We have recently developed a minimal system for generating long tubular nanostructures that resemble tubes observed in vivo with biological membranes. Here, we studied membrane tube pulling in ternary mixtures of sphingomyelin, phosphatidylcholine and cholesterol. Two salient results emerged: the lipid composition is significantly different in the tubes and in the vesicles; tube fission is observed when phase separation is generated in the tubes. This shows that lipid sorting may depend critically on both membrane curvature and phase separation. Phase separation also appears to be important for membrane fission in tubes pulled out of giant liposomes or purified Golgi membranes. 相似文献
Binary lipid monolayers consisting of equimolar proportions of a phosphoinositide and a nickel-chelating lipid formed helical tubular vesicular structures, which appeared to be induced and/or stabilized by myelin basic protein (MBP). Another basic polypeptide, poly-L-lysine, had a similar effect but not to as great a degree as MBP; the proteins thus appeared to act as polycations. Although, the nickel-chelating lipid is a synthetic product, other endogenous divalent cations such as Zn(2+), as well as phosphoinositides, are integral and dynamic components of the myelin sheath in vivo. There, comparable helical tubular structures might represent a means for sequestration of these lipids into domains of high local concentration, perhaps in regions where the membrane is greatly curved. 相似文献
In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models. 相似文献
Hemolymph calcium homeostasis in insects is achieved by the Malpighian tubules, primarily by sequestering excess Ca2+ within internal calcium stores (Ca‐rich granules) most often located within type I (principal) tubule cells. Using both the scanning ion‐selective electrode technique and the Ramsay secretion assay this study provides the first measurements of basolateral and transepithelial Ca2+ fluxes across the Malpighian tubules of an Orthopteran insect, the house cricket Acheta domesticus. Ca2+ transport was specific to midtubule segments, where 97% of the Ca2+ entering the tubule is sequestered within intracellular calcium stores and the remaining 3% is secreted into the lumen. Antagonists of voltage‐gated (L‐type) calcium channels decreased Ca2+ influx ≥fivefold in adenosine 3′,5′‐cyclic monophosphate (cAMP)‐stimulated tubules, suggesting basolateral Ca2+ influx is facilitated by voltage‐gated Ca2+ channels. Increasing fluid secretion through manipulation of intracellular levels of cAMP or Ca2+ had opposite effects on tubule Ca2+ transport. The adenylyl cyclase‐cAMP‐PKA pathway promotes Ca2+ sequestration whereas both 5‐hydroxytryptamine and thapsigargin inhibited sequestration. Our results suggest that the midtubules of Acheta domesticus are dynamic calcium stores, which maintain hemolymph calcium concentration by manipulating rates of Ca2+ sequestration through stimulatory (cAMP) and inhibitory (Ca2+) regulatory pathways. 相似文献
The action of selegiline, a selective and irreversible inhibitor of monoamine oxidase B, commonly applied in the therapy of Parkinson's disease, on glucose formation was investigated in isolated rabbit hepatocytes and kidney-cortex tubules, maintaining the whole body glucose homeostasis via gluconeogenic pathway activity. An intensive hepatic metabolism of selegiline resulted in formation of selegiline-N-oxide, desmethylselegiline, methamphetamine and amphetamine, whereas during slow degradation of the drug in freshly isolated renal tubules selegiline-N-oxide was mainly produced. At 100 μM concentration selegiline markedly diminished glucose synthesis in isolated renal tubules incubated with dihydroxyacetone or alanine + glycerol + octanoate (by about 60 and 30%, respectively), while at 5 μM concentration a similar degree of inhibition was achieved in renal tubules grown in primary culture under the same conditions (about 40 and 60%, respectively). Moreover, desmethylselegiline and selegiline-N-oxide considerably diminished glucose production in renal tubules whereas selegiline and its metabolites did not affect gluconeogenesis in hepatocytes. Contrary to control animals, following selegiline administration to alloxan-diabetic rabbits for 8 days (10 mg kg−1 body wt. daily) the blood glucose and serum creatinine levels were significantly diminished, suggesting a decrease in renal gluconeogenesis and improvement of kidney functions.
Since in renal tubules selegiline induced a decline in the intracellular levels of gluconeogenic intermediates and ATP content accompanied by a decrease in oxygen consumption in both kidney-cortex and hepatic mitochondria it seems possible that its inhibitory action on renal gluconeogenesis might result from an impairment of mitochondrial function, while an intensive selegiline metabolism in hepatocytes causes decrease of its concentration and in consequence no inhibition of gluconeogenesis. In view of these observations it is likely that an increased risk of selegiline-induced hypoglycemia might be expected particularly in patients exhibiting an impairment of liver function and following transdermal administration of this drug, i.e. under conditions of increased serum selegiline concentrations. 相似文献
Understanding renal function requires one to integrate the structural complexity of kidney nephrons and the dynamic nature of their cellular processes. Multi-photon fluorescence microscopy is a state-of-the-art imaging technique for in vivo analysis of kidney tubules structure and function in real time. This study presents visual evidence for several levels of heterogeneity of proximal tubular endocytic uptake in the superficial renal mouse cortex and illustrates the potential of multi-photon microscopy for providing a comprehensive and dynamic portrayal of renal function. 相似文献
Abstract. Crude trifluoroacetic acid extracts were prepared from different body parts of the forest ant, Formica polyctena Foerster, Hymenoptera, Formicidae, and the honeybee, Apis mellifera carnica Pollmann, Hymenoptera, Apidae. Extracts were prepurified by means of solid phase extraction over reversed-phase cartridges. The effects of the resulting fractions on fluid secretion rates were tested on single preparations of ant Malpighian tubules. Diuretic and antidiuretic factors were shown to be present. High-performance liquid chromatography allowed for the identification of a diuretic factor from the bee-head extract which had a similar elution profile to a previously purified diuretic peptide from ant-head extracts. An antidiuretic factor was also identified from the bee abdominal extract. Similar to an antidiuretic peptide, which was purified from ant abdominal extracts, this antidiuretic factor inhibited fluid secretion and depolarized the transepithelial potential of ant Malpighian tubules. 相似文献