Biomedical subjects
H Huitfeldt
Publications and source records attributed to H Huitfeldt.
Ploidy-dependent growth and binucleation in cultured rat hepatocytes.
The proliferative activity of rat hepatocytes, cultured in the presence of epidermal growth factor (EGF) and insulin, was examined by immunostaining of S-phase cells labeled with bromodeoxyuridine (BrdU) in culture. Proliferation rates of the different hepatocellular ploidy and nuclearity classes were measured by fluorescence image cytometry or by microscope counting of immunostained cells. Effects of EGF and insulin were largely additive, the binuclear cells being more growth factor-dependent (showing less growth in the absence of factors) than the mononuclear cells. A serial warm-washing procedure was used to remove excess BrdU from the culture medium, allowing the study of hepatocellular binucleation by a BrdU pulse-chase approach. A high rate of binucleation was detected (50%, possibly suggesting a quantal mechanism), indicating that the hormones induce a binucleating (polyploidizing) type of growth similar to that normally observed in the liver of growing rats. The highest proliferative activity (labeling index) in the hepatocyte cultures was found among the diploid cells, independent of the degree of mitogenic stimulation. The labeling index was inversely correlated with ploidy, suggesting that the ability of hepatocytes to proliferate decreases with increasing polyploidization.
BrdUrd incorporation studies for evaluation of spermatogenesis in the blue fox.
The utility of BrdUrd incorporation techniques for studies of spermatogenesis was investigated in the blue fox. BrdUrd was injected intraperitoneally followed by collection of testicular tissue by castration/hemicastration at intervals up to 35 days after pulse labelling. Fluorescent tagged monoclonal antibodies against BrdUrd allowed detection of cells with incorporated tracer in histological sections by fluorescent light microscopy as well as in isolated testicular cells by bivariate BrdUrd/DNA flow cytometry. The duration of the spermatogenic cycle was estimated by following the labelled cohort of preleptotene spermatocytes by immunofluorescence in sections through the various stages of maturation to the late spermatid stage. These data were confirmed by bivariate BrdUrd/DNA flow cytometry of testicular cells in suspensions. Furthermore, estimations of the S phase durations and length of the spermatogonial cell cycle were possible. A consistent and satisfactory fluorescence intensity of incorporated label throughout the study shows that degradation of the incorporated label is no practical problem for this type of study, and suggests that the method is an excellent tool for studying aspects of proliferation and maturation during normal as well as perturbed spermatogenesis. Advantages of the described method include avoidance of potential radiation influence on spermatogenesis from commonly used radiolabelled tracers, e.g., 3H-TdR, and that both large and small animals can be investigated at modest cost since the unlabelled BrdUrd is considerably less expensive than labelled tracers.
Chromosome site-specific immunohistochemical detection of DNA adducts in N-acetoxy-2-acetylaminofluorene--exposed Chinese hamster ovary cells.
In these studies a polyclonal antiserum elicited against a carcinogen-DNA adduct was used to explore the localization of DNA adducts in metaphase chromosomes of cultured cells. Morphological visualization of the adduct N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) in Chinese hamster ovary (CHO) cells exposed to the direct-acting carcinogen N-acetoxy-2-acetylaminofluorene (N-Ac-AAF) was accomplished by indirect immunofluorescence with an anti-G-C8-AF antiserum. At the same time the pattern of chromosomal DNA replication was determined by replicative incorporation of bromodeoxyuridine (BrdUrd) and chromosomal staining with anti-BrdUrd. Visualization of DNA in chromosomes was accomplished with Hoechst 33258 dye. When synchronized CHO cells were exposed to N-Ac-AAF for 0.5 h during early S phase, the chromosomal pattern of dG-C8-AF adduct formation was not random. Metaphase chromosome spreads from cells exposed to N-Ac-AAF in different experiments contained certain chromosome regions that had a consistently high adduct concentration. The regions of high DNA damage corresponded to the regions active in DNA synthesis when BrdUrd and the carcinogen were given simultaneously in early S phase. In addition, the patterns of high adduct concentration and replicative synthesis shifted when the carcinogen and BrdUrd were given simultaneously during late S phase. Thus, the stage of cell cycle in which adducts are induced is an important factor in the specific location of the highest concentrations of this type of DNA lesion.
Pleomorphic adenomas contain a keratin-negative population of HLA-DR-positive dendritic cells.
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Regulated expression of differentiation-associated keratins in cultured epidermal cells detected by monospecific antibodies to unique peptides of mouse epidermal keratins.
Monospecific antibodies to mouse epidermal keratins were generated in rabbits and guinea pigs by injecting synthetic peptides of unique keratin sequences. The sequences were deduced from nucleotide sequences of cDNA clones representing basal (K14) and suprabasal (K1 and K10) cell-specific and hyperproliferative (K6) keratins of both the type-I and type-II subclasses. By applying single-and double-label immunofluorescence analysis, the expression of keratin peptides was analyzed in cultured keratinocytes maintained in the basal or suprabasal cell phenotypes. These cell types were selected by growth in medium containing 0.05 mM Ca2+ (basal cell) or 1.4 mM Ca2+ (suprabasal cell). The cultured basal cells expressed K6 and K14, but less than 1% expressed K1 and K10. Within a few hours after being placed in 1.4 mM Ca2+, K1 expression was observed, and by 24 h, 10%-17% of the cells expressed K1. K10 expression appeared to lag behind K1 expression, with only 5%-10% of cells in 1.4 mM Ca2+ exhibiting K10 immunoreactivity. Double-labeling studies indicated that virtually all K10-positive cells also expressed K1, while only about one-half of the K1-positive cells expressed K10. The treatment of basal cells with retinoic acid at pharmacological concentrations prevented the expression of K1 and K10 when cells were challenged by 1.4 mM Ca2+. Similarly, the introduction of the v-rasH oncogene into basal cells by a defective retroviral vector prevented the expression of suprabasal keratins in 1.4 mM Ca2+ medium.(ABSTRACT TRUNCATED AT 250 WORDS)
Skin calcium-binding protein is a parvalbumin of the panniculus carnosus.
Skin calcium-binding protein (SCaBP) is a calcium binding protein purified from whole rat skin. It has a molecular weight of approximately 12,000 daltons but migrates at Mr 13,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gels. On nitrocellulose blots of SDS-polyacrylamide gels, 6 different antisera to SCaBP reacted equally well with SCaBP and parvalbumin (PV), an 11,500-dalton calcium-binding protein purified from rat skeletal muscle, which also migrates at Mr 13,000 on SDS-polyacrylamide gels. Rabbit antiserum to muscle PV also recognized both PV and SCaBP, and either protein absorbed specific antibodies against either antigen from both types of antisera. Soluble protein extracts from whole adult rat and mouse skin contained a Mr 13,000 protein which was recognized on nitrocellulose blots of SDS gels by both antisera. Blots of extracts from epidermis, dermis, whole skin, and skin scraped on the dermal side to remove hypodermal tissue revealed that the Mr 13,000 PV/SCaBP cross-reacting antigen was restricted to the hypodermal tissue removed by scraping. Immunofluorescent staining of Bouin-fixed skin sections with these antisera confirmed the localization of PV/SCaBP to the panniculus carnosus, a hypodermal muscle layer. Newborn mouse skin does not contain this antigen. Additional polypeptides of Mr 10,500 and 12,000 on SDS gels of extracts from the epidermis of newborn and adult rats and mice were found to be immunoreactive with anti-SCaBP serum. These polypeptides were not recognized by the PV antiserum, and the reactivity of anti-SCaBP for these antigens was not absorbed by purified PV or SCaBP. Our results indicate that SCaBP is antigenically indistinguishable from PV and is localized in the adult rodent panniculus carnosus, and that antisera to SCaBP are polyspecific, recognizing epidermal proteins in addition to SCaBP/PV.
Human heart muscle contains keratin-like material in intercalated discs.
Two different rabbit antisera to human epidermal keratins produced immunohistochemical staining of intercalated discs in myocardial fibres. This finding indicated that tonofilaments of the keratin-type may be associated with the transverse segments of these functional specializations.