The fate of prorenin during granulopoiesis in epithelioid cells. Immunocytochemical experiments with antisera against renin and different portions of the renin prosegment

Comparative immunocytochemical experiments with antisera directed against renin and three synthetical peptides (Pro 1, Pro 2A and Pro 3) covering almost the entire span of human renin prosegment were performed on human kidney tissue. With anti-Pro 1, i.e. the antiserum which recognizes the NH2 terminus of human prorenin, no clear immunolabeling of juxtaglomerular epithelioid cell secretory granules could be obtained. It is therefore concluded that the corresponding portion of human prorenin may be cleaved off in the Golgi complex. After application of anti-Pro 3, the antiserum which recognizes the COOH terminus of the prosegment, only the juvenile secretory granules of epithelioid cells were consistently labeled, whereas, in contrast, some of the intermediate and most of the mature secretory granules were anti-Pro 3-negative. As the immunoreactivity of mature renin increased remarkably from protogranules to mature secretory granules, it is suggested that the cleavage of the COOH terminus of the prosegment, i.e. the activation of renin, takes place in juvenile and intermediate granules during condensation of the enzyme. The immunoreactivity of Pro 2A, corresponding to the middle portion of the prosegment, disappeared in a somewhat earlier stage of granulopoiesis than that of Pro 3. It is therefore concluded that the corresponding segmental cleavage, the result of which is a truncated version of intact prorenin, occurs in the protogranules of epithelioid cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development and fate of the secretory granules of juxtaglomerular epithelioid cells

Summary The development and fate of the secretory granules in murine, rat and human juxtaglomerular epithelioid cells were examined using ultrastructural and immunocytochemical methods. The formation of mature renin granules occurs by fusion of rhomboid protogranules followed by coalescence of their paracrystalline contents, and by the fusion of roundish juvenile granules having an amorphous internum. Protogranules with paracrystalline contents are prominent in animals with stimulated renin synthesis, indicating an overcharge in processing and/or packaging of the secretory product, renin, under these conditions. Various similarities between lysosomes/multivesicular bodies (MVBs) and juvenile renin granules have been observed. With the exception of small MVBs, no renin-negative organelles that could be regarded as lysosomes were found in epithelioid cells of mice and rats. Therefore, we suggest that renin granules are modified lysosomes. Immunocytochemical findings indicate that juvenile secretory granules of epithelioid cells represent the converting and activating compartment for prorenin. Endocytosed foreign tracers such as HRP or cationized ferritin are preferentially internalized by juvenile renin granules, which hence appear to be outstanding by their fusogeneity. Consequently, juvenile granules are probably responsible for the secretion of prorenin, and mature granules for that of active renin.These studies were supported by the German Research Foundation within the Forschergruppe Niere/Heidelberg     

The fate of larval flagellated cells during metamorphosis of the sponge Halisarca dujardini

Sponge larval flagellated cells have been known to form the external layer of larva, but their subsequent fate and morphogenetic role are still unclear. It is actually impossible to follow flagellated cell developmental fate unless a specific marker is found. We used percoll density gradient fractionation to separate different larval cell types of Halisarca dujardini (Demospongiae, Halisarcida). A total of 5 fractions were obtained which together contained all cell types. Fraction 1 contained about 100% FC and its polypeptide composition was very different to that of the other fractions. Of all larval cell types, flagellated cells displayed the lowest in vitro aggregation capacity. We raised a polyclonal antibody against a 68 kDa protein expressed by larval flagellated cells. Its specificity was tested on total protein extract from adult sponges by Western blotting and proved to be suitable for immunofluorescence. By means of double immunofluorescence using both this polyclonal antibody and commercial anti-tubulin antibodies, we studied the distribution of the 68 kDa protein in larval flagellated cells and its fate at successive stages of metamorphosis. In juvenile sponges just after metamorphosis the choanocytes and the upper pinacoderm were labelled with both antibodies. In larval flagellated cells, the 68 kDa protein was found all over the cytoplasm appearing as granules, while in adult sponges, it was present in the apical part of choanocytes in the vicinity of collars. Direct participation of the larval flagellated cells in the development of definitive structures was demonstrated.     

The fate of desmosomal proteins in apoptotic cells

Activation of caspases results in the disruption of structural and signaling networks in apoptotic cells. Recent biochemical and cell biological studies have shown that components of the cadherin-catenin adhesion complex in epithelial adherens junctions are targeted by caspases during apoptosis. In epithelial cells, desmosomes represent a second type of anchoring junctions mediating strong cell-cell contacts. Using antibodies directed against a set of desmosomal proteins, we show that desmosomes are proteolytically targeted during apoptosis. Desmogleins and desmocollins, representing desmosome-specific members of the cadherin superfamily of cell adhesion molecules, are specifically cleaved after onset of apoptosis. Similar to E-cadherin, the desmoglein-3 cytoplasmic tail is cleaved by caspases. In addition the extracellular domains of desmoglein-3 and desmocollin-3 are released from the cell surface by a metalloproteinase activity. In the presence of caspase and/or metalloproteinase inhibitors, both cleavage reactions are almost completely inhibited. As reported previously, the desmosomal plaque protein plakoglobin is cleaved by caspase-3 during apoptosis. Our studies now show that plakophilin-1 and two other major plaque proteins, desmoplakin-1 and -2, are also cleaved by caspases. Immunofluorescence analysis confirmed that this cleavage results in the disruption of the desmosome structure and thus contributes to cell rounding and disintegration of the intermediate filament system.     

The fate of embryonal-carcinoma cells in mouse blastocysts

Double-labeled embryonal-carcinoma (ECa) cells were injected into blastocysts or incorporated into blastocysts by aggregation, and their fate after various periods of time in culture was investigated. ECa-247 cells labeled with fluorescent microscopheres were easily identified in whole blastocysts. These blastocysts were embedded in plastic, serially sectioned, and prepared for autoradiography. The 3H-thymidine label on the embryonal-carcinoma cells allowed precise localization of the cancer-derived cells. ECa-247 cells preferentially localized in the mural trophectoderm, with a few being seen in primitive endoderm and, even more rarely, in the inner cell mass. Selected autoradiograms were re-embedded and thin sectioned for transmission electron microscopy. The cancer-derived cells were found to have differentiated in accordance with their localization.     

The role of colony-stimulating factor in granulopoiesis

The proliferation and maturation of granulocytic-monocytic stem cells appears to be controlled by a series of closely related glycoproteins termed “colony-stimulating factors” (CSFs). Recently, we devised a 6-step scheme for the purification of murine fibroblast (L-cell)-derived CSF. Ten liter pools of conditioned media were concentrated by ultrafiltration, precipitated by ethanol, and separated on DEAE cellulose, Con-A Sepharose, and Sephadex G 150. The CSF was separated from trace contaminants, including endotoxin, by density gradient centrifugation. The purified material was radioiodinated and used to define the serum half-life and in vivo distribution. Following IV injection there was a biphasic serum clearance with a t½ of 24–40 min and 2–2½ hours in the first and second phases. Approximately 25% of the tracer was excreted in the urine at 6 h; however, urinary radioactivity was due to low molecular weight peptides. Simultaneous studies by radioimmunoassay showed a similar rapid serum clearance of unlabeled CSF but virtually no urinary CSF activity. Thus, assays for urinary CSF may not provide useful measures of in vivo CSF activity. Further in vitro studies have defined the interaction of CSF with responsive cells in the marrow. Varying doses of CSF were incubated with 10⁷ marrow cells for intervals of 24–48 h. The major increment in cell-associated radioactivity occurred between 6 and 16 h. The reaction was saturable with 1–2 ng/ml CSF. Binding was prevented by cold CSF, but not by other proteins. Irradiation yielded only a minimal reduction in CSF binding. The interaction of CSF with marrow cells appeared to require new protein synthesis, as binding was completely inhibited by cycloheximide and puromycin. Irradiated mice injected with antibodies to CSF showed an inhibition of granulopoiesis by marrow cells in peritoneal diffusion chambers; however, granulopoiesis in the intact bone marrow was unaffected. Granulpoiesis in long-term marrow cultures was also unaffected by anti-CSF. These different responses may be due to accelerated clearance of injected CSF in nonirradiated mice or to extensive stromal interactions that modulate and perhaps control granulocytic differentiation in the intact bone marrow microenvironment.     

The fate of p21 in cells surviving UV-irradiation

p21(CDKN1A) is a critical regulator of cell cycle progression in response to DNA damage. There are conflicting conclusions as to whether p21(CDKN1A) levels increase or decrease after ultraviolet (UV)-irradiation and recently it was even reported to disappear entirely following 2.5-30 Jm(-2) of UV-irradiation in the presence of growth medium. The latter would suggest an alternative mechanism for cell cycle arrest after UV-irradiation, since p21(CDKN1A) induction has been considered to be the major mediator of p53-mediated cell cycle arrest after DNA damage. Using physiological UV doses based on cell-killing, we previously observed and here verify that low doses (1.2-6 Jm(-2)) induce p21(CDKN1A) immediately after UV-irradiation, though higher doses cause a latency during which p21(CDKN1A) levels remain fairly constant before increasing. As expected, p53 induction preceded p21(CDKN1A) induction at all doses. Thus, p21(CDKN1A) levels after low doses of UV-irradiation may be controlled in a p53-dependent manner without severe reduction. We propose that physiological relevant UV doses should be determined for each target cell type prior to studying UV-induced responses and that p21(CDKN1A) is indeed critical for cell cycle arrest in cells that survive UV-irradiation.     

Immunoaffinity purification of human prorenin produced in Chinese hamster ovary cells

A simple immunoaffinity column chromatographic procedure is described whereby recombinant human prorenin secreted from Chinese hamster ovary cells may be isolated in a high state of purity from serum-free culture medium. Prorenin thus purified has been characterized by SDS-polyacrylamide gel electrophoresis and by partial sequence analysis which has revealed the expected N-terminal sequence. Trypsin treatment gives rise to renin, and reversible acid activation has also been demonstrated for the recombinant zymogen.     

Isolation and characterization of recombinant human prorenin in Chinese hamster ovary cells

Recombinant human prorenin (rh-prorenin) was purified from supernatants of Chinese hamster ovary (CHO) cell line transfected with the cDNA for rh-prorenin by employing a simple two-step procedure which consisted of ammonium sulfate precipitation and immunoaffinity chromatography using a monoclonal antibody specific for the profragment of human prorenin. About 100-fold purification with 35% recovery was achieved after the two steps. Purified rh-prorenin migrated as a single protein band with apparent molecular weights of 46,000-47,000 and about 50,000 on SDS-PAGE and gel filtration (HPLC), respectively, although it consisted of multiple components (pI values, 5.6-6.4) that could be resolved by isoelectric focusing (IEF). The treatment of rh-prorenin with endo-beta-N-acetylglucosaminidase converted the rather broad protein band to a sharp band on SDS-PAGE and reduced the number of multiple pI peaks on IEF. Amino-terminal sequence analysis of both the purified rh-prorenin and rh-renin revealed Leu-Pro-Thr-Asp- and Leu-Thr-Leu-Gly-, respectively, which agreed with those predicted from the base sequences of their cDNA. These data suggested that microheterogeneity of rh-prorenin is due to the carbohydrate moiety, but not to the protein moiety. Purified rh-prorenin was almost inactive, but was cleaved at the carboxyl end of a dibasic pair Lys-2-Arg-1 by trypsin and converted to active renin. However, at the early stage during trypsin activation, new intermediate forms between rh-prorenin and rh-renin were formed, suggesting multiple activation steps of rh-prorenin in addition to the one step activation.     

The fate of lipopolysaccharide in cultured rat Kupffer cells

Cultured rat Kupffer cells were incubated in presence of biologically tritiated Salmonella abortus equi lipopolysaccharide. Uptake of lipopolysaccharide increased rapidly during the first 2 h of incubation and then levelled off. Within the first h of incubation 10(6) Kupffer cells were able to ingest up to 18 micrograms lipopolysaccharide. Kupffer cells metabolised lipopolysaccharide and released lipopolysaccharide-related substances, but neither the cell-associated lipopolysaccharide nor the released lipopolysaccharide products were detoxified, as measured by the mouse lethality test.     

Inhibition of in vitro granulopoiesis by autologous allogeneic human NK cells

This study demonstrates the ability of human NK cells to inhibit in vitro granulopoiesis of autologous and allogeneic BM cells. NK lytic activity and GM-CFC inhibition was present among nonstimulated lymphocytes from healthy donors and could be increased by treatment of PBL with IFN. Both the cytotoxic NK cells and the GM-CFC inhibitory cells could be enriched for among nonadherent, low-density cells. High-density cells were not cytotoxic, only inhibitory to a small extent, and could become neither cytotoxic nor more inhibitory after IFN treatment. In contrast, low-density cells showed an increased cytotoxic and GM-CFC inhibitory capacity after IFN treatment. The NK mediated GM-CFC inhibition was dependent on cell contact with BM cells, increased with longer preincubation times, and was most efficient against 7-day GM-CFC as compared with 14 day GM-CFC progenitors. In conclusion, these data provide new information about the human NK cell as a potent inhibitor of in vitro granulopoiesis and also as a possible regulator of hematopoiesis in vivo.     

The fate of nutrients during fires in a tropical savanna

Abstract The nutrient loads contained in the grassy fuel before fires, and of ash subsequently, were compared to determine the fluxes of macronutrients, copper and zinc during fires at Kapalga in Kakadu National Park. The fluxes were estimated in three vegetation types: forest, woodland and open woodland. The magnitudes of the fluxes were greatest in the forest community where grassy fuel loads were highest at about 6.3 t ha^?1. In these sites, 54–94% of all measured nutrients in the fuel were transferred to the atmosphere during the fires. For each nutrient, the proportion transferred to the atmosphere as entrained ash was calculated by assuming that calcium was not volatilized during the fires. If the transfer of entrained ash represents local redistribution only, then rainfall accession and the deposition of these particu-lates should replace most of the losses of all nutrients except nitrogen (N). Estimated rates of biological fixation of N appear to be insufficient to replace the annual losses of N. It is therefore concluded that a regime of annual fires that completely burn the available grassy fuel would deplete N reserves in these savannas, unless there are other sources of biologically fixed N, which are unknown at present.