Membrane chloride transport measured using a chloride-sensitive fluorescent probe

Transport of chloride across cell membranes through exchange, cotransport, or conductive pathways is a subject of great biological importance. Current methods of measurement are restricted in their sensitivity, time resolution, and applicability. A new transport measurement technique has been developed on the basis of the fluorescence quenching by chloride of the dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). SPQ fluorescence quenching by chloride is rapid (less than 1 ms) and sensitive, with a greater than 50% decrease in fluorescence at 10 mM chloride. SPQ fluorescence is not altered by other physiological anions or by pH and can be used to measure both neutral and conductive transport processes. The high water solubility and membrane permeability properties of SPQ make it ideal for use in both membrane vesicles and cells. Chloride transport determined with SPQ was validated by measurement of erythrocyte chloride/anion exchange and membrane vesicle chloride conductance.     

Hepatic reactions in erythropoietic protoporphyria (author's transl)

The main hepatic change in erythropoietic protoporphyria is the deposition of protoporphyrin. Brown deposits of this pigment occur in bile canaliculi and ductules, discretely in hepatocytes, and secondarily in macrophages and Kupffer cells. The pigment is deposited in a crystalline form. Under the fluorescence microscope with a mercury maximum pressure burner (HO 50) at a wave length of 380--500 nm, it shows a typical red fluorescence even after paraffin embedding. Its crystalline structure results in a characteristic double refraction under the polarising microscope. Light-microscopically, hepatocellular reactions are characterised mainly by discrete alterations in the ergastoplasm. However, cell damage is indicated by diffusely distributed, hyaline single cell necrosis and by cytolytic piecemeal necrosis at the peripheries of hepatic lobules. Numerous, often disturbed mitoses produce binuclear and multinuclear hepatocytes. The obligatory secretion of protoporphyrin into the bile ducts leads to an alteration in the canalicular and ductular excretion apparatus which involves distinct ductular proliferation and accompanying fibrosis. Piecemeal necrosis is a further consequence of this process. The resulting histological picture is similar to sclerosing cholangitis with which it also has in common the slowly progressive development of hepatic cirrhosis.     

The Sodium/Proton Exchanger NHE8 Regulates Late Endosomal Morphology and Function

The pH and lumenal environment of intracellular organelles is considered essential for protein sorting and trafficking through the cell. We provide the first evidence that a mammalian NHE sodium (potassium)/proton exchanger, NHE8, plays a key role in the control of protein trafficking and endosome morphology. At steady state, the majority of epitope-tagged NHE8 was found in the trans-Golgi network of HeLa M-cells, but a proportion was also localized to multivesicular bodies (MVBs). Depletion of NHE8 in HeLa M-cells with siRNA resulted in the perturbation of MVB protein sorting, as shown by an increase in epidermal growth factor degradation. Additionally, NHE8-depleted cells displayed striking perinuclear clustering of endosomes and lysosomes, and there was a ninefold increase in the cellular volume taken up by LAMP1/LBPA-positive, dense MVBs. Our data points to a role for the ion exchange activity of NHE8 being required to maintain endosome morphology, as overexpression of a nonfunctional point mutant protein (NHE8 E225Q) resulted in phenotypes similar to those seen after siRNA depletion of endogenous NHE8. Interestingly, we found that depletion of NHE8, despite its function as a sodium (potassium)/proton antiporter, did not affect the overall pH inside dense MVBs.     

A high‐throughput method to quantify feeding rates in aquatic organisms: A case study with Daphnia

1. Food ingestion is one of the most basic features of all organisms. However, obtaining precise—and high‐throughput—estimates of feeding rates remains challenging, particularly for small, aquatic herbivores such as zooplankton, snails, and tadpoles. These animals typically consume low volumes of food that are time‐consuming to accurately measure.
2. We extend a standard high‐throughput fluorometry technique, which uses a microplate reader and 96‐well plates, as a practical tool for studies in ecology, evolution, and disease biology. We outline technical and methodological details to optimize quantification of individual feeding rates, improve accuracy, and minimize sampling error.
3. This high‐throughput assay offers several advantages over previous methods, including i) substantially reduced time allotments per sample to facilitate larger, more efficient experiments; ii) technical replicates; and iii) conversion of in vivo measurements to units (mL^‐1 hr^‐1 ind^‐1) which enables broad‐scale comparisons across an array of taxa and studies.
4. To evaluate the accuracy and feasibility of our approach, we use the zooplankton, Daphnia dentifera, as a case study. Our results indicate that this procedure accurately quantifies feeding rates and highlights differences among seven genotypes.
5. The method detailed here has broad applicability to a diverse array of aquatic taxa, their resources, environmental contaminants (e.g., plastics), and infectious agents. We discuss simple extensions to quantify epidemiologically relevant traits, such as pathogen exposure and transmission rates, for infectious agents with oral or trophic transmission.

     

β-Xylosidases from filamentous fungi: an overview

β-Xylosidases are hydrolytic enzymes which play an important role in xylan degradation, hydrolyzing xylobiose and xylooligosaccharides to xylose from the non-reducing end. Filamentous fungi are particularly interesting producers of this enzyme from an industrial point of view, due to the fact that they secrete β-xylosidases into the medium. Besides, fungal β-xylosidases are highly advantageous for their elevated activity levels and specificity. Interest in xylanolytic enzymes has been increasing, for their possible application in many biotechnological processes. This fact has driven the isolation, purification and characterization of several β-xylosidases. In this review, the mechanisms of action, substrate specificities, physicochemical characteristics, regulation at molecular level, molecular cloning and classification of filamentous fungal β-xylosidases are described. The potential industrial applications of fungal β-xylosidases will also be presented.     

Protective reaction of the myocardium in poisoning

Intensive synthesis of collagen-like substance was revealed in the rabbit myocardium during experimental diphtheria intoxication. It was more marked in the right ventricle 24 hours after the injection of diphtheria toxin. Since similar changes (the substance was mainly formed around blood vessels) have been observed in other cases of toxic myocardial alterations (i.e. ethanol intoxication, injection of pharmacological agents, etc.), it can be assumed that it is a standard protective reaction of the altered heart to the penetration of toxic agents from the blood into the myocardial tissue.     

Effects of EEG of the osmotic opening of the blood-brain barrier in rats

EEG activity was recorded in rats submitted to osmotic opening of the BBB by intracarotid mannitol infusion.This procedure produced an immediate short-lasting depression of the EEG and a tardive paroxysmal EEG activity. Both these phenomena were more relevant on the ipsilateral hemisphere. In some instances a tonico-clonic seizure was recorded.Pre-treatment with diazepam abolished the occurrence of the tardive EEG and behavioral modifications.In accord with previous findings, focal seizure activity is likely to be responsible for the metabolic abnormalities associated with osmotic opening of the BBB. This preparation therefore produces in the brain unphysiological states in respect to local metabolism and electrical function.