No abstract

The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), at nanomolar concentrations, induces rapid (t1/2 approximately 30 s) protrusion of multiple petal-shaped lamellae by neutrophil leucocytes. Lamellae are richly endowed with actin filaments as determined by the localization of rhodamine-phalloidin, suggesting extensive assembly at the cell cortex. Direct measurement of the proportion of total cell actin which is polymerized, by using a deoxyribonuclease I inhibition assay, indicates that the proportion of polymerized actin approximately doubles, and that assembly initiated by 30 nM TPA occurs with no obvious lag phase and with a t1/2 of about 30 s. A half-maximal response was induced at 2 nM TPA. Since both actin assembly and protrusion of lamellae are completely inhibited by 10(-6) M cytochalasin D, protrusion of lamellae is presumably dependent on actin filament assembly. To examine whether TPA induces actin assembly via changes in [Ca2+]i or pHi, these parameters were monitored in cells loaded with the fluorescent indicators quin2 and quene1 respectively. Addition of TPA caused no change in [Ca2+]i but a biphasic change in pHi. To examine further the potential role of ionic changes in regulation of actin assembly, the morphological responses of cells to TPA were monitored in severely Ca2+-depleted cells, or cells in which pHi had been experimentally raised or lowered by simultaneous additions of a weak base (NH4Cl) or weak acid (CH3COONa) respectively. The protrusion of lamellae induced by TPA was completely unaffected by these experimental manipulations indicating that TPA can directly regulate actin assembly, and hence morphology, by mechanisms essentially independent of [Ca2+]i and pHi.(ABSTRACT TRUNCATED AT 250 WORDS)

Summary Although intra-luminal epidermal growth factor (EGF) may stimulate cell proliferation in the upper gastrointestinal tract, its role in the large bowel has not been established. We have therefore studied the effect of intra-rectal EGF administration on both normal growth and carcinogenesis in the rat colon. Colonic cancer was induced in rats with azoxymethane (10 mg kg-' week-' for 12 weeks s.c.) and controls dosed with saline. In each group, animals were randomised to receive EGF (12 nM, 0.8 nM or saline control) in 0.5 ml saline via a rectal tube daily for 24 weeks. At this time, crypt cell production rates (CCPRs) were determined at two sites in the colon: one of maximal and another of minimal exposure to EGF (5 cm and 10 cm from the anal margin respectively). No effects of EGF were seen at 10 cm. The lower dose of EGF gave CCPRs that mirrored the control values. The higher dose of EGF in the animals not treated with azoxymethane stimulated mucosal growth. Azoxymethane increased in CCPR, but this was suppressed by the high dose of EGF. These results suggest that (1) luminal EGF and azoxymethane independently increase the colonic CCPR and their combined effect is not synergistic but antagonistic; (2) EGF may have a role in normal epithelial growth, but does not potentiate colonic carcinogenesis in this model.

Frozen sections, 0.5 micron thick, of the lactating mouse mammary gland have been used to localize immunoglobulins A and G and serum albumin throughout the connective tissue stroma, in the lumina of blood vessels, in milk stored in the alveoli and in the lateral spaces between adjacent epithelial cells. In addition, the immunoglobulins were localized to their specific plasma cells in the connective tissue stroma. Serum albumin was further identified within the mammary epithelial cells as small spots of fluorescence scattered throughout the cytoplasm. The immunoglobulins were not localized within these cells in untreated sections, but in sections treated with trypsin and Soybean trypsin inhibitor, it was possible to identify a similar distribution to that for serum albumin. The spots of fluorescence representing the intracellular localization of the immunoglobulins and serum albumin were frequently found in association with the periphery of intracellular lipid droplets.

Homozygous mutations of granulin precursor (GRN) lead to neuronal ceroid lipofuscinosis, a severe neurodevelopmental disease, in humans and neuroinflammation in mice. Haploinsufficiency of GRN almost invariably causes frontotemporal dementia (FTD). The GRN locus produces progranulin (PGRN), a lysosomal precursor protein that is cleaved to granulin peptides. Despite intensive investigation, the function of granulins and the reason why their absence causes neurodegeneration remain unclear. Here, we investigated PGRN function in lipid degradation, a major function of lysosomes. We show that PGRN knockout human cells, PGRN-deficient murine brain, and frontal lobes of human brains from subjects with PGRN deficient FTD have increased levels of gangliosides, highly abundant sialic acid containing glycosphingolipids (GSL) that are degraded in lysosomes. Probing how PGRN deficiency causes these changes, we found normal levels and activities of enzymes that catabolize gangliosides. However, levels of bis(monoacylglycero)phosphate (BMP), a lysosomal lipid required for ganglioside catabolism, were markedly reduced in PGRN deficient cells and brain tissues. These data indicate that granulins are required to maintain BMP levels, which regulate ganglioside catabolism, and that PGRN deficiency in lysosomes leads to gangliosidosis. This aberrant accumulation of gangliosides may contribute to neuroinflammation and neurodegeneration susceptibility.