Characterization of a newin vitro model for studies of reepithelialization in human partial thickness wounds

Summary Reepithelialization of artificial partial thickness wounds made in biopsies of human skin was determined after 3, 5, or 7 d of incubation, submerged or elevated to the air-liquid interface. The biopsies were reepithelialized within 5–7 d, with a more complete epidermal healing in wounds exposed to air. Both types of wounds showed similar time-course in deposition of basement membrane components, as detected by immunofluorescence labeling. Laminin and collagen type VII were deposited underneath the migrating tips, whereas collagen type IV was detected after reepithelialization. Markers of terminal differentiation showed a pattern close to normal in the air-liquid incubated wounds after reepithelialization. Involucrin was detected in the suprabasal regions of the migrating epidermis and thereafter in the upper half of neo-epidermis in the air-liquid incubated wound. Filaggrin could not be detected in the submerged wounds at any time during healing, whereas wounds exposed to air showed a well-differentiated epidermis by Day 7. Tritiated thymidine-incorporation indicated proliferation of epidermal and dermal cells during reepithelialization and a maintained viability, as shown by cultivation of endothelial- and fibroblast-like cells obtained from the dermis 7 d after wounding. Reepithelialization in this humanin vitro model is supported by a matrix close to normal with the possibility of extracellular influences and cell-cell interactions and, in addition, the technique is simple and reproducible. Therefore, we suggest this model for studies of regeneration in culture and as a complement toin vivo studies on epidermal healing.     

Chlorophyll a/b-binding proteins, pigment conversions, and early light-induced proteins in a chlorophyll b-less barley mutant.

Monospecific polyclonal antibodies have been raised against synthetic peptides derived from the primary sequences from different plant light-harvesting Chl a/b-binding (LHC) proteins. Together with other monospecific antibodies, these were used to quantify the levels of the 10 different LHC proteins in wild-type and chlorina f2 barley (Hordeum vulgare L.), grown under normal and intermittent light (ImL). Chlorina f2, grown under normal light, lacked Lhcb1 (type I LHC II) and Lhcb6 (CP24) and had reduced amounts of Lhcb2, Lhcb3 (types II and III LHC II), and Lhcb4 (CP 29). Chlorina f2 grown under ImL lacked all LHC proteins, whereas wild-type ImL plants contained Lhcb5 (CP 26) and a small amount of Lhcb2. The chlorina f2 ImL thylakoids were organized in large parallel arrays, but wild-type ImL thylakoids had appressed regions, indicating a possible role for Lhcb5 in grana stacking. Chlorina f2 grown under ImL contained considerable amounts of violaxanthin (2-3/reaction center), representing a pool of phototransformable xanthophyll cycle pigments not associated with LHC proteins. Chlorina f2 and the plants grown under ImL also contained early light-induced proteins (ELIPs) as monitored by western blotting. The levels of both ELIPs and xanthophyll cycle pigments increased during a 1 h of high light treatment, without accumulation of LHC proteins. These data are consistent with the hypothesis that ELIPs are pigment-binding proteins, and we suggest that ELIPs bind photoconvertible xanthophylls and replace "normal" LHC proteins under conditions of light stress.     

Characterization of the activity of fatty-acid epoxide hydrolase in seeds of castor bean (Ricinus communis L.)

Epoxide hydrolase (EC 3.3.2.3) activity was measured with [1-¹⁴C]cis-9,10-epoxystearic acid as the substrate. Homogenates were prepared from the endosperm tissue of germinating seeds of castor bean (Ricinus communis L. zanzibariensis). The activity of fatty-acid epoxide hydrolase was characterized with respect to dependence on time, amount of protein, pH and temperature. Analyses of enzyme distribution in endosperm, cotyledons, root and hypocotyl showed the highest total activity in the endosperm, less in the cotyledons and low activity in the root and hypocotyl. The specific activity was similar for cotyledons and endosperm. Analysis of the temporal expression of the enzyme in the endosperm during germination revealed high activity already in the imbibed seed. Activity was maximal between days four to six and then decreased at the end of one week. Subcellular fractionation of endosperm revealed a dual distribution of activity between the glyoxysomal and the cytosolic fractions.     

A high-resolution map of the vicinity of the R1 locus on chromosome V of potato based on RFLP and AFLP markers

The R1 allele confers on potato a race-specific resistance to Phytophthora infestans. The corresponding genetic locus maps on chromosome V in a region in which several other resistance genes are also located. As part of a strategy for cloning R1, a high-resolution genetic map was constructed for the segment of chromosome V that is bordered by the RFLP loci GP21 and GP179 and includes the R1 locus. Bulked segregant analysis and markers based on amplified fragment length polymorphisms (AFLP markers) were used to select molecular markers closely linked to R1. Twenty-nine of approximately 3200 informative AFLP loci displayed linkage to the R1 locus. Based on the genotypic analysis of 461 gametes, eight loci mapped within the GP21–GP179 interval. Two of those could not be seperated from R1 by recombination. For genotyping large numbers of plants with respect to the flanking markers GP21 and GP179 PCR based assays were also developed which allowed marker-assisted selection of plants with genotypes Rr and rr and of recombinant plants.     

Feeding the fairy shrimp Streptocephalus (Anostraca - Crustacea) with the rotifer Anuraeopsis

Laboratory-cultured Streptocephalus torvicornis were offered 8 concentrations (from 6 to 800 ind. ml^–1) of Anuraeopsis fissa for periods of 2 h 30 min. Two size classes, small (male: 14.7 mm± 1.6, female: 15.4 mm± 1.3) and large (male: 20.0 mm±2.0, female: 23.1 mm± 1.5), of S. torvicornis were used. Functional response for large S. torvicornis (both sexes) plateaued at 400 rotifers ml^–1, while in small specimens it did so at 200 prey ml^–1. Females consumed significantly more (30%) prey than males. Large males consumed maximum 4730 rotifers h^–1, females 6560 h^–1.     

Genetic relationships among species of hagfish revealed by protein electrophoresis

Hagfish species of the genera Myxine, Eptatretus and Paramyxine were analysed for genetic variation by allozyme electrophoresis and isoelectric focusing of general proteins. Large genetic differences were observed between samples of supposed conspecifics of Myxine circifrons from off the Californian coast, and also within one sample of Paramyxine sp. from Sagami Bay, Japan. The results are convincing evidence of the existence of additional sympatric species in these two areas. In general, the highest genetic identities were found between species within the subfamilies Myxininae and Eptatretinae. Within Eptatretinae, the Japanese species Eptatretus burgeri was genetically more similar to Japanese species of Paramyxine than to American species of Eptatretus . Thus, our data indicate that the generic status of Paramyxine should be reconsidered.     

Structure of an acidic microcapsular glycan from the reference strain (C.D.C. 866-57) for Serratia marcescens serogroup 01

The structure of the acidic polysaccharide from Serratia marcescens serogroup O1 has been investigated. NMR spectroscopy together with sugar and methylation analysis have been used as well as a uronic acid degradation. The polysaccharide consists of pentasaccharide repeating units having the following structure.

Full-size image

The polysaccharide also contains one equivalent of O-acetyl groups per repeating unit present on, inter alia, a hydroxymethyl group.     

Historical biogeography and morphological differentiation ofStreptocephalus torvicornis (Waga) since the Würm III-glaciation

We argue that the Würm III glaciation eradicated possible European populations ofS. torvicornis, and that today, a reconquest of Europe takes place on two fronts.A western wave has reached the Pyrenees, an eastern one now occupies most of eastern and northern Europe. The western route probably started in the Maghreb, the eastern one in the Levant and the Ponto-Caspian. Animals in the west had to move north by crossing, at right angles, a series of east-west oriented river valleys and progressed slowly; animals in the east could move up river valleys extending north-east, and moved quickly. Italy was not occupied, becauseS.torvicornis is a warm stenotherm, and by the time the climate had warmed sufficiently for it to reach the southern shore of the mediterranean (ca 6000 BP), the gap with Italy was probably too large for a crossing. Cold-loving species (of the generaBranchipus, Chirocephalus) conversely, and could freely flow across the Central Mediterranean at low sea-levels (ca 12000 BP), and now occur in Italy (and the rest of Europe) as well as in Northern Africa.A prediction of our hypothesis is that the pioneer populations in Spain and Central Europe should have been isolated longest. Thisis tested and confirmed by their comparative morphology, and two subspecies,S. t. torvicornis andS. t. bucheti are reinstated.A gap across the Nile Valley where onlyS. rubricaudatus seems to occur, deserves further study.     

High-resolution mapping by YAC fragmentation of a 2.5-Mb Xp22 region containing the human RS, KFSD and CLS disease genes

The disease loci for X-linked Retinoschisis (RS), Keratosis follicularis spinulosa decalvans (KFSD), and Coffin-Lowry syndrome (CLS) have been localized to the same, small region in Xp22 on the human X Chromosome (Chr). To generate a high-resolution map of the available contig in this area, we have used the YAC fragmentation vectors pBP108/ADE2 and pBP109/ADE2 and generated fragmented YACs from a 2.5-Mb YAC (y939H7) spanning the mentioned disease gene candidate regions. Forty-seven fragmented YACs were generated and analyzed, ranging in size from 170 kb to over 2400 kb. The resulting YAC fragmentation panel was used to construct a detailed restriction map of the region and has been used to bin clones and markers. As a deletion panel, it will present a valuable resource for further mapping. Received: 31 December 1996 / Accepted: 22 February 1997     

Somatic expansion of the (CAG) n repeat in Huntington disease brains

The mutation causing Huntington disease (HD) has been identified as an expansion of a polymorphic (CAG) _n repeat in the 5 part of the huntingtin gene. The specific neuropathology of HD, viz. selective neuronal loss in the caudate nucleus and putamen, cannot be explained by the widespread expression of the gene. Since somatic expansion is observed in affected tissue in myotonic dystrophy, we have studied the length of the (CAG) _n repeat in various regions of the brain. Although we have not found clear differences when comparing severely and mildly affected regions, we have observed a minor increase in repeat length upon comparison of affected brain samples with cerebellum or peripheral blood. Hence, although further somatic amplification seems to occur in affected areas of the brain, the differences between affected and unaffected regions are too small to make this mechanism an obvious candidate for the cause of differential neuronal degeneration in HD.