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.     

Purification and characterization of a membrane-bound protein kinase from spinach thylakoids

A protein kinase was isolated from spinach thylakoid membranes by solubilization with octyl glucoside and cholate. The enzyme was purified to apparent homogeneity by ammonium sulfate precipitation, gel filtration, and sucrose density centrifugation, followed by affinity chromatography on either Affi-Gel blue (yielding denatured enzyme) or on histone cross-linked to Sepharose (yielding active enzyme). Electrophoresis on denaturing polyacrylamide gels, followed by staining with silver, revealed the kinase as a single band corresponding to an apparent molecular mass of 64 kDa. The active enzyme underwent autophosphorylation and could be detected by autoradiography following incubation with [gamma-32P]ATP and Mg2+ ion. The specific phosphotransferase activity of purified kinase was approximately 30 nmol of phosphate min-1 (mg protein)-1 with lysine-rich histone (III-S or V-S) as substrate; casein was phosphorylated at approximately 30% of this rate. The physiological substrate for the kinase is presumed to be light-harvesting chlorophyll a/b protein complex. In solubilized form, this was phosphorylated at approximately 10% of the rate observed with histone III-S as substrate, or 10-100 times slower than the estimated rate of phosphorylation of the light-harvesting complex in situ. Possible reasons for this shortfall are considered. The kinase is proposed as the principal effector of thylakoid protein phosphorylation and associated State transition phenomena.     

Conus-like structure in the eye of the deep-sea teleost Radiicephalus elongatus (Pisces,Teleostei)

Summary A conus-like structure, the hyaloid conus, located on the optic nerve head of the mesopelagic deep-sea teleost Radiicephalus elongatus is described. The hyaloid conus consists of a tapering sheath of unpigmented, vascularized connective tissue enveloping the proximal part of the hyaloid artery which proceeds from the optic nerve head through the vitreous body to the ventrally located falciform process and lens muscles. The hyaloid artery passes through the hyaloid conus without giving off any branches. The conus vessels encircling the hyaloid artery receive arterial blood from the choroid via small arteries and are drained to the choroid by a single vein. The hyaloid conus is compared with the lacertilian conus papillaris. The function of the hyaloid conus is unknown. Because of its small dimensions relative to those of the eyeball and its few capillaries, it is unlikely that the hyaloid conus is a supplemental nutritive device for the retina.     

Electrophoretic evaluation of the taxonomic status of two species of sea anemone

Puerto Rican populations of two species of sea anemones (Bunodosoma cavernata and B. granulifera) which had previously been considered one were assayed electrophoretically for enzymes encoded by 12 loci. The two species shared no common allozymes at 6 of the 12 loci. Genetic distance and identity values based on these allozymes were computed for the Puerto Rican populations and for B. cavernata from Florida and B. granulifera from Panama. The Puerto Rican populations of both species had much higher genetic identities for their geographically distant conspecifics than for each other. These results indicate that the two species are reproductively isolated and should be considered as separate valid species. Average heterozygosities are presented which are the first published for coelenterate species.     

Structure—activity relationships in the mutagenicity of quinone methides of 7-hydroxyflavylium salts for Salmonella typhimurium

Several synthetic 7-hydroxyflavylium salts related to apigeninidin, a natural 3-deoxyanthocyanidin, have been studied in the Ames mutagenicity test using strain TA1537 of Salmonella typhimurium. Under the neutral pH conditions of the test, these flavylium salts are deprotonated through ionization of the C7-OH (pK_a′ = 4.2–4.4) to form quinone methides. Only the quinone methides of 4-methyl-7-hydroxyflavylium chloride and 4′-methoxy-4-methyl-7-hydroxy-flavylium chloride showed mutagenicity. Responses of 4–8 times the background were observed at the higher doses (1000 μg/plate), both with and without metabolic activation. It was concluded that the induction of frameshift mutagenicity by this group of compounds is caused by those quinone methides that have non-ionic, stable polycyclic structures at neutral pH.     

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.     

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.     

Absolute configuration and conformation of the pure opioid antagonist (+)-2,9 alpha-dimethyl-5-(m-hydroxyphenyl)morphan.

(+)-2,9 alpha-Dimethyl-5-(m-hydroxyphenyl)morphan is the only phenylmorphan analog whose affinity for opioid kappa-receptors is greater than its affinity for opioid mu-receptors. Pharmacologically, the compound is a pure opioid antagonist devoid of agonist activity in in vivo assays of antinociception. The absolute configuration of the compound has been determined to be (1R,5S,9R) from an X-ray crystallographic study of the chloride salt. Thus, the absolute configuration corresponds to that of the atypical opioid agonist (-)-phenylmorphan while the weak atypical agonist (-)-2,9 alpha-dimethyl-5-(m- hydroxyphenyl)morphan corresponds to the potent morphine-like (+)-phenylmorphan. The preferred orientations of the phenyl ring for the two stereoisomers were determined using the molecular mechanics program MM2-87 and found to vary from that of the two parent compounds. The atypical properties of the two 9 alpha-methyl analogs is consistent with an opioid ligand model which proposes that morphine-like properties require a particular range of phenyl orientations. There was good agreement between the structure obtained from X-ray crystallography and computed with the MM2-87 program.     

The position of the epistatic S locus in the halothane linkage group in pigs

The linkage of the Phi, Pgd, Po2, S, H and halothane sensitivity loci was followed in a Belgian Landrace family, heterozygous for these systems over 6 generations. Recombination next to the S locus occurred mainly in pigs belonging to this particular family. From this investigation the position of the S locus is proved to be outwith the Phi-Pgd region, next to Phi . Therefore the gene sequence S - Phi - Hal -H- Po2 -Pgd is proposed. Higher recombination rates were observed in the female parental line of the multiheterozygous family when compared to the male parental line. Additional data from animals, unrelated to this strain, confirm the evidence of close linkage of the S system to the nearest marker loci.