Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: keratin 19 expressing cells are differentially localized in function of anatomic sites, and their number varies with donor age and culture stage.

This study was undertaken to evaluate keratin 19 (K19) as a biochemical marker for skin stem cells in order to address some long standing questions concerning these cells in the field of cutaneous biology. We first used the well-established mouse model enabling us to identify skin stem cells as [3H]thymidine-label-retaining cells. A site directed antibody was raised against a synthetic peptide of K19. It reacted specifically with a 40 kDa protein (K19) on immunoblotting. It labelled the bulge area of the outer root sheath on mouse skin by immunohistochemistry. Double-labelling revealed that K19-positive-cells were also [3H]thymidine-label-retaining cells, suggesting that K19 is a marker for skin stem cells of hair follicles. K19-expression was then used to investigate the variation in mouse and human skin stem cells as a function of body site, donor age and culture time. K19 was expressed in the hair follicle and absent from the interfollicular epidermis at hairy sites (except for some K18 coexpressing Merkel cells). In contrast, at glabrous sites, K19-positive-cells were in deep epidermal rete ridges. K19 expressing cells also contained high levels of alpha 3 beta 1 integrin. The proportion of K19-positive-cells was greater in newborn than older foreskins. This correlated with keratinocyte culture lifespan variation with donor age. Moreover, it could explain clinical observations that children heal faster than adults. In conclusion, K19 expression in skin provides an additional tool to allow further characterization of skin stem cells under normal and pathological conditions in situ and in vitro.

Localization of Merkel cells at hairless and hairy human skin sites using keratin 18.

Merkel cells are neurosecretory cells of the skin with epithelial features such as desmosomes and expression of keratins 8, 18, 19, and 20. Merkel cells are scarcely distributed in adult human skin. Although they are present in hair follicles, their density is higher at hairless anatomic sites such as palms and soles. These cells are often innervated by sensory nerve fibers and are thought to be specialized mechanosensory skin receptor cells. However, their precise origin and function are not clearly established. The aim of this study was to localize Merkel cells in human hairless and hairy skin by immunohistochemistry with antibodies Ks18.174 and Ks19.1 directed against keratins 18 and 19, respectively. In glabrous skin of palm and sole, Merkel cells have been localized at the bottom of the rete ridges, in the epidermal basal layer. To study Merkel cell distribution at hairy anatomic sites, we have chosen breast skin, a tissue containing small hair follicles typical of those covering most of the body's surface. Merkel cells were present in the interfollicular epidermis. In hair follicles, they have been identified in the isthmus region.

1G5: a calmodulin-binding, vesicle-associated, protein kinase-like protein enriched in forebrain neurites.

We have characterized cDNA clones of 1G5, an mRNA highly enriched in the mammalian forebrain that encodes a 504-residue protein found in association with perikaryal membranes and neurites. The protein, which accumulates predominantly postnatally, is associated with vesicles in both axons and dendrites. The sequence of the 1G5 protein highly resembles those of protein kinases with serine/threonine specificity; however, although most residues universally conserved among protein kinases are present, a few signature residues are absent from the 1G5 protein. Furthermore, although recombinant 1G5 protein binds calmodulin in the presence of calcium, it lacks kinase activity with a sample substrate.

An endoplasmic reticulum-specific cyclophilin.

Cyclophilin is a ubiquitously expressed cytosolic peptidyl-prolyl cis-trans isomerase that is inhibited by the immunosuppressive drug cyclosporin A. A degenerate oligonucleotide based on a conserved cyclophilin sequence was used to isolate cDNA clones representing a ubiquitously expressed mRNA from mice and humans. This mRNA encodes a novel 20-kDa protein, CPH2, that shares 64% sequence identity with cyclophilin. Bacterially expressed CPH2 binds cyclosporin A and is a cyclosporin A-inhibitable peptidyl-prolyl cis-trans isomerase. Cell fractionation of rat liver followed by Western blot (immunoblot) analysis indicated that CPH2 is not cytosolic but rather is located exclusively in the endoplasmic reticulum. These results suggest that cyclosporin A mediates its effect on cells through more than one cyclophilin and that cyclosporin A-induced misfolding of T-cell membrane proteins normally mediated by CPH2 plays a role in immunosuppression.

Patterns of cerebral cortex mRNA expression.

A pool of 163 clones, isolated by screening 60,000 members of a Macaca fascicularis cerebral cortex cDNA library with a cortex-minus-cerebellum subtracted probe prepared by the phenol enhancement method, was analyzed by Northern blot hybridization studies. One hundred fifty-three of these clones corresponded to 22 RNAs whose abundance was at least 2-fold higher in cerebral cortex poly(A)+ RNA samples than in samples of cerebellar poly(A)+ RNA. Seven of these RNAs, represented by 131 clones, were undetectable in cerebellum. Only 10 of the 163 clones proved to be false positives. The abundance of several of these cortex-enriched RNAs was altered in Alzheimer's disease brains. Several RNAs that were present in cerebral cortex but undetectable in cerebellum were generally enriched in telencephalon, although none was restricted to the cortex. One of the cortex enriched RNAs, whose nucleotide sequence is presented, encoded monkey preprocholecystokinin. Overall, this study provides insights into the powers and limitations of subtractive hybridization and into the patterns of gene expression in the central nervous system.

Rat and mouse identifier sequences are preferentially but not exclusively located in cortical neuronal genes expressed postnatally.

A genetic element called the identifier (ID) sequence, highly repeated in the rat genome, has previously been reported to be located in the introns of some genes transcribed in the adult rat brain by RNA polymerase II (Pol II). We show that nuclear RNA isolated from neurons of cerebral hemispheres (cortex) of 14-day old rats is enriched more than 10-fold in ID sequences compared to nuclear RNA from liver, kidney, cerebellum, or cortical glia. The developmental onset of the difference is during the first 2 weeks after birth. Mouse cortical neuronal nuclear RNA is similarly enriched in an element related but not identical to the rat ID element, and the enrichment also has postnatal onset. The enriched appearance of ID sequences in transcripts whose expression is increased postnatally in cortical neurons correlates developmentally and spatially with the transcription of ID elements by RNA polymerase III (Pol III) and with a change in chromatin structure.

Modulation of rat pituitary gonadotrophin secretion by porcine granulosa cell 'inhibin', LH releasing hormone and sex steroids in rat anterior pituitary cells in culture.

The incubation of female rat adenohypophysial cells in primary culture with porcine granulosa cell culture medium (GCM) led to the complete inhibition of responses of LH and FSH to LH releasing hormone (LHRH) as well as to the inhibition of spontaneous release of FSH. These effects of GCM suggest the specificity of the 'inhibin'-like activity of this material. Granulosa cell culture medium completely reversed the stimulatory effect of oestradiol-17 beta on the responses of LH and FSH to LHRH, as well as reversing the stimulatory effect of progesterone, oestradiol or a combination of both steroids on the spontaneous release of FSH, while not affecting the spontaneous release of LH. The antioestrogenic effects of progesterone observed on the response of LH to 0.3 nM-LHRH were amplified in the presence of GCM while the stimulatory effects of progesterone, oestradiol or both on the response of FSH to 0.3 nM-LHRH were completely reversed by the medium. Moreover, the presence of GCM led to an additive inhibitory effect with dihydrotestosterone on the response of LH to LHRH while it completely reversed the stimulatory effect of the androgen on spontaneous and LHRH-induced FSH release. The present data show that the presence of porcine granulosa cell 'inhibin' activity can exert marked interactions with sex steroids in the control of gonadotrophin secretion. This 'inhibin' activity reversed all the stimulatory effects and potentiated all the inhibitory effects of sex steroids on gonadotrophin secretion. Although the physiological role of 'inhibin' remains to be defined well, the importance of this activity is clearly demonstrated in anterior pituitary cells in culture.