index ischemia produced by coronary artery occlusions (CAO) of varying duration (15-120 min) followed by reperfusion, without or with postconditioning produced by three cycles of 30-s reperfusion and reocclusion (3POC30). 3POC30 limited infarct size produced by 45-min CAO (CAO45) from 45 +/- 3% to 31 +/- 5%, and CAO60 from 60 +/- 3% to 47 +/- 6% (both P < or = 0.05). In contrast, 3POC30 increased infarct size produced by CAO15 from 3 +/- 1% to 19 +/- 6% and CAO30 from 36 +/- 6 to 48 +/- 4% (both P < or = 0.05). This deleterious effect of 3POC30 was not stimulus sensitive because postconditioning with 3POC5 and 3POC15 after CAO30 also increased infarct size. The cardioprotection by 3POC30 after CAO60 was accompanied by an increased stimulation of Akt phosphorylation at 7 min of reperfusion and a 36% lower superoxide production, measured by dihydroethidium fluorescence, after 2 h of reperfusion. Consistent with these results, cardioprotection by 3POC30 was abolished by phosphatidylinositol-3-OH-kinase inhibition, as well as nitric oxide (NO) synthase inhibition. The deleterious effect of 3POC30 after CAO15 was accompanied by an increased superoxide production with no change in Akt phosphorylation and was not affected by NO synthase inhibition. In conclusion, the effect of cardiac POC depends critically on the duration of the index ischemia and can be either beneficial or detrimental. These paradoxical effects of POC may be related to the divergent effects on Akt phosphorylation and superoxide production.     

Occurrence and Fate of Estrone, 17β-estradiol and 17α-ethynylestradiol in STPs for Domestic Wastewater

Estrone (E1), 17β-estradiol (E2) and 17α-ethynylestradiol (EE2) discharged from sewage treatment plants (STPs) into surface waters, are seen as a threat effecting aquatic life by its estrogenic character. Therefore, much research is conducted on the fate and removal of these compounds. Since these compounds are present in influents and effluents in the ng/l range, methods for detection deserve special attention. Most important processes that play a role in the removal of estrogens are: adsorption, aerobic degradation, anaerobic degradation, anoxic biodegradation and photolytic degradation. Halflifes tend to vary and are remarkably shorter when low initial concentrations are applied. In general anaerobic conditions result in longer halflifes then aerobic conditions. EE2 shows far most persistence of the compounds, thereby also the estrogenic effect in vitro is about 2–3-fold higher compared to E2. The three compounds show a higher affinity to sorb to sludge compared to other tested adsorption materials like sediment. Aerobic degradation is far the most efficient in removing these compounds, but adsorption seems to play a significant role in retaining the estrogens inside full-scale STPs. Removal rates in full scale plants depend on the HRT, SRT and loading rates, but lack of information on the exact dependency so far prevents an optimal design able to fully eliminate estrogens from wastewater.     

Design of vascular endothelium-specific drug-targeting strategies for the treatment of cancer

Tumor endothelial cells are actively involved in the neovascularization processes that accompany tumor growth. Their easy accessibility for systemically applied therapeutics makes them interesting targets for therapeutic intervention. Especially for drug targeting-based therapeutics that often consist of macromolecular moieties, the tumor endothelium is considered a much better target than the tumor cells located behind the vascular wall barrier. In this review, the general principles underlying the development and choices in the development of vascular drug-targeting strategies are discussed. An overview of target epitopes identified in the past two decades is followed by a summary of those strategies that directly or indirectly induced tumor blood flow blockade in vivo. The demonstrated therapeutic success in pre-clinical animal models in debulking large tumor masses and inhibiting tumor outgrowth warrant further development of these therapeutic approaches. Yet, more effort should be put in studies in which the efficacy of different effector activities aimed at the same target, of one effector activity aimed at different targets, and of multiple target strategies are be compared. Combining these data with proper inventories on the molecular basis of tumor endothelial heterogeneity in general will make possible the development of tumor vascular drug-targeting strategies towards clinical application.     

Two Routes of Metabolic Cross-Feeding between Bifidobacterium adolescentis and Butyrate-Producing Anaerobes from the Human Gut

Dietary carbohydrates have the potential to influence diverse functional groups of bacteria within the human large intestine. Of 12 Bifidobacterium strains of human gut origin from seven species tested, four grew in pure culture on starch and nine on fructo-oligosaccharides. The potential for metabolic cross-feeding between Bifidobacterium adolescentis and lactate-utilizing, butyrate-producing Firmicute bacteria related to Eubacterium hallii and Anaerostipes caccae was investigated in vitro. E. hallii L2-7 and A. caccae L1-92 failed to grow on starch in pure culture, but in coculture with B. adolescentis L2-32 butyrate was formed, indicating cross-feeding of metabolites to the lactate utilizers. Studies with [¹³C]lactate confirmed carbon flow from lactate, via acetyl coenzyme A, to butyrate both in pure cultures of E. hallii and in cocultures with B. adolescentis. Similar results were obtained in cocultures involving B. adolescentis DSM 20083 with fructo-oligosaccharides as the substrate. Butyrate formation was also stimulated, however, in cocultures of B. adolescentis L2-32 grown on starch or fructo-oligosaccharides with Roseburia sp. strain A2-183, which produces butyrate but does not utilize lactate. This is probably a consequence of the release by B. adolescentis of oligosaccharides that are available to Roseburia sp. strain A2-183. We conclude that two distinct mechanisms of metabolic cross-feeding between B. adolescentis and butyrate-forming bacteria may operate in gut ecosystems, one due to consumption of fermentation end products (lactate and acetate) and the other due to cross-feeding of partial breakdown products from complex substrates.