Transgenic markers for mammalian chimeras

Summary We describe the construction of aggregation chimeras between normal and transgenic embryos containing multiple copies of mouse -globin genes. The transgenic component of the chimeras is then detected in tissue sections by a DNA-DNA in situ hybridization technique, using a biotinylated DNA -globin probe and an avidin-linked alkaline phosphatase detection system. The general advantages of transgenic markers for chimeras are discussed.     

Untersuchung der eiweißspindeln des kakteen-X-Virus mit fluoreszierenden Antikörpern

A?koliv na zá kladě mnoha pokus? se p? edpokládalo, ?e tzv. bÍlkovinná v?etena v buňkách tzn. buně?né inkluse X-viru kaktus? (Ca XV), jsou slo?ena z ?etních prodlou?ených ?ásti Ca XV, p?esto to dosud nebylo proká zá no. Proto jsme se pokusili pomocÍ fluoreskujÍcÍch protilátek doká ?at, ?e bilkovinná v?etena jsou skute?ně agregáty virových ?ástic. V těto práci jsme pouzili tzv. nep?Ímé metody. Nejprve jsme p? sobili na buňky obsahujÍci tato v?etena homologiokým antisé rem proti Ca XV, zÍskanym imunizacÍ králÍk? a teprve potom jsme buňky vlo?ili do roztoku fluoreskujicÍch protilátek proti králicimu γglobulinu. BÍlkovinná v?etena svitila potom ve fluorescen?nÍm mikroskopu silně ?lutozeleně (bylopou?ito fluoresceinisothiocyaná tu). Tato fluorescence ná m uká zala, ?e nastala pozitivnÍ reakce a ?e bÍlkovianá v?etena jsou slo?ena z virových ?ástic. ?etné kontrolnÍ pokusy potvrdily ná? základnÍ pokus.     

Identification of noncollagenous basement membrane glycopolypeptides synthesized by mouse parietal entoderm and an entodermal cell line

Using two-dimensional gel electrophoresis, we have identified two noncollagenous basement membrane (BM) glycopolypeptides which are synthesized by the mouse teratocarcinoma-derived parietal yolk sac (PYS) cell line. These glycopolypeptides have molecular weights of about 200,000 and isoelectric points of about 5.6. Polypeptides with identical parameters are synthesized by the parietal entodermal cells of mouse embryos and are found in Reichert's membrane. Pluripotent embryonal carcinoma cells (ECC) synthesize considerable amounts of the two polypeptides, whereas the yield from nullipotent ECC is negligible. The treatment of nullipotent F9 cells with retinoic acid, which induces entodermal differentiation, activates the synthesis of these polypeptides. These results indicate that the two polypeptides can be used as markers of parietal entoderm differentiation.     

The effect of phosphatidylcholine depletion on biochemical and physical properties of a Neurospora crassa membrane mutant

By using the choline starvation process it is possible to deplete the membranes of Neurospora crassa choline auxotroph $\text{chol-1}$ of phosphatidylcholine, without affecting the viability of germinated spores or whole mycelium. Spin label probes were used to examine the possible dependence of the physical state of cellular lipids on the presence of phosphatidylcholine in the membranes.Increased freedom of rotational motion of lipid soluble probes was regularly detected in choline-starved mycelium. The accumulation of neutral lipids (mostly triglycerides) in bulk form was also observed during the choline starvation process. The experiments with isolated and separated lipid classes indicated that the observed increase in fluidity of lipids in choline-starved mycelium is partly due to the difference in physical properties between bulk lipids and membrane lipids. Spin label probe 2N4 ($\text{2-}\text{propyl}\text{-2,5,5-}\text{trimethyl-oxazolidine}\text{-N-}\text{oxyl}$), which can partition at the membrane-water interface, exhibited easier partitioning among membrane lipids of choline-starved mycelium.     

Regulation of phenotype in somatic cell hybrids derived by fusion of teratocarcinoma cell lines with normal or tumor-derived mouse cells

The phenotypes of somatic cell hybrids between murine embryonal carcinoma cell lines, F9 BrdU 7C12 and PCC4 aza 1, and normal murine splenic lymphocytes or thymoma-derived cell lines were compared. Analysis of morphology in vivo and in vitro of cell surface markers and of the karyotype of these cloned hybrid cells did not reveal any simple mechanism for the regulation of the phenotype of such hybrids. Hybrids of either the embryonal carcinoma cell phenotype or of a differentiated morphology (resembling neither parental cell) but not of lymphoid morphology can be derived from fusions of this type. Moreover, transition from one phenotype to the other (ECC → differentiated cell and differentiated cell → ECC) can be found with passage of clonally derived hybrid cell lines. Coordinate control of the phenotypic markers of the state of differentiation in these hybrid cells was found.     

The induction of antigenic changes in a teratocarcinoma stem cell line (F9) by retinoic acid

Treatment of embryonal carcinoma cells F9 with retinoic acid results in the appearance of epithelioid cells resembling endoderm which synthesize basement membrane protein and plasminogen activator. Concomitant with the appearance of these properties of differentiated cells, the epithelial cells cease to express SSEA-1, an antigenic determinant characteristic of teratocarcinoma stem cells and early mouse embryos. Our evidence indicates that the phenotypic changes that accompany retinoic acid treatment of embryonal carcinoma cells are irreversible and a consequence of the differentiation of the cells into endoderm.     

Eiweißkristalloide inOpuntia inermis

Zusammenfassung In den Epidermiszellen der Blätter vonOpuntia inermis wurden neben den X-Körpern und Eiweißspindeln auch echte Eiweißkristalle in Form rhombenförmiger Plättchen gefunden. Diese Kristalle sind vermutlich im Zellsafte lokalisiert.Die spindelhaltigen Exemplare vonOpuntia inermis unterschieden sich auch dadurch von anderen spindelführenden Kakteen, daß sie auch in Schließzellen Eiweißspindeln enthielten. Das Ausbleiben der Eiweißspindeln in den Schließzellen der Kakteen ist demnach keine regelmäßige Erscheinung.Prof. Dr. V.Vouk zum 70. Geburtstag gewidmet.     

Inhibition of both BRAF and MEK in BRAFV600E mutant melanoma restores compromised dendritic cell (DC) function while having differential direct effects on DC properties

Purpose

Dendritic cells (DCs) can induce strong tumor-specific T-cell immune responses. Constitutive upregulation of the mitogen-activated protein kinase (MAPK) pathway by a BRAF^V600 mutation, which is present in about 50 % of metastatic melanomas, may be linked to compromised function of DCs in the tumor microenvironment. Targeting both MEK and BRAF has shown efficacy in BRAF^V600 mutant melanoma.

Methods

We co-cultured monocyte-derived human DCs with melanoma cell lines pretreated with the MEK inhibitor U0126 or the BRAF inhibitor vemurafenib. Cytokine production (IL-12 and TNF-α) and surface marker expression (CD80, CD83, and CD86) in DCs matured with the Toll-like receptor 3/Melanoma Differentiation-Associated protein 5 agonist polyI:C was examined. Additionally, DC function, viability, and T-cell priming capacity were assessed upon direct exposure to U0126 and vemurafenib.

Results

Cytokine production and co-stimulation marker expression were suppressed in polyI:C-matured DCs exposed to melanoma cells in co-cultures. This suppression was reversed by MAPK blockade with U0126 and/or vemurafenib only in melanoma cell lines carrying a BRAF^V600E mutation. Furthermore, when testing the effect of U0126 directly on DCs, marked inhibition of function, viability, and DC priming capacity was observed. In contrast, vemurafenib had no effect on DC function across a wide range of dose concentrations.

Conclusions

BRAF^V600E mutant melanoma cells modulate DC through the MAPK pathway as its blockade can reverse suppression of DC function. MEK inhibition negatively impacts DC function and viability if applied directly. In contrast, vemurafenib does not have detrimental effects on important functions of DCs and may therefore be a superior candidate for combination immunotherapy approaches in melanoma patients.