Increased phosphorylation of nuclear substrates for rat brain protein kinase C in regenerating rat liver nuclei.

Protein phosphorylation catalysed by rat brain protein kinase C (PKC) has been studied in nuclei isolated from normal and regenerating rat liver. Histone H1 and a 40,000 molecular weight protein were hyperphosphorylated at all the explored regeneration times, ranging from 3 to 22 h after partial hepatectomy. Phosphorylation of the two substrates was totally dependent on calcium and lipids and was abolished by low concentration of staurosporine. The observed early change of phosphate content of histone H1 and of the 40,000 molecular weight protein on the time scale of liver regeneration suggests that PKC might be involved in the initial nuclear events leading to cell proliferation.     

Cell detachment mediated by hyaluronic acid

Exogenous hyaluronic acid (HA) added to a confluent monolayer of 3T3 BALB cells facilitates cell detachment which can be enhanced by gently pipetting. When HA is added to a cell culture with the cell inoculum, the cells are able to grow and form a confluent monolayer, but the cellular density is lower than in the control cultures, in a concentration-dependent way. This difference seems due to the ease of detachment promoted by HA on the cells near confluency. In fact only near confluency is the amount of the detached cells greater in the culture plates containing HA than in controls. Culture dishes containing substrate-attached material (SAM) left behind by the confluent 3T3 BALB cells have been prepared by removing the cells with different detaching agents. The SAM-containing dishes have been incubated in the presence of HA for 24 h and, after washing, were used for cell cultures. The cells grown on such HA-treated dishes show a very low density at confluency and in some cases are prevented from forming a confluent monolayer. When the SAM-containing dishes are treated with Streptomyces hyaluronidase, the effect of HA is abolished and the cells are able to grow normally. Among the chondroitin sulphates, only chondroitin sulphate C shows the same effects as HA, whereas A and B are ineffective.     

Further considerations on the thermal stabilization of the nuclear matrix in mouse erythroleukemia cells.

The morphology and the polypeptide composition of the nuclear matrix obtained from 37 degrees C incubated nuclei has been studied in mouse erythroleukemia cells. From a structural point of view, in the absence of heat treatment, the matrix lacked identifiable nucleolar remnants and the internal fibrogranular meshwork whereas a peripheral lamina was seen. On the contrary, the matrix obtained from heat exposed nuclei displayed very electrondense nucleolar remnants and an abundant inner network. These results were obtained irrespective of the type of extracting agent (2M NaCl or 0.2 M (NH4)2SO4) used to remove histones and other soluble proteins. The heat stabilization of the matrix could not be prevented by sulfhydryl blocking chemicals such as iodoacetamide and n-ethylmaleimide, thus suggesting that heat does not stabilize the matrix by inducing the formation of disulfide bonds. Only limited differences in the polypeptide pattern of matrix isolated under different conditions were seen using one-dimensional pore gradient polyacrylamide gels stained with both Coomassie Brilliant Blue and silver despite the fact that the matrix fraction from heat treated nuclei retained about three fold more protein in comparison with controls. The same results were obtained also by means of two-dimensional non-equilibrium gel electrophoresis.     

The effect of in vitro heating on the distribution of nuclear matrix polypeptides in HeLa cells

The in situ nuclear matrix was obtained from HeLa cells. After permeabilization with nonionic detergent, the resulting structures were incubated for 1h at 37°C to determine whether or not such an incubation might result in the redistribution of nuclear polypetides which resisted extraction with buffers of high-ionic strength (1.6 M NaCl or 0.25 M (NH₄)2SO₄ as well as DNase I digestion. Using indirect immunofluorescence experiments and monoclonal antibodies we show that heating to 37° C changes the distribution of a 160 kDa protein previously shown to be a component of the inner matrix network. On the other hand, a 125 kDa polypeptide was not affected at all by the incubation. Our results clearly indicate that the inclusion of a 37°C incubation (for example during digestion with DNase I) in the protocol to obtain the in situ nuclear matrix can result in the formation of in vitro artifacts.     

Translocation of Akt/PKB to the nucleus of osteoblast-like MC3T3-E1 cells exposed to proliferative growth factors

An active phosphatidylinositol 3-kinase (PI3K) has been shown in nuclei of different cell types. The products of this enzyme, i.e. inositides phosphorylated in the D3 position of the inositol ring, may act as second messengers themselves. Nuclear PI3K translocation has been demonstrated to be related to an analogous translocation of a PtdIns(3,4,5)P(3) activated PKC, the zeta isozyme. We have examined the issue of whether or not in the osteoblast-like clonal cell line MC3T3-E1 there may be observed an insulin-like growth factor-I- (IGF-I) and platelet-derived growth factor- (PDGF) dependent nuclear translocation of an active Akt/PKB. Western blot analysis showed a maximal nuclear translocation after 20 min of IGF-I stimulation or after 30 min of PDGF treatment. Both growth factors increased rapidly and transiently the enzyme activity of immunoprecipitable nuclear Akt/PKB on a similar time scale and after 60 min the values were slightly higher than the basal levels. Enzyme translocation was blocked by the specific PI3K inhibitor, LY294002, as well as cell entry into S-phase. Confocal microscopy showed an evident increase in immunostaining intensity in the nuclear interior after growth factor treatment but no changes in the subcellular distribution of Akt/PKB when a LY294002 pre-treatment was administered to the cells. These findings strongly suggest that the intranuclear translocation of Akt/PKB is an important step in signalling pathways that mediate cell proliferation.     

Rab3A and Rab3D control the total granule number and the fraction of granules docked at the plasma membrane in PC12 cells

Rab proteins are Ras-like GTPases that regulate traffic along the secretory or endocytic pathways. Within the Rab family, Rab3 proteins are expressed at high levels in neurons and endocrine cells where they regulate release of dense core granules and synaptic vesicles. Immuno-electron microscopy shows that Rab3A and Rab3D can coexist on the same granule before and after docking. Using electron microscopy of transfected PC12 cells, we report that expression of wild-type Rab3A (or Rab3D) increases the total number of granules and the percentage that is docked at the plasma membrane. Mutated Rab3A N135I (or Rab3D N135I) decreases the total granule number and the fraction of granules docked to the plasma membrane. These data show that at least one of the functions of Rab3A and Rab3D proteins is to control the number of granules docked at the plasma membrane.     

Behavior of nucleolar proteins during the course of apoptosis in camptothecin-treated HL60 cells

By means of immunofluorescence and immunoelectron microscopy we have studied the fate of different nucleolar components during the apoptotic process in camptothecin-treated HL60 cells. We have found that RNA polymerase I disappeared while UBF was associated with previously described fibrogranular threaded bodies. In contrast, fibrillarin, C23/nucleolin, and B23/nucleophosmin remained detectable in granular material present amid micronuclei of late apoptotic cells. Double immunolabeling experiments showed colocalization of both C23 and B23 with fibrillarin. Immunoblotting analysis showed that UBF was proteolytically degraded, whereas fibrillarin, C23/nucleolin, and B23/nucleophosmin were not. These results may help explain the presence of anti-nucleolar antibodies seen in various pathological disorders.     

Fine structure of the development of Sarcocystis singaporensis in Python reticulatus from macrogamont to sporulated oocyst stage

Three, 4-month old reticulated pythons (Python reticulatus) hatched from eggs laid by a newly caught female from Singapore Island, were fed on muscles of Sarcocystis singaporensis-infected Rattus rattus caught in Singapore. Snakes were sacrificed five, six and eight days later. The infected tissues were studied by transmission electron microscope. The present communication summarizes findings on macrogamont and oocyst stages. In the premature stages, rough endoplasmic reticulum consolidate into a large rectangular array; the electron-dense wall-forming-like bodies reveal a laminar structure. Macrogamont parasitophorous vacuoles became filled with granular matrix and electron-dense strands, which later on consolidate into a coat around the fertilized zygote. The oocyst wall is constructed from several formed membranes combined with deposited substance. All development to the sporulated oocyst stage occurs in the mucosal epithelium.     

Rabphilin localizes with the cell actin cytoskeleton and stimulates association of granules with F-actin cross-linked by {alpha}-actinin

In endocrine cell, granules accumulate within an F-actin-rich region below the plasma membrane. The mechanisms involved in this process are largely unknown. Rabphilin is a cytosolic protein that is expressed in neurons and neuroendocrine cells and binds with high affinity to members of the Rab3 family of GTPases localized to synaptic vesicles and dense core granules. Rabphilin also interacts with alpha-actinin, a protein that cross-links F-actin into bundles and networks and associates with the granule membrane. Here we asked whether rabphilin, in addition to its granule localization, also interacts with the cell actin cytoskeleton. Immunofluorescence and immunoelectron microscopy show that rabphilin localizes to the sub-plasmalemmal actin cytoskeleton both in neuroendocrine and unspecialized cells. By using purified components, it is found that association of rabphilin with F-actin is dependent on added alpha-actinin. In an in vitro assay, granules, unlike endosomes or mitochondria, associate with F-actin cross-linked by alpha-actinin. Rabphilin is shown to stimulate this process. Rabphilin enhances by approximately 8-fold the granule ability to localize within regions of elevated concentration of cross-linked F-actin. These results suggest that rabphilin, by interacting with alpha-actinin, organizes the cell cytoskeleton to facilitate granule localization within F-actin-rich regions.