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Biomedical subjects

P Steinert

Publications and source records attributed to P Steinert.

31 records · Page 2Linked to original sources
PubMed1987

Cultivation of murine hair follicles as organoids in a collagen matrix.

Techniques are described for the isolation and cultivation of functionally intact mouse hair follicles. Follicles were isolated by collagenase digestion of dermis from 5-day-old mice and purified by differential centrifugation and filtration. Purified follicles were cultured in a Type 1 collagen matrix using Medium 199 and 8% fetal calf serum as the basic nutrient. Viability of follicles was maintained in culture since the cultures incorporated thymidine into DNA and methionine into proteins for at least 7 days. Furthermore, follicles isolated from the collagen matrix after 7 days could reattach to a plastic culture substrate or be further cultivated in a fresh collagen matrix. Functional integrity of cultured follicles was maintained since some follicle-specific cytoskeletal proteins were synthesized in vitro, and follicles isolated from the collagen matrix after 7 days formed a haired skin when recombined with dermal fibroblasts and grafted to a skin site on nude mice. Only a minority of follicles appeared to produce a mature hair shaft in vitro by morphologic criteria, however, and synthesis of the total complement of hair proteins was not observed. Cholera toxin was a strong mitogen for cultured follicles, whereas epidermal growth factor was slightly mitogenic. Epidermal growth factor stimulated the release of a Type 1 collagenase by follicle cells, however. This model system provides an opportunity for the systematic analysis of factors required for the induction of hair growth and the underlying physiology of hair follicle development. This model should also be useful for studying the role of the hair follicle in skin carcinogenesis.

Animals↗
PubMed1982

Retinoic acid induces transglutaminase activity but inhibits cornification of cultured epidermal cells.

In cultured mouse epidermal basal cells, retinoic acid is a potent inducer of transglutaminase, the enzyme responsible for isodipeptide bond formation in protein cross-linking in the production of the cornified membrane during terminal differentiation. Paradoxically retinoic acid also inhibits the formation of the cross-linked envelope and greatly reduces the level of dipeptide bond formation in epidermal cells induced to differentiate by calcium. These results suggest a novel mechanism by which retinoids can modify transglutaminase activity and epidermal differentiation.

Animals↗
PubMed1980

Calcium regulation of growth and differentiation of mouse epidermal cells in culture.

Modification of the ionic calcium concentration in the culture medium markedly alters the pattern of proliferation and differentiation in cultured mouse epidermal cells. When medium calcium is lowered to 0.05--0.1 mM, keratinocytes proliferate rapidly with a high growth fraction and do not stratify, but continue to synthesize keratin. The cells grow as a monolayer for several months and can be subcultured and cloned in low Ca++ medium. Ultrastructural examination of cells cultured under low Ca++ conditions reveals widened intercellular spaces, abundant microvilli and perinuclear organization of tonofilaments and cellular organelles. Desmosomes are absent. Epidermal cells growing as a monolayer in low Ca++ can be induced to terminally differentiate by adding calcium to the level normally found in the culture medium (1.2 mM). Cell-to-cell contact occurs rapidly and desmosomes form within 2 hr. The cells stratify by 1--2 days and terminally differentiate with cell sloughing by 3--4 days. After Ca++ addition, DNA synthesis decreases with a lag of 5--10 hr and is totally inhibited within 34 hr. In contrast, RNA and protein synthesis continue at 40--50% of the low Ca++ level at day 3, a time when many cells are detaching from the culture dish. Keratin synthesis is unaffected by the Ca++ switch.

Animals↗
PubMed1980

Growth and differentiation of mouse epidermal cells in culture: effects of extracellular calcium.

The pattern of proliferation and differentiation in cultured mouse epidermal cells in markedly altered by modifying the ionic calcium concentration in the culture medium. When medium calcium is lowered from 1.44 mM to 0.05-0.1 mM, keratinocytes proliferate rapidly with a high growth fraction, do not stratify, but continue to synthesize keratin. The cells grow as a monolayer for several months and can be subcultured in low Ca++ medium. Ultrastructural examination of cells cultured under low Ca++ conditions reveals widened intercellular spaces with an absence of desmosomes. Microvilli are numerous, and tonofilaments and cellular organelles are organized perinuclearly. Epidermal cells growing as a monolayer in low Ca++ can be identified to terminally differentiate by adding calcium to the level normally found in the culture medium. Contact between cells occurs rapidly and desmosomes form within 2 hours. The cells stratify in 1-2 days and terminally differentiate in 3-4 days. After Ca++ addition, DNA synthesis decreases after a lag of 5-10 hours and is totally inhibited within 36 hours. In contrast, RNA and protein synthesis continue at 40-50% of the control level at Day 3, a time when many cells are detaching from the culture dish. Keratin synthesis is unaffected by the Ca++ switch. Manipulation of epidermal proliferation and differentiation by altering extracellular calcium levels should enhance the usefulness of epidermal cell cultures in the study of differentiation and carcinogenesis.

Animals↗
PubMed1978

Biochemical evidence for keratinization by mouse epidermal cells in culture.

More than 70 percent of the urea-extractable proteins from mouse stratum corneum or from differentiated cells of mouse epidermis grown in culture are two proteins of molecular weight 68,000 (keratin I) and 60,000 (kerae are two proteins of molecular weight 68,000 (keratin 1) and 60,000 (keratin 2), which are present in equimolar amounts on polyacrylamide gels. These proteins are the subunits of the keratin filaments, because when isolated from stratum corneum or cells grown in culture they form native-type epidermal keratin filaments in vitro. These observations provide biochemical evidence that epidermal cells grown in culture synthesize the major differentiation products of the epidermis.

Animals↗
PubMedDate not supplied

Sequence, heterologous expression and functional characterization of a novel tryparedoxin from Crithidia fasciculata.

Tryparedoxin has recently been discovered as a constituent of the trypanosomal peroxidase system catalysing the reduction of a peroxiredoxin-type peroxidase by trypanothione [Nogoceke et al. (1997) Biol. Chem. 378, 827-836] and has attracted interest as a potential molecular target for the development of trypanocidal agents. Here we describe the first isolation of a novel gene from Crithidia fasciculata encoding a different tryparedoxin designated tryparedoxin II. The deduced amino acid sequence of tryparedoxin II (accession number AF055986) differs substantially from the partial sequence reported for the tryparedoxin described previously and now renamed tryparedoxin I. It shares the sequence motif Vx3FSAxWCPPCR shown to represent the catalytic site in tryparedoxin I [Gommel et al. (1997) Eur. J. Biochem. 248, 913-918] with mouse nucleoredoxin (accession number X92750), and a thioredoxin-like gene product of Caenorhabditis elegans (accession number U23511). Depending on which ATG is considered functional as translation start codon, tryparedoxin II, with 150 or 165 amino acid residues, is 50% larger than the typical thioredoxins. The tryparedoxins appear phylogenetically related to the thioredoxins, but sequence similarities are restricted to the active site motifs and their intimate neighbourhood. His-tagged tryparedoxin II expressed in E. coli exhibited ping-pong kinetics in the trypanothione:peroxiredoxin assay with kinetic parameters (KM peroxiredoxin = 4.2 microM, KM trypanothione = 33 microM, Vmax/[E] = 952 min(-1)) similar to those reported for tryparedoxin I [Gommel et al. (1997) Eur. J. Biochem. 248, 913-918]. The co-existence of two distinct tryparedoxins in C. fasciculata suggests diversified biological roles of this novel type of protein, which in trypanosomatids may substitute for the pleiotropic redox catalyst thioredoxin.

Amino Acid Sequence↗
PubMedDate not supplied

An all sulfur analogue of the smallest subunit of F420-non-reducing hydrogenase from Methanococcus voltae--metal binding and structure.

The 25 amino acid long subunit VhuU of the F420-non-reducing hydrogenase from Methanococcus voltae contains selenocysteine within the consensus sequence of known [NiFe] hydrogenases DP(C or U)CxxCxxH (U = selenocysteine). The sulfur-analogue VhuUc was chemically synthesized, purified and its metal binding capability, the catalytic properties, and structural features were investigated. The polypeptide was able to bind nickel, but did not catalyse the heterolytic activation of H2. 2D-NMR spectroscopy revealed an alpha-helical secondary structure for the 15 N-terminal amino acids in 50% TFE. Nickel only binds to the C-terminus, which contains the conserved amino acid motif. Structures derived from the NMR data are compatible with the participation of both sulfur atoms from the conserved cysteine residues in a metal ion binding. Structures obtained from the data sets for Ni.VhuUc as well as Zn.VhuUc showed no further ligands. The informational value for Ni.VhuUc was low due to paramagnetism.

Amino Acid Sequence↗