Normal ageing processes are associated with an accumulation of mutations within the mitochondrial (mt) DNA. The most frequent mutation is a 4977 base pair (bp) deletion known as common deletion. In order to test the hypothesis that chronically sun-exposed skin is characterized by an increased mutation frequency of mtDNA, the mutation frequency of the common deletion between skin and another replicating tissue (the hematopoietic system) and chronically sun-exposed versus sun-protected skin was compared in the same individuals. This was done by comparing the amount of mutated mtDNA molecules with the whole mitochondrial genome in the same specimen with a semiquantitative polymerase chain reaction method, thus allowing direct comparison of different tissues. In all skin specimens the common deletion could be observed. In contrast only 3 of 10 blood samples revealed detectable amounts of the common deletion. Comparison of sun-exposed versus sun-protected skin exhibited a higher content of the common deletion in sun-exposed skin in 7 of 10 individuals. Additionally, a hitherto undescribed mtDNA mutation was detected exclusively in human skin. These studies indicate that exposure of human skin to solar radiation leads to an accumulation of mtDNA mutations, possibly via oxidative damage, which may play an important role in photoageing.
Chronic inflammatory conditions of human skin, such as prurigo lesions of atopic dermatitis, are characterized clinically by intense pruritus and histologically by increased innervation. Regulation of skin innervation is thought to depend on neurotrophic factors. In this study, human skin cells were identified as a source of neurotrophins. Cultured keratinocytes expressed neurotrophin-4, whereas dermal fibroblasts expressed neurotrophin-3. In vitro stimulation with interferon-gamma, a marker cytokine for atopic eczema, induced keratinocyte neurotrophin-4 production, which was able to support growth of a neuroglioblastoma-derived cell line. In vivo, immunohistochemistry of human skin for neurotrophins showed neurotrophin-4 staining in the epidermal layer and neurotrophin-3 staining in the dermal compartment. Neurotrophin-4 but not neurotrophin-3 expression was markedly increased in interferon-gamma-injected skin. Prurigo lesions of atopic dermatitis skin were characterized by intense epidermal staining for neurotrophin-4, suggesting a pathophysiologic role for this neurotrophin in the increased innervation characteristic for these skin lesions. This study demonstrates differential expression and regulation of neurotrophins in human skin. It also identifies keratinocyte-derived neurotrophin-4 as a possible link between the immune and the nerve system of human skin.
We have undertaken a study of phosphofructokinase (PFK; E.C. 2.7.1.11) in the yeast Kluyveromyces lactis. Like other eukaryotic PFKs, the K. lactis enzyme is activated by the allosteric effectors AMP and fructose-2,6-bisphosphate. PFK activity is induced in cells grown on glucose as compared to ethanol-grown cells, in contrast to the constitutive expression of PFK in Saccharomyces cerevisiae. We show here that phosphofructokinase of the yeast K. lactis is composed of two non-identical types of subunits, encoded by the genes KIPFK1 and KIPFK2. We have cloned and sequenced both genes. KIPFK1 and KIPFK2 encode the alpha- and the beta-PFK subunits with deduced molecular weights of 109.336 Da and 104.074 Da, respectively. Sequence analysis indicates that the genes evolved from a double duplication event. Null mutants in either of the genes lack detectable PFK activity in vitro and the respective subunits cannot be detected on Western blots. In contrast to the situation in S. cerevisiae, Klpfk1 Klpfk2 double mutants retain the ability to grow on glucose. However, Klpfk2 mutants and the double mutants do not grow on glucose, when respiration is blocked. These data suggest that the pentose phosphate pathway and respiration play a substantial role in glucose utilization by K. lactis. The K. lactis PFK genes can be expressed independently in S. cerevisiae and each of them complements the glucose-negative phenotype of pfk1 pfk2 double deletion mutants in this yeast. Expression of both K. lactis PFK genes simultaneously in S. cerevisiae pfk double deletion mutants complements for PFK activity. However, expression of a combination of PFK genes from K. lactis and S. cerevisiae does not lead to the production of a functional enzyme.
Mammalian intestinal organoids are multicellular structures that closely resemble the structure of the intestinal epithelium and can be generated in vitro from intestinal stem cells under appropriate culture conditions. This technology has transformed pharmaceutical research and drug development in human medicine. For the insect gut, no biotechnological platform equivalent to organoid cultures has been described yet. Comparison of the regulation of intestinal homeostasis and growth between insects and mammals has revealed significant similarities but also important differences. In contrast to mammals, the differentiation potential of available insect cell lines is limited and can not be exploited for in vitro permeability assays to measure the uptake of insecticides. The successful development of in vitro models could be a result of the emergence of molecular mechanisms of self-organization and signaling in the intestine that are unique to mammals. It is nevertheless considered that the technology gap is a consequence of vast differences in knowledge, particularly with respect to culture conditions that maintain the differentation potential of insect midgut cells. From the viewpoint of pest control, advanced in vitro models of the insect midgut would be very desirable because of its key barrier function for orally ingested insecticides with hemolymphatic target and its role in insecticide resistance.
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