Susanne Kölare

Munc-18 associates with syntaxin and serves as a negative regulator of exocytosis in the pancreatic β-cell

The Munc-18 protein (mammalian homologue of theunc-18 gene; also called nSec1 or rbSec1) has been identified as an essential component of the synaptic vesicle fusion protein complex. The cellular and subcellular localization and functional role of Munc-18 protein in pancreatic β-cells was investigated. Subcellular fractionation of insulin-secreting HIT-T15 cells revealed a 67-kDa protein in both cytosol and membrane fractions. Immunohistochemistry showed punctate Munc-18 immunoreactivity in the cytoplasm of rat pancreatic islet cells. Direct double-labeling immunofluorescence histochemistry combined with confocal laser microscopy revealed the presence of Munc-18 immunoreactivity in insulin-, glucagon-, pancreatic polypeptide-, and somatostatin-containing cells. Syntaxin 1 immunoreactivity was detected in extracts of HIT-T15 cells, which were immunoprecipitated using Munc-18 antiserum, suggesting an intimate association of Munc-18 with syntaxin 1. Administration of Munc-18 peptide or Munc-18 antiserum to streptolysin O-permeabilized HIT-T15 cells resulted in significantly increased insulin release, but did not have any significant effect on voltage-gated Ca2+ channel activity. The findings taken together show that the Munc-18 protein is present in insulin-secreting β-cells and implicate Munc-18 as a negative regulator of the insulin secretory machinery via a mechanism that does not involve syntaxin-associated Ca2+ channels.

Serum Factors Induce C- fos Expression and Rapid Cell Proliferation in Adolescent But Not in Infant Rat Proximal Tubule Cells

ABSTRACT: Kidney epithelial cells in short-term primary culture have been studied with regard to proliferative rate and expression on the c-fos protooncogene. The experiments were performed on subconfluent renal proximal tubule cells isolated from infant and adolescent rats. Proliferation was determined by 3H-thymidine autoradiography and nuclear content of c-fos protein by semiquantitative immunofluorescence. The basal proliferative rates in infant and adolescent renal proximal tubule cells were the same after 48 h of primary culture in Dulbeccos modified Eagles medium with 10% FCS. Serum deprivation for 24 h caused a significant growth inhibition in both infant and adolescent cells. C-fos was expressed to the same extent in infant and adolescent serum-deprived cells. The rapid response to the addition of serum was markedly different in infant and adolescent cells. In adolescent cells, addition of serum led to a transient significant increase in the nuclear expression of c-fos protein, reaching a peak at 60 rain. No increase hi c-fos was seen in infant cells. In adolescent cells, the rate of proliferation increased 11-fold and 3H-thymidine labeling index reached 26.7 ± 4.3%. In infant cells, the proliferative response to serum addition was significantly lower; the labeling index reached only 4.2 ± 1.2%. It could be excluded that the attenuated response in infant cells was due to cell death or impaired metabolic function. The results imply that the principles of growth regulation change postnatally.

Studies on thymocyte subpopulations in guinea pigs: in vivo differentiation of bromodeoxyuridine labelled cells, with special reference to rosette-forming ability

Cycling thymocytes were labelled by an intracardial injection of bromodeoxyuridine (BrdUrd) in a total of 32 guinea pigs and the incorporation into DNA studied in subpopulations of cells defined by buoyant density and rosette-forming ability. The labelling pattern was compared at different times after injection (0.5 h to 120 h). A marked shift of labelled cells from large, low density cells (population 1a) to small, high density cells (population 2) was observed. During the first 48 h, the ratio between labelling indices of cell populations 1a and 2 decreased from 10 to 0.5. The number of labelled cells forming rosettes with rabbit erythrocytes (RFC+) increased while the number of labelled non-rosetting cells (RFC-) decreased from 0.5 h to 48 h, probably representing transformation of RFC- to RFC+. Then, after a decreased labelling in all cell populations at 72 h, an increase in both RFC+ and RFC- populations occurred at 96 h. The labelling in RFC- cells at 96 h was nearly as high as immediately after labelling. This second labelling of RFC- cells could represent immigration of precursor cells, a wave of proliferation in initially labelled precursors, and/or the formation of mature cells from the initially labelled precursors. The results indicate that a great majority of proliferating cells differentiate into small, high density cells within 48 h and that rosetting ability is acquired in many cells during this period. A model of intrathymic differentiation which fits the observations is presented.

Studies on Thymocyte Subpopulations in Guinea Pigs VI. Differentiation of Precursor Cells in vivo and in Vitro

The relation between six subpopulations of guinea pig thymocytes, separated on the basis of PNA binding and buoyant density, was studied. Incubation in vitro of either unseparated thymocytes or isolated large, low density cells, caused a shift towards smaller cells with high buoyant density. In order to determine whether these changes reflect a normal differentiation we pulsed labelled thymocytes with 3H-Thymidine and traced the labelling in the separate subpopulations after different intervals by autoradiography. Both in vivo and in vitro, the thymidine was initially incorporated mainly into PNA+ cells (85% of all labelled cells), particularly into PNA+ low-density cells (called PNA+Ia), which constitute 14% of all thymus cells (1). Correlation with autoradiographic sections of the thymus indicated that these cells were mainly located in the subcapsular and juxtamedullary cortex. At 24 h after labelling in vivo, more than 95% of the labelled cells were smaller, high density cells, both PNA+ and PNA-. At this time, labelled cells were present throughout the thymus with no preferential localization. Labelling and incubation in vitro were accompanied by similar changes after a 24-h time period when cells were cultured in the presence of serum. We conclude that the labelled PNA+Ia cells represent precursor cells, located in the subcapsular and juxtamedullary parts of the cortex. Within 24 h these cells differentiate into high density cells, some of which are PNA-. The corresponding results obtained in vitro indicate that these differentiation steps may also occur in the absence of a thymic microenvironment, and the study of histological sections indicate that these steps were not associated with any major relocalization within the thymus.

Monosialoganglioside GM1 increases survival of thymocytes

Gangliosides are normal constituents of the plasma membrane. Exogenous gangliosides can be incorporated into the membrane and extensive research in nervous tissue has demonstrated a beneficial effect of gangliosides on the functional recovery of lesioned neurons and protection against neurotoxins. This paper shows that the effect of gangliosides is not restricted to neurons. The monosialoganglioside GM1 efficiently increases the survival of thymocytes and protects them against both the lytic effect of the glucocorticoid prednisolone and the effect of a thymocytotoxic serum. The protective effect of GM1 was achieved bothin vitro andin vivo.

Studies on Thymocyte Subpopulations in Guinea Pigs

Thymocytes were separated according to increasing buoyant density into the three subpopulations la (25% of recovered cells), lb (20%) and II (55%), and according to binding to peanut agglutinin (PNA) into PNA+ (65 %) and PNA– cells (35 %). The frequency of PNA+ was 56% in la, 60% in lb and 66% in population II. Electronic cell volume determinations disclosed mean volumes of 160 fl for la, 130 fl for lb and 100 fl for population II. PNA+ and PNA– cells were very similar as regards cell volume. Thus, PNA+ and PNA– cells are remarkably uniformly distributed among cell categories of different density and cell volume. The rapidly cycling thymocytes, regarded as the most immature cells in the thymus, and the target cells for a thymocyte growth factor both belonged to the PNA+ cells of population la. The mitogen-responsive thymocytes also belonged to population la, but were PNA-. The largest subpopulation of thymocytes, apparently corresponding to the small, non-cycling cortical cells, were recovered as PNA+ cells of population II.

Effect of Growth Factors on the Proliferation of Thymocytes in Different Functional and Anatomical Compartments

The intrathymic location of cells responding to the thymocyte growth peptide (TGP) was investigated in guinea pigs. Thymus cells were labelled by topical application of a fluorescein isothiocyanate solution and the percentage of fluorescent cells analyzed by flow cytometry. Large cells forming rosettes with rabbit erythrocytes, known to respond to TGP, had a lower percentage of fluorescent cells than non-rosetting cells which do not respond to TGP. If the TGP-responding cells are anatomically segregated in the guinea pig thymus, this might indicate that they are preferentially located in the deep, juxtamedullary part of the cortex, where one major proliferating compartment is situated.