Molecular and functional characterization of human pendrin and its allelic variants.

Pendrin (SLC26A4, PDS) is an electroneutral anion exchanger transporting I(-), Cl(-), HCO(3)(-), OH(-), SCN(-) and formate. In the thyroid, pendrin is expressed at the apical membrane of the follicular epithelium and may be involved in mediating apical iodide efflux into the follicle; in the inner ear, it plays a crucial role in the conditioning of the pH and ion composition of the endolymph; in the kidney, it may exert a role in pH homeostasis and regulation of blood pressure. Mutations of the pendrin gene can lead to syndromic and non-syndromic hearing loss with EVA (enlarged vestibular aqueduct). Functional tests of mutated pendrin allelic variants found in patients with Pendred syndrome or non-syndromic EVA (ns-EVA) revealed that the pathological phenotype is due to the reduction or loss of function of the ion transport activity. The diagnosis of Pendred syndrome and ns-EVA can be difficult because of the presence of phenocopies of Pendred syndrome and benign polymorphisms occurring in the general population. As a consequence, defining whether or not an allelic variant is pathogenic is crucial. Recently, we found that the two parameters used so far to assess the pathogenic potential of a mutation, i.e. low incidence in the control population, and substitution of evolutionary conserved amino acids, are not always reliable for predicting the functionality of pendrin allelic variants; actually, we identified mutations occurring with the same frequency in the cohort of hearing impaired patients and in the control group of normal hearing individuals. Moreover, we identified functional polymorphisms affecting highly conserved amino acids. As a general rule however, we observed a complete loss of function for all truncations and amino acid substitutions involving a proline. In this view, clinical and radiological studies should be combined with genetic and molecular studies for a definitive diagnosis. In performing genetic studies, the possibility that the mutation could affect regions other than the pendrin coding region, such as its promoter region and/or the coding regions of functionally related genes (FOXI1, KCNJ10), should be taken into account. The presence of benign polymorphisms in the population suggests that genetic studies should be corroborated by functional studies; in this context, the existence of hypo-functional variants and possible differences between the I(-)/Cl(-) and Cl(-)/HCO(3)(-) exchange activities should be carefully evaluated.

Senescence and apoptosis in yeast mother cell-specific aging and in higher cells: a short review.

It is our intention to give the reader a short overview of the relationship between apoptosis and senescence in yeast mother cell-specific aging. We are studying yeast as an aging model because we want to learn something of the basic biology of senescence and apoptosis even from a unicellular eukaryotic model system, using its unrivalled ease of genetic analysis. Consequently, we will discuss also some aspects of apoptosis in metazoa and the relevance of yeast apoptosis and aging research for cellular (Hayflick type) and organismic aging of multicellular higher organisms. In particular, we will discuss the occurrence and relevance of apoptotic phenotypes for the aging process. We want to ask the question whether apoptosis (or parts of the apoptotic process) are a possible cause of aging or vice versa and want to investigate the role of the cellular stress response system in both of these processes. Studying the current literature, it appears that little is known for sure in this field and our review will therefore be, for a large part, more like a memorandum or a program for future research.

Curcumin affects cell survival and cell volume regulation in human renal and intestinal cells.

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1E,6E-heptadiene-3,5-dione or diferuloyl methane) is a polyphenol derived from the Curcuma longa plant, commonly known as turmeric. This substance has been used extensively in Ayurvedic medicine for centuries for its anti-oxidant, analgesic, anti-inflammatory and antiseptic activity. More recently curcumin has been found to possess anti-cancer properties linked to its pro-apoptotic and anti-proliferative actions. The underlying mechanisms of these diverse effects are complex, not fully elucidated and subject of intense scientific debate. Despite increasing evidence indicating that different cation channels can be a molecular target for curcumin, very little is known about the effect of curcumin on chloride channels. Since, (i) the molecular structure of curcumin indicates that the substance could potentially interact with chloride channels, (ii) chloride channels play a role during the apoptotic process and regulation of the cell volume, and (iii) apoptosis is a well known effect of curcumin, we set out to investigate whether or not curcumin could (i) exert a modulatory effect (direct or indirect) on the swelling activated chloride current ICl(swell) in a human cell system, therefore (ii) affect cell volume regulation and (iii) ultimately modulate cell survival. The ICl(swell) channels, which are essential for regulating the cell volume after swelling, are also known to be activated under isotonic conditions as an early event in the apoptotic process. Here we show that long-term exposure of a human kidney cell line to extracellular 0.1-10 µM curcumin modulates ICl(swell) in a dose-dependent manner (0.1 µM curcumin is ineffective, 0.5-5.0 µM curcumin increase, while 10 µM curcumin decrease the current), and short-term exposure to micromolar concentrations of curcumin does not affect ICl(swell) neither if applied from the extracellular nor from the intracellular side - therefore, a direct effect of curcumin on ICl(swell) can be ruled out. Furthermore, we show that curcumin exposure induces apoptosis in human kidney cells, and at a concentration of 5.0-10 µM induces the appearance of a sub-population of cells with a dramatically increased volume. In these cells the regulation of the cell volume seems to be impaired, most likely as a consequence of the ICl(swell) blockade. Similarly, 50 µM curcumin induced apoptosis, caused cell cycle arrest in G1-phase and increased the volume of human colorectal adenocarcinoma HT-29 cells. The cell cycle arrest in G1 phase may be the mechanism underlying the volume increase observed in this cell line after exposure to curcumin.

The mitochondrial ribosomal protein of the large subunit, Afo1p, determines cellular longevity through mitochondrial back-signaling via TOR1.

Yeast mother cell-specific aging constitutes a model of replicative aging as it occurs in stem cell populations of higher eukaryotes. Here, we present a new long-lived yeast deletion mutation,afo1 (for aging factor one), that confers a 60% increase in replicative lifespan. AFO1/MRPL25 codes for a protein that is contained in the large subunit of the mitochondrial ribosome. Double mutant experiments indicate that the longevity-increasing action of the afo1 mutation is independent of mitochondrial translation, yet involves the cytoplasmic Tor1p as well as the growth-controlling transcription factor Sfp1p. In their final cell cycle, the long-lived mutant cells do show the phenotypes of yeast apoptosis indicating that the longevity of the mutant is not caused by an inability to undergo programmed cell death. Furthermore, the afo1 mutation displays high resistance against oxidants. Despite the respiratory deficiency the mutant has paradoxical increase in growth rate compared to generic petite mutants. A comparison of the single and double mutant strains for afo1 and fob1 shows that the longevity phenotype of afo1 is independent of the formation of ERCs (ribosomal DNA minicircles). AFO1/MRPL25 function establishes a new connection between mitochondria, metabolism and aging.