Slow Growth and Increased Spontaneous Mutation Frequency in Respiratory Deficient afo1- Yeast Suppressed by a Dominant Mutation in ATP3

A yeast deletion mutation in the nuclear-encoded gene, AFO1, which codes for a mitochondrial ribosomal protein, led to slow growth on glucose, the inability to grow on glycerol or ethanol, and loss of mitochondrial DNA and respiration. We noticed that afo1- yeast readily obtains secondary mutations that suppress aspects of this phenotype, including its growth defect. We characterized and identified a dominant missense suppressor mutation in the ATP3 gene. Comparing isogenic slowly growing rho-zero and rapidly growing suppressed afo1- strains under carefully controlled fermentation conditions showed that energy charge was not significantly different between strains and was not causal for the observed growth properties. Surprisingly, in a wild-type background, the dominant suppressor allele of ATP3 still allowed respiratory growth but increased the petite frequency. Similarly, a slow-growing respiratory deficient afo1- strain displayed an about twofold increase in spontaneous frequency of point mutations (comparable to the rho-zero strain) while the suppressed strain showed mutation frequency comparable to the repiratory-competent WT strain. We conclude, that phenotypes that result from afo1- are mostly explained by rapidly emerging mutations that compensate for the slow growth that typically follows respiratory deficiency

From Mice to Men: An Evolutionary Conserved Breakdown of the Epidermal Calcium Gradient and Its Impact on the Cornified Envelope

In previous publications, we could establish that a hallmark of human skin aging is the breakdown of the epidermal calcium gradient. This redistribution of calcium has many implications, including a restructuring of the cornified envelope, a reduced epidermal barrier function, a change in lipid composition, a reduced skin hydration, and an increased skin pH. Especially the age-dependent change in the cornified envelope composition was solely studied in human foreskin samples. The aim of this study was to confirm that this effect is neither restricted to UV-protected skin area nor limited to a specific sex. In addition, we wanted to show that the collapse of the epidermal calcium gradient is not only a hallmark of human skin aging, but is also evolutionarily conserved in mammals. By using such techniques as IHC, Western blot analysis, and RT-PCR, we could demonstrate the following: (1) A change in the epidermal calcium gradient is in fact the most important sign of epidermal aging in mammals (as shown in female human eyelids and mouse skin samples of the external ear-shell); (2) The disturbed calcium homeostasis affects the expression and crosslinking of most cornified-envelope-specific genes such as loricrin and filaggrin. In this way, we could establish that the age-dependent altered composition of the cornified envelope is a typical sign of skin aging not only in humans, but in mice, too. This makes the mouse an important model organism to study these changes

Biomolecules / Oxidative stress in aging human skin

Oxidative stress in skin plays a major role in the aging process. This is true for intrinsic aging and even more for extrinsic aging. Although the results are quite different in dermis and epidermis, extrinsic aging is driven to a large extent by oxidative stress caused by UV irradiation. In this review the overall effects of oxidative stress are discussed as well as the sources of ROS including the mitochondrial ETC, peroxisomal and ER localized proteins, the Fenton reaction, and such enzymes as cyclooxygenases, lipoxygenases, xanthine oxidases, and NADPH oxidases. Furthermore, the defense mechanisms against oxidative stress ranging from enzymes like superoxide dismutases, catalases, peroxiredoxins, and GSH peroxidases to organic compounds such as L-ascorbate, -tocopherol, beta-carotene, uric acid, CoQ10, and glutathione are described in more detail. In addition the oxidative stress induced modifications caused to proteins, lipids and DNA are discussed. Finally age-related changes of the skin are also a topic of this review. They include a disruption of the epidermal calcium gradient in old skin with an accompanying change in the composition of the cornified envelope. This modified cornified envelope also leads to an altered anti-oxidative capacity and a reduced barrier function of the epidermis.(VLID)160767

From Mice to Men : An Evolutionary Conserved Breakdown of the Epidermal Calcium Gradient and Its Impact on the Cornified Envelope

In previous publications, we could establish that a hallmark of human skin aging is the breakdown of the epidermal calcium gradient. This redistribution of calcium has many implications, including a restructuring of the cornified envelope, a reduced epidermal barrier function, a change in lipid composition, a reduced skin hydration, and an increased skin pH. Especially the age-dependent change in the cornified envelope composition was solely studied in human foreskin samples. The aim of this study was to confirm that this effect is neither restricted to UV-protected skin area nor limited to a specific sex. In addition, we wanted to show that the collapse of the epidermal calcium gradient is not only a hallmark of human skin aging, but is also evolutionarily conserved in mammals. By using such techniques as IHC, Western blot analysis, and RT-PCR, we could demonstrate the following: (1) A change in the epidermal calcium gradient is in fact the most important sign of epidermal aging in mammals (as shown in female human eyelids and mouse skin samples of the external ear-shell); (2) The disturbed calcium homeostasis affects the expression and crosslinking of most cornified-envelope-specific genes such as loricrin and filaggrin. In this way, we could establish that the age-dependent altered composition of the cornified envelope is a typical sign of skin aging not only in humans, but in mice, too. This makes the mouse an important model organism to study these changes.(VLID)284485

Cell Death Discovery / Clearing the outer mitochondrial membrane from harmful proteins via lipid droplets

In recent years it turned out that there is not only extensive communication between the nucleus and mitochondria but also between mitochondria and lipid droplets (LDs) as well. We were able to demonstrate that a number of proteins shuttle between LDs and mitochondria and it depends on the metabolic state of the cell on which organelle these proteins are predominantly localized. Responsible for the localization of the particular proteins is a protein domain consisting of two -helices, which we termed V-domain according to the predicted structure. So far we have detected this domain in the following proteins: mammalian BAX, BCL-XL, TCTP and yeast Mmi1p and Erg6p. According to our experiments there are two functions of this domain: (1) shuttling of proteins to mitochondria in times of stress and apoptosis; (2) clearing the outer mitochondrial membrane from pro- as well as anti-apoptotic proteins by moving them to LDs after the stress ceases. In this way the LDs are used by the cell to modulate stress response

The Human NADPH Oxidase, Nox4, Regulates Cytoskeletal Organization in Two Cancer Cell Lines, HepG2 and SH-SY5Y

NADPH oxidases of human cells are not only functional in defense against invading microorganisms and for oxidative reactions needed for specialized biosynthetic pathways but also during the past few years have been established as signaling modules. It has been shown that human Nox4 is expressed in most somatic cell types and produces hydrogen peroxide, which signals to remodel the actin cytoskeleton. This correlates well with the function of Yno1, the only NADPH oxidase of yeast cells. Using two established tumor cell lines, which are derived from hepatic and neuroblastoma tumors, respectively, we are showing here that in both tumor models Nox4 is expressed in the ER (like the yeast NADPH oxidase), where according to published literature, it produces hydrogen peroxide. Reducing this biochemical activity by downregulating Nox4 transcription leads to loss of F-actin stress fibers. This phenotype is reversible by adding hydrogen peroxide to the cells. The effect of the Nox4 silencer RNA is specific for this gene as it does not influence the expression of Nox2. In the case of the SH-SY5Y neuronal cell line, Nox4 inhibition leads to loss of cell mobility as measured in scratch assays. We propose that inhibition of Nox4 (which is known to be strongly expressed in many tumors) could be studied as a new target for cancer treatment, in particular for inhibition of metastasis