In vivo and in vitro evidence of dermal fibroblasts influence on human epidermal pigmentation

Using chimeric human epidermal reconstructs, we previously demonstrated that epidermal pigmentation is dependent upon the phototype of melanocytes. We report here several lines of experimental evidence for dermal modulation of human epidermal pigmentation. First, phototype II-III epidermal reconstructs grafted on the back of immunotolerant Swiss nu/nu mice developed a patchy pigmentation dependent on the presence of colonizing human or mouse fibroblasts. Similarly, human white Caucasoid split-thickness skin xenografted on the same mouse strain became black within 3 months and histochemistry revealed a phototype VI pattern of melanin distribution. In vitro, human fibroblasts colonizing human dead de-epidermized dermis (DDD) induced a decrease in epidermal pigmentation whereas mouse (Swiss nu/nu) fibroblasts increased epidermal pigmentation. Conditioned medium from mice (Swiss nu/nu) fibroblasts also increased pigmentation whereas conditioned medium from human fibroblasts had no significant effect. Lastly, epidermal reconstructs made with normal or vitiligo keratinocytes and/or normal or vitiligo melanocytes from the same donor grown on DDD originating from several donors of the same clinical phototype did not pigment similarly and no specific dermal influence was noted for vitiligo. Thus, fibroblast secretion and acellular dermal connective tissue itself significantly influence melanocyte proliferation and melanin distribution/degradation. Our study suggests that murine fibroblasts are more potent than human fibroblasts in secreting soluble factors which can act directly on pigmentation, such as SCF, or activate keratinocytes to produce basement membrane proteins or melanogenic factors.     

Trans-species polymorphism of the Mhc class II DRB-like gene in banded penguins (genus Spheniscus)

The Major Histocompatibility Complex (Mhc) class II DRB locus of vertebrates is highly polymorphic and some alleles may be shared between closely related species as a result of balancing selection in association with resistance to parasites. In this study, we developed a new set of PCR primers to amplify, clone, and sequence overlapping portions of the Mhc class II DRB-like gene from the 5′UTR end to intron 3, including exons 1, 2, and 3 and introns 1 and 2 in four species (20 Humboldt, six African, five Magellanic, and three Galapagos penguins) of penguin from the genus Spheniscus (Sphe). Analysis of gene sequence variation by the neighbor-joining method of 21 Sphe sequences and 20 previously published sequences from four other penguin species revealed overlapping clades within the Sphe species, but species-specific clades for the other penguin species. The overlap of the DRB-like gene sequence variants between the four Sphe species suggests that, despite their allopatric distribution, the Sphe species are closely related and that some shared DRB1 alleles may have undergone a trans-species inheritance because of balancing selection and/or recent rapid speciation. The new primers and PCR assays that we have developed for the identification of the DRB1 DNA and protein sequence variations appear to be useful for the characterization of the molecular evolution of the gene in closely related Penguin species and might be helpful for the assessment of the genetic health and the management of the conservation and captivity of these endangered species. The nucleotide sequence and amino acid sequence data reported in this paper have been submitted to the DDBJ database and have been assigned the accession numbers AB301478, AB301944–AB301950, AB302087–AB302090, AB302190–AB302192, AB302843, AB302844, and AB303942–AB303945.     

A proteomic approach to identify differentially expressed plasma proteins between the fed and prolonged fasted states

Prolonged fasting is characterized by consecutive phases, a short period of adaptation (phase 1), phase 2 (P2) characterized by fat oxidation, and phase 3 (P3) during which energy requirements are mostly derived from increased protein utilization. At this latter stage, food seeking behavior is induced. Very few circulating biomolecules have been identified that are involved in the response to prolonged fasting. To this end, rat plasma samples were compared by a proteomic approach, using 2‐DE. The results revealed a selective variation of the levels of apolipoprotein A‐IV, A‐I, and E, haptoglobin, transthyretin, plasma retinol binding‐protein, and vitamin D binding‐protein in P2 and P3. The variations in protein levels were confirmed by ELISA. Changes in mRNA levels encoding these proteins did not systematically correlate well with protein concentrations, and tissue‐specific regulation of mRNA expression was observed, underlining the complex metabolic regulation in response to food deprivation. In late fasting, the marked reduction of apolipoprotein A‐IV levels could contribute to the alarm signal that triggers refeeding. The variations of the other differentially expressed proteins are more likely related to lipid metabolism and insulin signaling alterations.     

Stoichiometry of Base Excision Repair Proteins Correlates with Increased Somatic CAG Instability in Striatum over Cerebellum in Huntington's Disease Transgenic Mice

Huntington''s disease (HD) is a progressive neurodegenerative disorder caused by expansion of an unstable CAG repeat in the coding sequence of the Huntingtin (HTT) gene. Instability affects both germline and somatic cells. Somatic instability increases with age and is tissue-specific. In particular, the CAG repeat sequence in the striatum, the brain region that preferentially degenerates in HD, is highly unstable, whereas it is rather stable in the disease-spared cerebellum. The mechanisms underlying the age-dependence and tissue-specificity of somatic CAG instability remain obscure. Recent studies have suggested that DNA oxidation and OGG1, a glycosylase involved in the repair of 8-oxoguanine lesions, contribute to this process. We show that in HD mice oxidative DNA damage abnormally accumulates at CAG repeats in a length-dependent, but age- and tissue-independent manner, indicating that oxidative DNA damage alone is not sufficient to trigger somatic instability. Protein levels and activities of major base excision repair (BER) enzymes were compared between striatum and cerebellum of HD mice. Strikingly, 5′-flap endonuclease activity was much lower in the striatum than in the cerebellum of HD mice. Accordingly, Flap Endonuclease-1 (FEN1), the main enzyme responsible for 5′-flap endonuclease activity, and the BER cofactor HMGB1, both of which participate in long-patch BER (LP–BER), were also significantly lower in the striatum compared to the cerebellum. Finally, chromatin immunoprecipitation experiments revealed that POLβ was specifically enriched at CAG expansions in the striatum, but not in the cerebellum of HD mice. These in vivo data fit a model in which POLβ strand displacement activity during LP–BER promotes the formation of stable 5′-flap structures at CAG repeats representing pre-expanded intermediate structures, which are not efficiently removed when FEN1 activity is constitutively low. We propose that the stoichiometry of BER enzymes is one critical factor underlying the tissue selectivity of somatic CAG expansion.     

Adverse effects of instrumentation in incubating Adélie penguins (Pygoscelis adeliae)

The use of data-loggers has permitted to explore the biology of free-ranging animals. However, this method has also been reported to reduce reproductive success while the reasons of this deleterious effect remain poorly documented. In this study, we aimed to identify critical periods of the breeding cycle of Adélie penguins (Pygoscelis adeliae) when the reproductive success may decrease because of instrumentation. For this purpose, we monitored 40 pairs, where one parent was instrumented before egg laying and 30 pairs without devices (controls). These pairs were followed at least during the incubation period but the majority was monitored during the entire breeding season. Reproductive success was affected in pairs where males were instrumented. This was not due to extra chick mortality during chick rearing but to a significantly lower hatching success. Moreover, the use of artificial eggs recording incubation temperatures and egg rotation indicated that in instrumented incubating males, eggs spent as much time at optimal incubation temperatures as control eggs but were rotated at a higher frequency. In Adélie penguins, males initiate incubation and it has been established that the early stage of incubation is one of the most critical periods for embryonic development. The low hatching rate observed in instrumented males was associated with a higher egg rotation rate, perhaps as a stress response to the presence of the instrument. Even though the causal effects remain unclear, instrumentation severely affected hatching success. For these reasons, we recommend equipping birds after the early incubation.     

Aggressive behavior in female golden hamsters: development and the effect of repeated social stress

In male golden hamsters, agonistic behavior matures during puberty, changing from play fighting to adult-like aggression. In addition, this transition is accelerated by repeated social subjugation early in puberty. However, little is known about the development of agonistic behavior in females. In the present study, we compared the development of agonistic behavior in male and female golden hamsters. Furthermore, we also tested the effects of repeated social subjugation on the development of agonistic behavior during puberty. Hamsters were tested for agonistic behavior in the presence of a smaller intruder at different intervals during puberty. Several observations were made. First, the frequency of attacks remained stable in females, while varying in males. Second, the transition from play fighting to adult-like aggression occurred at earlier time periods in females than in males. Finally, a clear transitional period marked by attacks focused on the flanks was observable in males around mid-puberty. However, this transitional period was not apparent in females. In addition, juvenile females were exposed to aggressive adult males or females. In both cases, repeated exposure to stress had no statistically significant effect on the development of agonistic behavior. After 2 weeks of subjugation, exposure to aggressive adults had no effect on serum cortisol levels, indicating that juvenile females habituate to repeated social stress. These data show significant sex differences in the development of agonistic behavior and adaptation to repeated stress in juvenile golden hamsters.     

A critical appraisal of vitiligo etiologic theories. Is melanocyte loss a melanocytorrhagy?

Common generalized vitiligo is an acquired depigmenting disorder characterized by a chronic and progressive loss of melanocytes from the epidermis and follicular reservoir. However, the mechanism of melanocyte disappearance has never been clearly understood, and the intervention of cellular and humoral autoimmune phenomena as primary events remains unproven. In this review, is discussed the data supporting the major theories of vitiligo, namely melanocyte destruction (autoimmune, neural and impaired redox status) and melanocyte inhibition or defective adhesion. Based on recent morphologic findings in vivo supporting a chronic detachment and transepidermal loss of melanocytes in common generalized vitiligo, a new theory is suggested proposing melanocytorrhagy as the primary defect underlying melanocyte loss, integrating most of the possible triggering/precipitating/enhancing effects of other known factors.     

Identification and functional analysis of the gene encoding methionine-gamma-lyase in Brevibacterium linens

The enzymatic degradation of L-methionine and subsequent formation of volatile sulfur compounds (VSCs) is believed to be essential for flavor development in cheese. L-methionine-gamma-lyase (MGL) can convert L-methionine to methanethiol (MTL), alpha-ketobutyrate, and ammonia. The mgl gene encoding MGL was cloned from the type strain Brevibacterium linens ATCC 9175 known to produce copious amounts of MTL and related VSCs. The disruption of the mgl gene, achieved in strain ATCC 9175, resulted in a 62% decrease in thiol-producing activity and a 97% decrease in total VSC production in the knockout strain. Our work shows that L-methionine degradation via gamma-elimination is a key step in the formation of VSCs in B. linens.     

Ability of thermophilic lactic acid bacteria to produce aroma compounds from amino acids

Although a large number of key odorants of Swiss-type cheese result from amino acid catabolism, the amino acid catabolic pathways in the bacteria present in these cheeses are not well known. In this study, we compared the in vitro abilities of Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, and Streptococcus thermophilus to produce aroma compounds from three amino acids, leucine, phenylalanine, and methionine, under mid-pH conditions of cheese ripening (pH 5.5), and we investigated the catabolic pathways used by these bacteria. In the three lactic acid bacterial species, amino acid catabolism was initiated by a transamination step, which requires the presence of an alpha-keto acid such as alpha-ketoglutarate (alpha-KG) as the amino group acceptor, and produced alpha-keto acids. Only S. thermophilus exhibited glutamate dehydrogenase activity, which produces alpha-KG from glutamate, and consequently only S. thermophilus was capable of catabolizing amino acids in the reaction medium without alpha-KG addition. In the presence of alpha-KG, lactobacilli produced much more varied aroma compounds such as acids, aldehydes, and alcohols than S. thermophilus, which mainly produced alpha-keto acids and a small amount of hydroxy acids and acids. L. helveticus mainly produced acids from phenylalanine and leucine, while L. delbrueckii subsp. lactis produced larger amounts of alcohols and/or aldehydes. Formation of aldehydes, alcohols, and acids from alpha-keto acids by L. delbrueckii subsp. lactis mainly results from the action of an alpha-keto acid decarboxylase, which produces aldehydes that are then oxidized or reduced to acids or alcohols. In contrast, the enzyme involved in the alpha-keto acid conversion to acids in L. helveticus and S. thermophilus is an alpha-keto acid dehydrogenase that produces acyl coenzymes A.