ATP-dependent strontium uptake by basolateral membrane vesicles from rat renal cortex in the absence or presence of calcium

ATP-dependent Sr²⁺ transport was examined in vitro using basolateral membrane (BLM) vesicles isolated from rat renal cortex to clarify the discrimination mechanisms between strontium (Sr) and calcium (Ca) in renal tubules during reabsorption. ATP-dependent Sr²⁺ uptake and Ca²⁺ uptake were observed in renal BLM vesicles and were inhibited by vanadate. Hill plots indicate similar kinetic behavior for Ca²⁺ and Sr²⁺ uptake. The apparentK _m andV _max of ATP-dependent Sr²⁺ uptake were both higher than those for Ca²⁺ uptake. ATP-dependent Sr²⁺ uptake by BLM vesicles diminished in the presence of 0.1 μM Ca²⁺ and was more markedly inhibited by 1 μM Ca²⁺. Hill plots of Sr²⁺ uptake data with and without 0.1 μM Ca²⁺ showed that the cooperative behavior of Sr²⁺ uptake was not changed by Ca²⁺. In the presence of 0.1 μM Ca²⁺, the affinity of the transport system for Sr²⁺ and the velocity of Sr²⁺ uptake in the BLM were both decreased. However, the rate of Ca²⁺ uptake was not diminished by Sr²⁺ concentrations of <1.6 μM. These results suggest that Ca²⁺ is preferentially transported in the renal cortex BLM when Ca²⁺ and Sr²⁺ are present at the same time.     

A simple method forin situ freezing of anchorage-dependent cells including rat liver parenchymal cells

We developed a simple method for freezing anchorage-dependent cells, including primary cultured rat liver parenchymal cells, without detaching the cells from the culture dish. The method consists of preculture of the cells to confluence, changing the growth medium to a conventional freezing medium, packaging in a container, and storage at –80°C. After thawing and changing the freezing medium to regular growth medium, cell growth was nearly identical to that of cells freshly seeded into a new dish.     

Characterization of ganglioside GM4 lactones isolated from the whale brain

Two novel ganglioside lactones were isolated from the brain of Bryde's whale (Balaenoptera edeni) and characterized. These gangliosides migrated above GM4 and were designated M4-1 and M4-2 in the order of location from the bottom of the TLC plate. These gangliosides were converted to GM4 by mild alkali treatment. Although M4-1 and M4-2 contained 1 mol each of N-acetylneuraminic acid and galactose, they behaved as neutral lipids on ion-exchange chromatography. Inner ester linkages were found in these gangliosides by secondary ion mass spectrometry and infrared spectroscopy. Two-dimensional J-correlated proton NMR spectra revealed that an inner ester linkage was formed in M4-1 between the sialic acid carboxyl group and the C-4 hydroxyl group of the galactosyl residue and another inner ester linkage was formed in M4-2 between the sialic acid carboxyl group and the C-2 hydroxyl group of galactose.     

Studies on the origin of the tip potential of glass microelectrode

1. Tip potential (TP) of glass microelectrodes filled with 3 M KCl increased remarkably with the increase in the storage period in 3 M KCl solution at 37? C, while the electrode resistances decreased gradually.
2. The electrical conductivity through the thin glass wall near the tip was found to increase in parallel with the TP increase.
3. The e.m.f. across the thin glass wall in the tip region was directly measured. This seems to contribute to the TP generation of the microelectrode when the conductivity of the glass wall is significantly high in the tip region.
4. Effects of the acid treatment of glass employed and the acidification of fillant electrolyte solution suggested that fixed negative charges on the glass wall play a fundamental role in the TP formation.
5. Based on these experimental results, it was concluded that not only the diffusion potential through the tip pore but also the interfacial potential through the thin glass wall near the tip contributes to the TP generation, and the contribution of the latter increases with a long exposure period of the electrodes to electrolyte solution.
6. In this connection, technical problems related to reduction of the tip potential were also discussed.

     

Heligmosomoides polygyrus infection reduces severity of type 1 diabetes induced by multiple low-dose streptozotocin in mice via STAT6- and IL-10-independent mechanisms

Some parasitic helminths are known to protect their hosts from allergic and autoimmune disorders. Here, we tested the effects of a gastrointestinal nematode, Heligmosomoides polygyrus (Hp), on streptozotocin (STZ)-induced type 1 diabetes (T1D) in mice. Hp infection significantly suppressed hyperglycemia induced by multiple low-dose administration of STZ, but did not affect hyperglycemia induced by single high-dose administration of STZ. In the multiple low dose model, Hp infection prevented a decrease in pancreatic islet size. The augmentation of TNF-α and IL-1β expression in the pancreas was abrogated by Hp infection. The genetic absence of IL-10 or STAT6 did not abrogate the anti-hyperglycemic effect of Hp. Hp has a suppressive effect on immune mechanism-mediated experimental T1D via Th2 polarization-independent mechanisms.     

Endothelin regulates neural crest deployment and fate to form great vessels through Dlx5/Dlx6-independent mechanisms

Endothelin-1 (Edn1), originally identified as a vasoconstrictor peptide, is involved in the development of cranial/cardiac neural crest-derived tissues and organs. In craniofacial development, Edn1 binds to Endothelin type-A receptor (Ednra) to induce homeobox genes Dlx5/Dlx6 and determines the mandibular identity in the first pharyngeal arch. However, it remains unsolved whether this pathway is also critical for pharyngeal arch artery development to form thoracic arteries. Here, we show that the Edn1/Ednra signaling is involved in pharyngeal artery development by controlling the fate of neural crest cells through a Dlx5/Dlx6-independent mechanism. Edn1 and Ednra knock-out mice demonstrate abnormalities in pharyngeal arch artery patterning, which include persistent first and second pharyngeal arteries, resulting in additional branches from common carotid arteries. Neural crest cell labeling with Wnt1-Cre transgene and immunostaining for smooth muscle cell markers revealed that neural crest cells abnormally differentiate into smooth muscle cells at the first and second pharyngeal arteries of Ednra knock-out embryos. By contrast, Dlx5/Dlx6 knockout little affect the development of pharyngeal arch arteries and coronary arteries, the latter of which is also contributed by neural crest cells through an Edn-dependent mechanism. These findings indicate that the Edn1/Ednra signaling regulates neural crest differentiation to ensure the proper patterning of pharyngeal arch arteries, which is independent of the regional identification of the pharyngeal arches along the dorsoventral axis mediated by Dlx5/Dlx6.