Modulation of Kir4.1 and Kir4.1-Kir5.1 channels by extracellular cations

This work demonstrates that extracellular Na⁺ modulates the cloned inwardly rectifying K⁺ channels Kir4.1 and Kir4.1-Kir5.1. Whole-cell patch clamp studies on astrocytes have previously indicated that inward potassium currents are regulated by external Na⁺. We expressed Kir4.1 and Kir4.1-Kir5.1 in Xenopus oocytes to disclose if Kir4.1 and/or Kir4.1-Kir5.1 at the molecular level are responsible for the observed effect of [Na⁺]_o and to investigate the regulatory mechanism of external cations further. Our results showed that Na⁺ has a biphasic modulatory effect on both Kir4.1 and Kir4.1-Kir5.1 currents. Depending on the Na⁺-concentration and applied voltage, the inward Kir4.1/Kir4.1-Kir5.1 currents are either enhanced or reduced by extracellular Na⁺. The Na⁺ activation was voltage-independent, whereas the Na⁺-induced reduction of the Kir4.1 and Kir4.1-Kir5.1 currents was both concentration-, time- and voltage-dependent. Our data indicate that the biphasic effect of extracellular Na⁺on the Kir4.1 and Kir4.1-Kir5.1 channels is caused by two separate mechanisms.     

Kinetics studies of the reaction of the ruthenium porphyrin Ru(OEP)(CO) with the S-nitrosothiol N-acetyl-1-amino-2-methylpropyl-2-thionitrite.

The reaction of the S-nitrosothiol compound N-acetyl-1-amino-2-methylpropyl-2-thionitrite (RSNO) with the model metalloporphyrin complex Ru(II)(OEP)(CO) (OEP = octaethylporphyrinato dianion) gives the addition product trans-Ru(II)(OEP)(NO)(SR). Here we report the details of a stopped flow kinetics investigation which demonstrates the rapid equilibrium formation of an intermediate concluded to be S-bound RSNO complex Ru(II)(OEP)(RSNO)(CO), which undergoes a rate-limiting step, presumably S-NO bond cleavage to give a second intermediate Ru(III)(OEP)(SR)(CO) too short lived for direct observation. Notably, this is different from the nitrogen coordination pathway often proposed and represents an alternative mechanism by which S-nitrosothiols may be formed or decomposed in the presence of redox active metal centers. Also reported is a brief study of the quantitative photochemistry of RSNO, the photodecomposition of which complicates the kinetics studies by spectroscopic techniques.     

Lysosomal degradation of receptor-bound urokinase-type plasminogen activator is enhanced by its inhibitors in human trophoblastic choriocarcinoma cells.

We have studied the effect of plasminogen activator inhibitors PAI-1 and PAI-2 on the binding of urokinase-type plasminogen activator (u-PA) to its receptor in the human choriocarcinoma cell line JAR. With 125I-labeled ligands in whole-cell binding assays, both uncomplexed u-PA and u-PA-inhibitor complexes bound to the receptor with a Kd of approximately 100 pM at 4 degrees C. Transferring the cells to 37 degrees C led to degradation to amino acids of up to 50% of the cell-bound u-PA-inhibitor complexes, whereas the degradation of uncomplexed u-PA was 15%; the remaining ligand was recovered in an apparently intact form in the medium or was still cell associated. The degradation could be inhibited by inhibitors of vesicle transport and lysosomal hydrolases. By electron microscopic autoradiography, both 125I-u-PA and 125I-u-PA-inhibitor complexes were located over the cell membrane at 4 degrees C, with the highest density of grains over the membrane at cell-cell interphases, but, after incubation at 37 degrees C, 17 and 27% of the grains for u-PA and u-PA-PAI-1 complexes, respectively, appeared over lysosomal-like bodies. These findings suggest that the u-PA receptor possesses a clearance function for the removal of u-PA after its complex formation with a specific inhibitor. The data suggest a novel mechanism by which receptor-mediated endocytosis is initiated by the binding of a secondary ligand.     

A rapid technique for separation of thymocytes from suspensions by centrifugation through silicone oil

A method is described for rapid separation of thymocytes from suspensions by centrifugation of the cells through silicone oil. The usefulness of several radioactive compounds as markers for the trapped extracellular water space and the total water space of the cell pellets was investigated. According to the results obtained with [³H]inulin and [³H]methoxyinulin, the extracellular water space comprised approximately 12% of the total water space as determined by ³H₂O or C³⁵S(NH₂)₂. The thymocytes appeared to be undamaged after the separation as judged by their oxygen consumption, sodium content, and dexamethasone binding.     

The phospholipid code: a key component of dying cell recognition,tumor progression and host–microbe interactions

A significant effort is made by the cell to maintain certain phospholipids at specific sites. It is well described that proteins involved in intracellular signaling can be targeted to the plasma membrane and organelles through phospholipid-binding domains. Thus, the accumulation of a specific combination of phospholipids, denoted here as the ‘phospholipid code'', is key in initiating cellular processes. Interestingly, a variety of extracellular proteins and pathogen-derived proteins can also recognize or modify phospholipids to facilitate the recognition of dying cells, tumorigenesis and host–microbe interactions. In this article, we discuss the importance of the phospholipid code in a range of physiological and pathological processes.     

Epidemiological models with age structure,proportionate mixing,and cross-immunity

Infection by one strain of influenza type A provides some protection (cross-immunity) against infection by a related strain. It is important to determine how this influences the observed co-circulation of comparatively minor variants of the H1N1 and H3N2 subtypes. To this end, we formulate discrete and continuous time models with two viral strains, cross-immunity, age structure, and infectious disease dynamics. Simulation and analysis of models with cross-immunity indicate that sustained oscillations cannot be maintained by age-specific infection activity level rates when the mortality rate is constant; but are possible if mortalities are age-specific, even if activity levels are independent of age. Sustained oscillations do not seem possible for a single-strain model, even in the presence of age-specific mortalities; and thus it is suggested that the interplay between cross-immunity and age-specific mortalities may underlie observed oscillations.     

Computerized analysis of the acetylcholinesterase distribution in the rabbit hippocampal region

Summary The distribution of acetylcholinesterase (AChE) was analysed in the hippocampal region of the rabbit, employing computerized optical densitometry (COD). AChE was demonstrated histochemically according to a modification of the copper thiocholine method, and computerized analysis was performed on a selected section with the use of a graphic operating processor, resulting in images with pixel values ranging between 0 and 255, measuring 512 × 512 pixels. Examples of the densitometric analysis include presentation of pixel values in single points, in profiles through selected areas and in slicing procedures. The results of the densitometric analysis basically agreed with the subjective visual impression of different staining intensities in the sections and corresponding photomicrographs. However, the densitometric analysis provided an objective and more exact expression of the relative AChE content of the different subfields and layers of the hippocampal region. In particular, zones with the same enzyme content as well as zones differing only minimally in activity can be recognized easily and unequivocally. In view of this, the promising future uses of COD are considered briefly.     

Furosemide kinetics and dynamics in rats and humans.

1. Increasing doses of furosemide (F) were given intravenously to rats and humans and initial pharmacokinetics and pharmacodynamics were compared. 2. Weight-related initial renal excretion rate of F was twice as high in rats and serum concentration at 30 min was twice as high in humans (P less than 0.01). 3. Volume of distribution for F was 44% larger in rats (P less than 0.01). 4. Maximal weight-related diuretic and natriuretic responses were, like the theoretical maximal efficiency, 5-6 times higher in the rat. The potency was 230 times lower in the rats. 5. On a molecular basis species differences in kinetics disappeared when standardization was based on ERPF and species differences in dynamics disappeared when standardization was based on GFR.