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Biomedical subjects

H J Stark

Publications and source records attributed to H J Stark.

27 records · Page 2Linked to original sources
PubMed1990

Keratins 1 and 10 or homologues as regular constituents of inner root sheath and cuticle cells in the human hair follicle.

Morphological maturation of the inner root sheath (IRS) and cuticle of the human hair follicle reveals analogies to differentiation processes in other keratinizing epithelia. Detailed biochemical analysis of respective differentiation products, however, has been largely restricted by their low solubility. Herein we provide further evidence for the existence of K1 and K10-derivatives in IRS and hair cuticle based on protein analysis of isolated fractions and immunofluorescence in situ, substantiating our earlier data (Stark, H. J., et al. Differentiation 35, 236-248 (1987)). Extracts from both compartments showed on two-dimensional (2D)-polyacrylamide gels a group of presumptive K1 and K10-turnover products in a wide pI (basic to acidic) and Mr range (56,000-65,000), named IC-I to III and IC-IV, respectively. These components (also found in nail plate) reacted with specific antibodies (to K1 and K10) on Western blots. Weak but distinctive radiolabeling of presumptive precursor spots close to authentic K1 and K10, respectively, and their presence in lower follicle fractions (distant from infundibulum) largely precluded epidermal contamination. Two-dimensional tryptic peptide maps of excised 2D spots from the IC-I to III series revealed high homology to K1, and those from IC-IV components to K10. Immunodetection in frozen sections was improved by trypsin pretreatment and showed distinguished staining for K1 and K10 in IRS ranging from the lower bulbus region up to the "keratinizing zone" of the follicle. Above, the reaction was abruptly abolished which coincides with ultrastructural "melting" of distinct filaments in the intracellular matrix. Thus, our data suggest that differentiation in these follicular compartments (IRS and cuticle) might follow common principles of keratinization.

Fluorescent Antibody Technique↗
PubMed1989

Density-dependent modulation of synthesis of keratins 1 and 10 in the human keratinocyte line HACAT and in ras-transfected tumorigenic clones.

The spontaneous human keratinocyte line HaCaT and c-Ha-ras oncogene-transfected cell clones are capable of expressing an unusually broad spectrum of keratins, not observed so far in epithelial cells. This expression is, however, strongly modulated by environmental conditions, including cell density. Both cells of the nontumorigenic HaCaT line and the tumorigenic HaCaT-ras clones, I-7 and II-3 (giving rise to benign and malignant tumors, respectively), constitutively expressed the keratins K5, K6, K14, K16 and K17, which are also common in cultures of normal keratinocytes. In addition keratins K7, K8, K18 and K19, generally associated with simple epithelia, were synthesized (to a most pronounced extent in sparse cultures), while keratins K4, K13 and K15 appeared at confluence, presumably with the onset of stratification. Moreover, in both HaCaT and HaCaT-ras clones the epidermal "suprabasal" keratins, K1 and K10, were expressed in conventional submerged cultures (at normal vitamin A levels), markedly rising with cell density, but not strictly correlated with the degree of stratification. This property was maintained in HaCaT cells up to the highest passages. According to immunofluorescence, this was due to increasing numbers of strongly stained cells, and not due to a gradual increase in all cells. Most strikingly, there was a significant delay in the appearance of K10 compared to K1, and this dissociation of expression was most evident in dispase-detached cell sheets (submerged cultures) and organotypic cultures of the ras clones (grown at the air-liquid interface). While on frozen sections bright staining for K1 was seen in some basal and virtually all suprabasal cell layers, K10 was largely restricted to the uppermost layers. Thus, obviously synthesis of K1 and K10 can be regulated independently, although generally in this given sequence. The apparent compatibility of K1 synthesis with proliferation and particularly the extended delay of K10 expression (as a postmitotic event) might be causally related to altered growth control and as such imply the significance of this disturbance. Finally, the highly preserved epidermal characteristics, in terms of expression of keratins (and other differentiation markers [5]) and their regulation, makes these cell lines excellent candidates for studying external modulators of differentiation and also underlying molecular mechanisms.

Antibody Specificity↗
PubMed1988

The proliferation rate of intracranial tumors as defined by the monoclonal antibody KI 67. Application of the method to paraffin embedded specimens.

60 intracranial tumors have been studied immunohistochemically to determine the proliferation rate by staining for the monoclonal antibody KI-67, which recognizes a nuclear antigen expressed by cells in proliferation. In gliomas a clear correlation of stained nuclei to the histologically determined degree of malignancy was found: slow growing astrocytomas and oligodendrogliomas had an average proliferation rate of 1%, more malignant forms of 7-10%. Glioblastomas were found to have a growth fraction of 15%. Metastases had an even higher rate of 20% proliferating cells. In meningiomas the proliferation rate was mainly about 1%, but in three cases it was between 5% and 7%. Whether this is indicative for a higher risk of tumor recurrence, remains to be correlated to the clinical course. Hemangiopericytomas had a proliferation rate of 9% and 16%, respectively, the latter recurring within four months. It may be concluded from the results of this study, that investigation of intracranial tumors with KI 67 may be of prognostic value and can possibly contribute to an individualized tumor therapy.

Antibodies, Monoclonal↗
PubMed1987

Keratins of the human hair follicle: "hyperproliferative" keratins consistently expressed in outer root sheath cells in vivo and in vitro.

Keratins produced by morphologically distinct compartments of the human hair folicle (hHF) were analysed and compared to those produced by cultured hHF and interfollicular keratinocytes. Five of the major keratins, the basic keratins nos. 5 and 6 (apparent mol. mass 60 and 58 kDa) and the acidic keratins nos. 14, 16, and 17 (51, 49 and 48 kDa), could be labelled in intact hHF and were found in all fractions of the outer root sheath (ORS). The other major keratins, which were not labelled under these conditions (basic-neutral hHbI and -II; 60-62 kDa and acidic hHaI and -II; 40-42 kDa) were associated with hair shaft (hHS) both in the follicle and, virtually unchanged, in the distal part of the hair. Another, previously undescribed, group of proteins with keratin-like properties exhibiting a broad pI-spectrum (basic to slightly acidic: hIC-I, -II, -III, 64-67 kDa; distinctly acidic: hIC-IV, about 54 kDa) was detected in isolated inner root sheath (IRS), in the cuticular material shed from denuded hHS, and also in nail plates. In our experiments only ORS cells grew readily in culture irrespective of their origin from peripheral (mesenchyme-adjacent) or more central ORS-cell layers. In contrast to keratinocytes from interfollicular epidermis (IFE) the cultured ORS cells expressed a keratin set virtually identical to that expressed in vivo. This set also closely resembled that expressed by IFE keratinocyte cultures. The identity of the respective keratins (nos. 5, 6, 14, 16, and 17) present in all these cells in vivo and in vitro was confirmed by tryptic peptide mapping. The data indicated that the microenvironment (in situ) directs the differentiation of ORS cells in a manner comparable to the way it is directed by conventional culture conditions, with consistent expression of the "basal" and "hyperproliferative" set of keratins. This, however, does not exclude the possibility that other types of environmentally induced response may occur, as seen for example during the reepithelialization of superficial skin wounds by ORS cells.

Cells, Cultured↗
PubMedDate not supplied

Quantitation of amanitins in Amanita verna with calf thymus RNA polymerase B.

A procedure utilizing the specific inhibition of calf thymus DNA-directed RNA polymerase B has been applied to the quantitation of amanitins. This procedure has permitted the accurate quantitation of alpha-amanitin in amounts as low as 0.05 nanogram, a sensitivity 2000-fold greater than chemical detection methods used following tlc. Analysis of extracts of specimens of Amanita verna identified by morphological criteria has demonstrated that while toxin concentration is variable, some specimens are practically devoid of amanitins and may represent a variety of A. verna or a distinct species.

Agaricales↗
PubMedDate not supplied

Immunohistochemical demonstration of the KI-67-antigen in paraffin-embedded tumor biopsies.

10 tumor specimens were processed according to a modification of the AMeX method, which allows for paraffin embedding plus immunostaining of the slices. We used this method to determine the growth fraction of tumors using the monoclonal antibody KI-67. Results were essentially the same as those obtained when studying frozen sections. The quality of the histological slices is equal to frozen sections, but greater areas of tumor can be examined. Furthermore, this method allows for compilation of a stock of tumor specimens which can be used for further immunohistochemical studies whenever needed.

Antibodies, Monoclonal↗