Restoration of cutaneous pigmentation by transplantation to mice of isogeneic human melanocytes in dermal–epidermal engineered skin substitutes

Autologous engineered skin substitutes (ESS) containing melanocytes (hM) may restore pigmentation and photoprotection after grafting to full‐thickness skin wounds. In this study, normal hM were isolated from discard skin, propagated with or without tyrosinase inhibitors, cryopreserved, recovered into culture, and added to ESS (ESS‐P) before transplantation. ESS‐P were incubated in either UCMC160/161 or UCDM1 medium, scored for hM densities, and grafted to mice. The results showed that sufficient hM can be propagated to expand donor tissue by 100‐fold; incubation of hM in tyrosinase inhibitors reduced pigment levels but did not change hM recovery after cryopreservation; hM densities in ESS‐P were greater after incubation in UCDM1 than UCMC160 medium; hM were localized to the dermal–epidermal junction of ESS‐P; and UCDM1 medium promoted earlier pigment distribution and density. These results indicate that hM can be incorporated into ESS‐P efficiently to restore cutaneous pigmentation and UV photoprotection after full‐thickness skin loss conditions.     

海水酸化及升温对刺参稚参生长和变色的影响

海洋酸化和海洋变暖是当下及未来海洋生物及其依存生态系统面临的主要环境压力和生态问题。当前,海洋生物早期发育气候变化生物学的研究主要集中于海洋酸化的影响,为了更好地探究海洋气候变化对海洋生物的影响,有必要研究海洋酸化和变暖联合作用下海洋生物的生态响应。以受精后24天的刺参稚参为研究对象,通过模拟当前和本世纪末海洋环境,观察海水酸化和升温对刺参稚参在体色发育关键时期生长、发育及体色变化的影响。实验设置对照组(大连近海水温,pCO₂ 400 mg·kg^-1)、升温组(对照组水温+2℃,pCO₂ 400 mg·kg^-1)、酸化组(对照组水温,pCO₂ 1000 mg·kg^-1)、酸化升温组(对照组水温+2℃,pCO₂ 1000 mg·kg^-1)。结果表明:温度升高2℃能够显著提高稚参发育速率,体色变化加快; pH值降低0.23个单位显著延迟稚参生长,体色变化减缓,个体间体重差异变大;升温2℃能抵消pH降低0.23个单位对...     

Simultaneous cell death and desquamation of the embryonic diffusion barrier during epidermal development

The periderm is an epithelial layer covering the emerging epidermis in early embryogenesis of vertebrates. In the chicken embryo, an additional cellular layer, the subperiderm, occurs at later embryonic stages underneath the periderm. The questions arose what is the function of both epithelial layers and, as they are transitory structures, by which mechanism are they removed. By immunocytochemistry, the tight junction (TJ) proteins occludin and claudin-1 were localized in the periderm and in the subperiderm, and sites of close contact between adjacent cells were detected by electron microscopy. Using horseradish peroxidase (HRP) as tracer, these contacts were identified as tight junctions involved in the formation of the embryonic diffusion barrier. This barrier was lost by desquamation at the end of the embryonic period, when the cornified envelope of the emerging epidermis was formed. By TUNEL and DNA ladder assays, we detected simultaneous cell death in the periderm and the subperiderm shortly before hatching. The absence of caspases-3, -6, and -7 activity, key enzymes of apoptosis, and the lack of typical morphological criteria of apoptosis such as cell fragmentation or membrane blebbing point to a special form of programmed cell death (PCD) leading to the desquamation of the embryonic diffusion barrier.     

A variation of pigmentation in the glabrous skin of dogs

The usual pigmentation pattern in mammalian skin consists of fixed melanocytes in the basal layer of the epidermis, supplying keratinocytes with melanosomes. We observed that the glabrous skin (rhinaria and footpads) of dogs deviates from this pattern. In dogs, melanocytes are found in both the dermis and epidermis. The epidermal melanocytes are situated in the intercellular spaces of the basal and spinous layers. They are characterized by a quantity of cytoplasm containing a centriole, also developing melanosomes, and in some cases annulate lamellae. There is a high frequency of closely apposed melanocytes in the epidermis. Melanosomes in different stages of formation are also abundant. The morphology of the glabrous skin of dogs suggests transport of melanocytes from the dermis into the epidermis and formation of melanosomes in the epidermis. A distributed and intense pigment formation may be necessary to achieve the black noses of many dog breeds and wild canids, as well as dark footpads despite heavy abrasion and rapid skin renewal.     

氧化应激对肠屏障功能障碍发病的影响

机体累积过量的活性氧自由基所导致的氧化应激是多种肠道疾病发生的共同病理生理基础。肠上皮细胞间的紧密连接是维持肠屏障功能的重要结构基础之一。近年来研究表明,氧化应激能通过多种途径破坏肠上皮细胞间的紧密连接,导致肠上皮屏障功能障碍。本文对蛋白激酶C、丝裂原活化蛋白激酶、蛋白质的修饰以及缺氧诱导因子-1(HIF-1)在肠屏障功能障碍中的作用机制进行简要概述,旨在为肠屏障功能障碍的治疗和预后提供新的思路。     

Chk1 is essential for the development of murine epidermal melanocytes

Embryonic deletion of mouse Chk1 is lethal; however, whether Chk1 is essential in all individual tissues is unknown. By breeding C57Bl/ 6 mice homozygous for a conditional allele of Chk1 (Chk1fl/fl) and bearing melanocyte‐specific Tyr::Cre and DCT:: LacZ transgenes, we investigated the consequences of Chk1 deletion in the melanocytic lineage. We show that adult Tyr::Cre; Chk1fl/fl mice lack coat pigmentation and epidermal melanocytes in the hair follicles, but retain eye pigmentation in the retinal pigmented epithelium (RPE). Melanoblasts formed normally during embryogenesis in Tyr::Cre; Chk1fl/fl mice at early times (embryonic day 10.5; E10.5) but were completely absent by stage E13.5, most probably as a consequence of spontaneous DNA damage and apoptosis. By contrast, melanoblast numbers were only slightly reduced in heterozygous Tyr::Cre; Chk1fl/ + embryos, and these mice exhibited normal coat pigmentation as adults. Thus, Chk1 is essential for the developmental formation of murine epidermal melanocytes but hemizygosity has little, if any, permanent developmental consequence in this cell type.     

A Lactobacillus mutant capable of accumulating long-chain polyphosphates that enhance intestinal barrier function

Inorganic polyphosphate (polyP) was previously identified as a probiotic-derived substance that enhances intestinal barrier function. PolyP-accumulating bacteria are expected to have beneficial effects on the human gastrointestinal tract. In this study, we selected Lactobacillus paracasei JCM 1163 as a strain with the potential to accumulate polyP, because among the probiotic bacteria stored in our laboratory, it had the largest amount of polyP. The chain length of polyP accumulated in L. paracasei JCM 1163 was approximately 700 phosphate (Pi) residues. L. paracasei JCM 1163 accumulated polyP when Pi was added to Pi-starved cells. We further improved the ability of L. paracasei JCM 1163 to accumulate polyP by nitrosoguanidine mutagenesis. The mutant accumulated polyP at a level of 1500 nmol/mg protein—approximately 190 times that of the wild-type strain. PolyP extracted from the L. paracasei JCM 1163 significantly suppressed the oxidant-induced intestinal permeability in mouse small intestine. In conclusion, we have succeeded in breeding the polyP-accumulating Lactobacillus mutant that is expected to enhance intestinal barrier function.     

Evidence for the effectiveness of the blood--CSF barrier in the fetal rat choroid plexus. A freeze-fracture and peroxidase diffusion study

The blood--cerebrospinal fluid (CSF) barrier in the choroid plexus is principally constituted of apical junctional complexes between epithelial cells. The effectiveness of this barrier was studied during the fetal development in the rat. Choroid plexuses from fetuses (14th and 18th embryonic day) and newborn (1 and 6 day old) rats were examined after intravascular administration of a proteic tracer (horseradish peroxidase) and investigated by freeze-fracture. From the 14th day of fetal life, apical junctions were seen to constitute a barrier that prevents the passage of peroxidase from blood to CSF; the tight junctions were morphologically similar to those of the mature animals; the junctional fibrils appeared continuous on complementary replicas. These data suggest that, from the 14th day of fetal development, the blood--CSF barrier is both morphologically and physiologically mature.     

A scan for signatures of positive selection in candidate loci for skin pigmentation in humans

Although the combination of pale skin and intense sun exposure results in an important health risk for the individual, it is less clear if at the population level this risk has possessed an evolutionary meaning. In this sense, a number of adaptive hypotheses have been put forward to explain the evolution of human skin pigmentation, such as photoprotection against sun-induced cancer, sexual selection, vitamin D synthesis or photoprotection of photolabile compounds, among others. It is expected that if skin pigmentation is adaptive, we might be able to see the signature of positive selection on some of the genes involved. In order to detect this signature, we analyze a battery of 81 candidate loci by means of phylogenetic and population genetic tests. Our results indicate that both light and dark skin may possess adaptive value. Of the main loci presenting this signature, TP53BP1 shows clear evidence of adaptive selection in Africans, whereas TYRP1 and SLC24A5 show evidence of adaptive selection in Caucasians. Although we cannot offer a mechanism that based on these genes explains the advantage of light skin, if TP53BP1, and perhaps RAD50, have truly conferred an adaptive value to the African population analyzed, photoprotection against sun-induced skin damage/cancer might be proposed as a mechanism that has driven the evolution of human skin pigmentation.     

An ultrastructural study of the blood/air barrier in the guinea-pig

Horseradish peroxidase (HRP) was intravenously injected into guinea-pigs to ultrastructurally examine the permeability of the blood/air barrier. Adults were given 300 mg/kg of the tracer in a small volume of saline, anesthetized and sacrificed at intervals by either intratracheal filling or right ventricular perfusion with 3% glutaraldehyde. The reaction product had passed through endothelial clefts and accumulated in the interstitium as early as 1.5 min after injection. This same degree of penetration occurred with either fixation method used. Tight junctions between pneumocytes prevented passage of the reaction product into alveoli. Pinocytotic vesicles were numerous in both endothelial and epithelial cells, but did not significantly contribute to tracer transport. Ten minutes post-injection was selected as optimal for this model since the highest concentration of tracer was found in the tissues at this time.     

Evidence that short-term regulation of nodule permeability does not occur in the inner cortex

Regulation of nodule permeability in response to short-term changes in environmental and physiological conditions is thought to occur by occlusion of intercellular spaces in the nodule inner cortex. To test this hypothesis, the permeability of legume nodules was altered by adapting them to either 20 or 80% O₂ over a 2.5-h period. The nodules were then rapidly frozen, cryo-planed and examined under cryo-scanning electron microscopy for differences in the number, area or shape factor of intercellular spaces. Comparisons were made between whole nodules and specific nodule zones (outer cortex, middle cortex, inner cortex and central zone) in each treatment. Gas analysis measurements indicated that nodules equilibrated at 20% O₂ had a 6.6-fold higher permeability than those equilibrated at 80% O₂ However, no significant differences were observed between pO₂ treatments in the number of open intercellular spaces, the cross-sectional area of those spaces, or the proportion of the tissue area present as open space in whole nodules or any nodule zone. Also, although nodules in both treatments possessed a boundary layer of tightly packed cells in the inner cortex, the total area of intercellular spaces between cells bordering this layer did not differ between treatments. Together these observations do not support the currently favored hypothesis that nodule permeability is regulated by opening or occlusion of intercellular spaces in the nodule inner cortex. Highly significant differences (P= 0.0006) were observed between O₂ treatments in the shape factor of the open intercellular spaces in all nodule zones. Nodules equilibrated at 80% O₂ had significantly more isodiametric spaces while those equilibrated at 20% O₂ had more long, narrow spaces. This observation suggests that the critical step in the regulation of nodule permeability to O₂ may be localized in the central, infected zone and involve changes in the ratio of the surface area of the intercellular space to the volume of the infected cell.     

Phylogenetic analysis of the Cardini group of Drosophila with respect to changes in pigmentation

Phenotypic variability is the engine that drives future diversification with the expectation that polymorphic ancestors give rise to descendants harboring a subset of the ancestral variation. Here we examine evolutionary transitions from polymorphism to monomorphism in a visually striking New World radiation of fruit flies, the Drosophila cardini group. This group is distributed across the Americas and the Caribbean islands and exhibits a wide spectrum of abdominal pigmentation variation. Specifically, the D. dunni subgroup consists of Caribbean island endemics, each of which is monomorphic for its pigmentation pattern, with an interspecific cline of pigmentation across the islands. The D. cardini subgroup consists of American continental species with wide-ranging distributions and intraspecifically variable abdominal pigmentation. We determined the phylogeny of 18 species and subspecies using three nuclear and three mitochondrial regions analyzed with maximum parsimony, maximum likelihood, and Bayesian methods. The topology produced from a combined dataset exhibited high support values at all nodes, and differed from earlier phylogenetic hypotheses based on polytene chromosome inversion patterns and isozyme data. We find that the D. dunni subgroup species, with the exception of D. belladunni, are derived from a single source not of direct South American origin and their dispersal across the islands of the Caribbean does not follow a simple stepping-stone model. Morphological changes in pigmentation across the island species are incongruent with the colonization history of the group indicating that natural selection may have played a role in the determination of this character. Finally, we demonstrate that monomorphic species have arisen independently from polymorphic ancestors two to three times.     

Delayed epidermal permeability barrier formation and hair follicle aberrations in Inv-Cldn6 mice

Homozygous mice overexpressing Claudin-6 (Cldn6) exhibit a perturbation in the epidermal differentiation program leading to a defective epidermal permeability barrier (EPB) and dehydration induced death ensuing within 48 h of birth [Turksen, K., Troy, T.C., 2002. Permeability barrier dysfunction in transgenic mice overexpressing claudin 6. Development 129, 1775-1784]. Their heterozygous counterparts are also born with an incomplete EPB; however, barrier formation continues after birth and normal hydration levels are achieved by postnatal day 12 allowing survival into adulthood. Heterozygous Inv-Cldn6 mice exhibit a distinct coat phenotype and histological analysis shows mild epidermal hyperkeratosis. Expression of K5 and K14 is aberrant, extending beyond the basal layer into the suprabasal layer where they are not co-localized suggesting that their expression is uncoupled. There is also atypical K17 and patchy K15 expression in the basal layer with no K6 expression in the interfollicular epidermis; together with marked changes in late differentiation markers (e.g. profilaggrin/filaggrin, loricrin, transglutaminase 3) indicating that the normal epidermal differentiation program is modified. The expression compartment of various Cldns is also perturbed although overall protein levels remained comparable. Most notably induction of Cldn5 and Cldn8 was observed in the Inv-Cldn6 epidermis. Heterozygous Inv-Cldn6 animals also exhibit subtle alterations in the differentiation program of the hair follicle including a shorter anagen phase, and altered hair type distribution and length compared to the wild type; the approximately 20% increase in zig-zag hair fibers at the expense of guard hairs and the approximately 30% shorter guard hairs contribute to coat abnormalities in the heterozygous mice. In addition, the transgenic hair follicles exhibit a decreased expression of K15 as well as some hair-specific keratins and express Cldn5 and Cldn18, which are not detectable in the wild type. These data indicate that Cldn6 plays a role in the differentiation processes of the epidermis and hair follicle and supports the notion of a link between Cldn regulation and EPB assembly/maintenance as well as the hair cycle.     

Permeation of blood-borne IL15 across the blood-brain barrier and the effect of LPS

Interleukin15 (IL 15) is a proinflammatory cytokine with elevated concentrations in autoimmune diseases involving the periphery (e.g. rheumatoid arthritis) and CNS (e.g. multiple sclerosis). Its interactions with the blood-brain barrier (BBB) were studied in normal and lipopolysaccharide (LPS)-treated mice. ¹²⁵I-IL15 remained intact for at least 10 min after i.v. injection and reached CNS parenchyma with regional differences between brain and spinal cord. Both in vivo and in situ brain perfusion of ¹²⁵I-IL15 showed that its permeation of the BBB was non-saturable. LPS induced a significant increase of IL15 uptake by the brain and spinal cord, partly related to a higher general permeability of the BBB. The results suggest that the BBB is an interface for blood-borne IL15 to interact with the CNS in the basal state and during inflammation.     

Bh (black at hatch) gene appears to be expressed in melanocytes of feather germs in the Japanese quail (Coturnix coturnix japonica)

The Bh (black at hatch) gene was examined to determine whether it is expressed in plumage melanocytes by analyzing pigmentation patterns of Bh melanocytes placed in the micro-environment of the feather germs of quail embryos with pink eyes. These host quails genetically lack a large part of plumage melanin. The Bh locus in these almost white quails is wild-type. When Bh neural crest cells were transplanted orthotopically into the host embryos, wild-type and Bh /+ melanocytes, which differentiated from the transplanted neural crest cells, formed plumage pigmentation patterns characteristic of each genotype in the micro-environment of the host feather germs. Brown plumage pigmentation, which was very similar to that of 10-day Bh / Bh embryos, was also observed in the feather germs of host embryos that received Bh neural crest cells, although the genotype of the donors could not be determined. These donors died before pigmentation of their feather germs occurred. The results demonstrate that pigmentation patterns of Bh menalocytes are not altered in the micro-environment of the host germs, suggesting that the Bh gene is autonomous in Bh melanocytes and is expressed in melanocytes of both Bh and the host feather germs, and that it causes the normal pigmentation pattern to be altered.     

Influence of surgical pain stress on the blood-brain barrier permeability in rats

Effect of surgical pain stress on the blood-brain barrier permeability was investigated in rats. The animals were divided into four groups: Group 1: control, Group 2: immobilization stress, Group 3: acute hypertension, Group 4: immobilization stress + surgical pain stress.Bilateral hid paw surgical wounds for cannulations were applied in animals' inguinal regions under diethyl-ether anesthesia, then the animals were awaken from anesthesia to produce surgical pain stress. Evans-blue was used as a blood-brain barrier tracer. There is no significantly blood-brain barrier breakdown after short-time immobilization stress, but after adrenalin hypertension blood-brain barrier permeability was increased especially on frontal and occipital cortices in 50% of the animals. Surgical pain stress increased blood-brain barrier permeabiliy in comparison to acute adrenalin-induced hypertension (p < 0.01). In surgical pain stress-induced animals distinct Evans-blue leakage was observed in the occipital, frontal and parieto-temporal cortices.     

The production of chimeric rats and their use in the analysis of the hooded pigmentation pattern

New, improved media and procedures for making rat chimeric embryos and culturing them in vitro have been developed. We have produced 27 rat chimeras: 20 males and 7 females. This ratio of males to females is consistent with that seen in mouse chimeras, suggesting that rat sex chimeras develop as phenotypic males. By aggregating embryos containing appropriate genetic markers for pigment cell differentiation, it is possible to produce chimeras that elucidate the site of action of the hooded gene. The coat color patterns of black ? black hooded chimeras display a white belly spot. In black ? albino hooded chimeras, small patches of white hair appear on the head and a large white spot occurs on the belly. Black ? agouti hooded chimeras display both agouti and nonagouti pigmentation over the entire surface of the chimera. These animals are fully pigmented with no white spots. In black ? albino non-hooded chimeras, rather small irregular patches of black and white hairs are distributed throughout the pelage. Histological examination of sections of hair follicles obtained from the white areas in the head of black ? albino hooded chimeras revealed amelanotic melanocytes. On the other hand, hair bulbs from the white belly spots do not contain any such melanocytes. Thus the white hairs of the head are due to the presence of albino melanocytes, but the white hairs of the belly are due to the total absence of melanocytes. All these observations are consistent with the conclusion that the hooded gene acts within melanoblasts, probably to retard their migration from the neural crest and/or to prevent their entrance into the hair follicles of the white areas of hooded rats.     

Role of lactadherin in intestinal barrier integrity in experimental neonatal necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is one of the most widespread and devastating gastrointestinal diseases in neonates. Destruction of the intestinal barrier is the main underlying cause of NEC. The aim of this study was to determine the role of lactadherin in preventing NEC in a neonatal rat model and investigate the molecular mechanism of lactadherin-mediated protection of the intestinal barrier. Neonatal rats were divided into three groups: dam feeding (DF), NEC (NEC), and NEC supplemented with 10 μg/(g·day) recombinant human lactadherin (NEC+L). Intestinal permeability, tissue damage, and cell junction protein expression and localization were evaluated. We found that lactadherin reduced weight loss caused by NEC, reduced the incidence of NEC from 100% to 46.7%, and reduced the mean histological score for tissue damage to 1.40 compared with 2.53 in the NEC group. Intestinal permeability of lactadherin-treated rats was significantly reduced when compared with that of the NEC group. In addition, the expression levels of JAM-A, claudin 3, and E-calcium in the ileum of NEC group animals increased compared with those in the ileum of DF group animals, and these levels decreased in the NEC+L group. Lactadherin changed the localization of claudin 3, occludin, and E-cadherin in epithelial cells. The mechanism underlying lactadherin-mediated protection of the intestinal barrier might be restoring the correct expression levels and localization of tight junction and adherent junction proteins. These findings suggest a new candidate agent for the prevention of NEC in newborns.     

Eggshell pigmentation pattern in relation to breeding performance of blue tits Cyanistes caeruleus

1. We test the consequences, in terms of breeding success and parental effort, of eggshell pigmentation pattern in a hole-nesting bird, the blue tit Cyanistes caeruleus that lays eggs asymmetrically speckled with reddish spots (maculated eggs). 2. We assess the effect of distribution of spots (pigment 'spread') and spot size and pigment intensity (pigment 'darkness') on eggshell physical properties and breeding parameters concerning nestling condition, investment of parents in offspring care and reproductive output in two different habitat types: a deciduous oakwoodland and an evergreen forest. 3. Blue tit clutches with more widely distributed spots showed a thicker eggshell, a shorter incubation period, a lesser amount of mass loss per day and a higher hatching probability than those with spots forming a 'corona' ring. While eggs with larger and darker (more pigment intensity) spots showed a thicker eggshell and a shorter incubation period. In the light of 'signal function hypothesis', these egg traits may reflect female health status and, consequently, this could affect male parental effort. 4. Here we show supports for some of the necessary assumptions of this hypothesis. We found a positive relationship between egg pigment 'spread' and male but not female provisioning rates per day. On the other hand, pigment 'darkness' of blue tits' clutches was positively related to female tarsus length, while pigment 'spread' was positively related to clutch size, male body mass and nestling tarsus length. Our study shows that eggshell pigment 'spread' can be used as an indicator of clutch quality. Further investigations are needed to understand the role of calcium availability as possible causal agent of deviant eggs and its relation to the maculation phenomenon.