Appearance of clasmatosis in the normal and pathological liver

It was established that clasmatosis of cytoplasmic fragments toward sinusoids occurred under normal physiological conditions (embryogenesis of chick liver, the liver of starved grass carp and silver carp) and pathological conditions (disturbance of rat hepato-intestinal circulation). The clasmosomes of rat and chick liver cells contained free ribosomes and small vesicles while those in the liver of starved fish consisted of glycogen. It was also shown that mitochondria with the signs of complete clasmatosis appeared in the hepatocyte cytoplasm immediately after the beginning of intensive bile secretion to the bile canaliculus (in liver cells of rat and chick embryo and in those of frogs after complete metamorphosis). Such mitochondria were partially disintegrated and were located near the bile canaliculi. It is assumed that clasmatosis of the fragments of the liver cell cytoplasm or mitochondria takes places where it is necessary to rapidly supply the body or cell with some products of metabolism or to remove something from the cell as is the case with erythroblasts, i. e. clasmatosis is one of the mechanisms of the adaptation of the cell and its organelles to changes in the environment.     

Formation of "ghost" bodies and calcification in experimental atherosclerosis in nonhuman primates. An ultrastructural study

Aortic lesions from African green monkeys fed a cholesterol diet for up to 24 months were studied by electron microscopy. The lesions, grossly classified as fatty dots and fatty streaks consisted of foam cells, increased amounts of interstitial connective tissues and osmiophilic lipid material. In addition, in the interstitial spaces, there were membrane-bound detached cytoplasmic fragments and deeply osmiophilic calcium spherules. The smooth muscle cells had a frayed appearance and bulbous cytoplasmic pseudopodlike processes. Figures suggestive of transition between these cell processes and the detached cytoplasmic fragments were observed. The detached cytoplasmic fragments or ghost bodies often contained lipid droplets, myelin-like figures and calcific material. The process of budding off cytoplasmic fragments was interpreted as a form of clasmatosis enabling smooth muscle cells to eliminate substances which could not be degraded intracellularly. It is proposed that material presented within the ghost bodies may become a nucleation site for calcium salts deposition. Cell necrosis was not a feature observed in this material.     

Clasmatosis: Certain qualitative and quantitative peculiarities of this phenomenon in the lymphoid organs of the rat

Summary Clasmatosis in the spleen, thymus, submaxillary lymph nodes, Peyer's patches and bone marrow has been studied in Wistar male rats by standard cytological methods, and a method of obtaining a fraction of cytoplasmic fragments (leptons) from the spleen has been elaborated. A conjectural scheme of the successive stages of this phenomenon has been drawn up; according to this scheme, the lepton detached from the clasmatocyte undergoes a series of transformations and again splits off a fragment from itself (secondary clasmatosis).Micrometric measurements of the diameters of free lymphocytes, clasmatocytes, and bound and free leptons have made it possible to establish that: 1. the size of the lepton does not depend on the size of its producer cell; 2. the largest percentage of clasmatocytes occurs among cells with a diameter of 9 (about 35%); 3. the lymphocytes of the spleen are smaller than the clasmatocytes of the same organ, and the free leptons are smaller than the bound leptons. The assumption is made that this difference is due in the former case to clasmatosis itself, and in the latter to formation of secondary leptons.Clasmatosis is traced in all stages of differentiation of lymphoid cells, which lose their pyroninophilic granularity along with their loss of cytoplasm.The different lymphoid organs have been compared according to their relative content of clasmatocytes and free leptons. The second, more distinct index increases in the following succession: thymus, submaxillary lymph nodes, spleen, Peyer's patches. The difference between the first and the last is 15.In the bone marrow of intact rats clasmatosis is practically absent.The results have been discussed in relation to the possible connection of clasmatosis with the differentiation of lymphoid cells.The authors are very grateful to N. S.Nikishova for her help in the cytological part of this work and to M. L.Kopnova for the microphotographs.     

Role of clasmatosis of the phagocytes in immunogenesis]

With the aid of cytological and cytochemical studies it was shown that clasmatosis of phagocytes (micro- and macrophages) was primarily characteristic of active phagocytising cells, and not of dying cells as considered formerly. Experiements were conducted on guinea pigs in the course of vaccinal and infectious processes in brucellosis. As noted, clasmatosis of phagocytes known in intact animals was considerably enhanced during vaccinal and infectious processes. Pieces of cytoplasm separating from phagocytes were full of acid phosphatase, brucella antigen; fragments of macrophage cytoplasm in addition contained RNA. There are reasons to suppose that with the aid of clasmocytosis of phagocytes became intensified and the antigenic information spread rapidly in the animal organism.     

Physiological and reparative regeneration of the mitochondria]

The ultrastructure of mitochondria of hepatocytes in normal and pathological conditions was studied. It was shown that the process of regeneration of the ultrastructure of swollen mitochondria with a lucent matrix up to the normal state was completed in hepatocytes of the rat and chick embryos within one day. It was established that one of the ways of intraorganoid regeneration of mitochondria in hepatocytes of chick embryos and of mice after injections of CCl4 twice a week for 5 months was clasmatosis of the destroyed mitochondria fragments and their removal through the partially disintegrated exterior membrane of mitochondria followed by the membrane restoration. The process of mitochondrial regeneration after clasmatosis of its fragments was shown to require two days in the chick embryo hepatocytes.     

Ultrastructural alteration of cytolytic T lymphocytes following their interaction with target cells. III. Plasmalemma, "membranosomas".

Ultrastructure of the plasma membranes of cytolytical T lymphocytes (CTL) in their interaction with target cells (TC) was studied. Thirty to sixty minutes after the beginning of interaction shedding of the CTL plasma membrane was observed: its fragments shedded from a local (50–100 nm in diam.) area on the lymphocyte surface at the site opposite to the CTL contact region with TC. Oval structures of high electron density 10 to 40 nm in diam. were detected on the CTL surface. We designated them as “membranosomas” (MS). MS were in close apposition to the inner surface of the plasma membrane and showed projections of 2 to 3 nm in diam. and 5 to 6 nm long towards the outer surface of the plasma membrane. MS were separated from the CTL surface during clasmatosis or as component parts of “shedding” plasma membranes.     

Distribution of cytoplasmic determinants in unfertilized eggs of the ascidian Halocynthia roretzi

 Cytoplasmic determinants that specify the fate of endoderm, muscle and epidermis cells are known to be localized in specific areas of fertilized eggs of ascidians. The presence of such cytoplasmic determinants in unfertilized eggs was demonstrated in previous studies, but no information has yet been proved about their distribution. To investigate the distribution of cytoplasmic determinants in unfertilized eggs, we devised a method for distinguishing the polarity of unfertilized eggs using vital staining and we performed cytoplasmic-transfer experiments by fusing blastomeres and cytoplasmic fragments from various identified regions of unfertilized eggs. Cytoplasmic fragments, that contained cortical and subcortical material, from five different positions along the animal-vegetal axis were prepared, and they were fused with a4.2 (presumptive-epidermis) or A4.1 (non-epidermis) blastomeres. The ectopic development of endoderm, muscle and epidermis cells that was promoted by the transplanted cytoplasm was assessed by examining the expression of alkaline phosphatase (ALP), myosin and epidermis-specific antigen, respectively. Differentiation of endoderm and muscle was observed at higher frequencies as cytoplasmic fragments closer to the vegetal pole were transplanted. Conversely, formation of epidermis was observed at higher frequencies as cytoplasmic fragments closer to the animal pole were transplanted. The results suggest that, in cortical and subcortical regions of unfertilized ascidian eggs, endoderm and muscle determinants are widely distributed along a gradient, with maximum activity at the vegetal pole, whilst epidermis determinants are also distributed along a gradient but with maximum activity at the animal pole. Recieved: 10 June 1996 / Accepted: 12 September 1996     

Proteolytic dissection of band 3, the predominant transmembrane polypeptide of the human erythrocyte membrane.

Band 3 is the major, membrane-spanning, approximately90 000 dalton polypeptide of the human erythrocyte membrane. To facilitate the analysis of its structural integration into the membrane, we have cleaved this protein in situ into large fragments and ascertained their disposition. Digestion of intact cells with chymotrypsin yielded band 3 fragments with apparent molecular weights of 38 000 and 55 000. Both fragments resisted elution by NaOH and acetic acid, suggesting that they are anchored in the apolar core of the membrane. Both pieces communicate with the extracellular space, and the 55 000 dalton species extends to the cytoplasmic surface as well. Digestion of unsealed ghosts with chymotrypsin produced a hydrophobic 17 000 dalton species, a segment of the 55 000 dalton fragment, which spans and is firmly anchored in the core of the membrane. Trypsin and papain at low concentration generated integral band 3 fragments of 52 000 daltons and released major band 3 fragments of less than or equal to 41 000 daltons from the cytoplasmic side of the membrane. The latter water-soluble polypeptides remained associated in discrete complexes which retained the capacity to bind glyceraldehyde-3-phosphate dehydrogenase. An interchain disulfide bond, which can be induced only at the cytoplasmic surface, cross-linked intact band 3, and certain of its water-soluble fragments. Finally, fragments of 23 000 daltons were generated from the innersurface domain by reacting disulfide-linked band 3 dimers with cyanide or reduced polypeptides with 2-nitro-5-thiocyanobenzoate. A provisional ordering of these fragments is proposed.     

Solubilization of the Cytoplasmic Membrane of Escherichia coli by Triton X-100

Treatment of a partially purified preparation of cell walls of Escherichia coli with Triton X-100 at 23 C resulted in a solubilization of 15 to 25% of the protein. Examination of the Triton-insoluble material by electron microscopy indicated that the characteristic morphology of the cell wall was not affected by the Triton extraction. Contaminating fragments of the cytoplasmic membrane were removed by Triton X-100, including the fragments of the cytoplasmic membrane which were normally observed attached to the cell wall. Treatment of a partially purified cytoplasmic membrane fraction with Triton X-100 resulted in the solubilization of 60 to 80% of the protein of this fraction. Comparison of the Triton-soluble and Triton-insoluble proteins from the cell wall and cytoplasmic membrane fractions by polyacrylamide gel electrophoresis after removal of the Triton by gel filtration in acidified dimethyl formamide indicated that the detergent specifically solubilized proteins of the cytoplasmic membrane. The proteins solubilized from the cell wall fraction were qualitatively identical to those solubilized from the cytoplasmic membrane fraction, but were present in different proportions, suggesting that the fragments of cytoplasmic membrane which are attached to the cell wall are different in composition from the remainder of the cytoplasmic membrane of the cell. Treatment of unfractionated envelope preparations with Triton X-100 resulted in the solubilization of 40% of the protein, and only proteins of the cytoplasmic membrane were solubilized. Extraction with Triton thus provides a rapid and specific means of separating the proteins of the cell wall and cytoplasmic membrane of E. coli.     

An ultrastructural and cytochemical study of the digestive system in Oxyposthia praedator (Turbellaria,Acoela)

The central parenchyma in Oxyposthia praedator consists of multifunctional cells. These cells digest food material intracellularly, can effect extracellular digestion through release of digestive enzymes by cell lysis and clasmatosis, and synthesize reserve nutritional substances.     

The cytoplasmic tail of NSP4, the endoplasmic reticulum-localized non-structural glycoprotein of rotavirus, contains distinct virus binding and coiled coil domains.

The final steps in the assembly of rotavirus occur in the lumen of the endoplasmic reticulum (ER). Targeting of the immature inner capsid particle (ICP) to this compartment is mediated by the cytoplasmic tail of NSP4, a non-structural virus glycoprotein located in the ER membrane. To delineate structural and functional features of NSP4, soluble fragments of the cytoplasmic tail have been expressed and purified. Our analysis combines a functional assay for ICP binding with biochemical and CD spectroscopic studies to examine the secondary and quaternary structure. The ICP-binding domain is located within the C-terminal 20 amino acids of the polypeptide. A second region, distinct from this receptor domain, adopts an alpha-helical coiled coil structure and mediates the oligomerization of the virus binding domains into a homotetramer. The domain organization of the cytoplasmic fragments of NSP4 suggests a novel structure for an icosahedral virus receptor protein in which C-terminal binding sites for immature rotavirus particles are connected to an alpha-helical coiled coil stalk which projects from the ER membrane.     

Insertion of a signal peptide-derived hydrophobic segment into the mature domain of OmpC, an outer membrane protein, does not interfere with the export of the following polypeptide chain across the cytoplasmic membrane of E. coli

The effects of a hydrophobic peptide segment inserted into the amino-terminal region of the mature domain of OmpC, an outer membrane protein, on its translocation across the cytoplasmic membrane was studied. Both the intact OmpC and central domain-deleted OmpC were examined. The hydrophobic segment was derived from the signal peptide of OmpF. Secretory translocation across the cytoplasmic membrane was examined by means of proteinase K treatment. Four monoclonal antibodies that recognize different regions of OmpC were used to characterize proteinase K-resistant fragments. Insertion of the hydrophobic segment did not appreciably prevent the translocation of these proteins across the cytoplasmic membrane, larger parts of them being found as mature forms, which were mostly localized outside the cytoplasmic membrane. Circumstantial evidence supports the view, on the other hand, that the inserted hydrophobic domain was retained in the cytoplasmic membrane. It is concluded, therefore, that the hydrophobic segment, although it is not exported across the cytoplasmic membrane, does not prevent the secretion of the following polypeptide chain. The secretion was dependent on the amino-terminal signal peptide. Insertion of positive charges immediately after the hydrophobic segment resulted in suppression of the translocation. Based on these results possible mechanisms by which the secretion of the polypeptide chain after the hydrophobic segment are discussed.     

Cytoplasmic versus nuclear localization of Fos-related proteins in the frog,Rana esculenta,testis: in vivo and direct in vitro effect of a gonadotropin-releasing hormone agonist

Evidence has been accumulated indicating that GnRH-like peptides are present in a variety of extrabrain areas of mammalian and nonmammalian vertebrates. A pioneer study carried out in the frog, Rana esculenta, demonstrated that testicular GnRH induced spermatogonial proliferation. Recently, we have shown that in proliferating spermatogonia (SPG) of frogs, a change of localization of the oncoprotein Fos, from the cytoplasm to the nucleus, occurs. This leads to the hypothesis that one or more testicular GnRH peptides may regulate SPG proliferation through Fos family proteins. Therefore, in vivo experiments in intact R. esculenta and in vitro incubations of testis fragments have been carried out using GnRH agonist (GnRHa; buserelin) and GnRH antagonist (D-pGlu(1),D-Phe(2),D-Trp(3,6)-GnRH). Cytoplasmic and nuclear Fos-like protein localization has been found by Western blot analysis in testicular extracts. Immunocytochemistry confirmed that cytoplasmic immunostaining was restricted to SPG; change of localization into the nuclear compartment was observed after GnRHa treatment. Northern blot analysis showed that treatments of testis fragments with GnRHa did not modify testicular c-fos mRNA expression. On the contrary, a Fos-like protein of 52 kDa, while not affected in vivo, disappeared from testicular cytosolic extracts after in vitro treatment with GnRHa. Contemporaneously, a 55-kDa Fos-related signal appeared in nuclear extracts. The GnRH antagonist counteracted the effects of GnRHa. Furthermore, in vivo treatments showed that GnRHa acted negatively on a 43-kDa nuclear Fos-related signal and that gonadotropins caused the decrease of 52-kDa cytoplasmic signal. In conclusion, we show, to our knowledge for the first time, that Fos is regulated by GnRHa directly (not through the pituitary) at the testicular level. The main effect appears to be related to Fos translocation from cytoplasmic to nuclear compartments of SPG.     

利用RNA指纹技术分析大白菜胞质雄性不育相关基因的差异表达

采用改进的RNA指纹技术(RAP-PCR)对两套大白菜胞质雄性不育材料及其杂种F1花蕾的总RNA进行了分析, 通过对186条随机引物的筛选, 获得4个重复性较好的差异片段S47-412, S93-622, S176-343, S199-904, 并对其克隆、测序。根据各差异条带的测序结果合成长引物进行验证, S47, S93所对应的差异消失, 而S176, S199长引物验证的结果与RAP-PCR相似, 序列分析表明: S176-343, S199-904两差异片段均和油菜Polima不育胞质线粒体基因组orf224/atp6位点有着极高的同源性, 两差异片段序列部分重叠, 这说明两差异片段可能和大白菜胞质雄性不育有很高的相关性。     

Comparison of the Restriction Endonuclease Digestion Patterns of Mitochondrial DNA from Normal and Male Sterile Cytoplasms of ZEA MAYS L

High resolution gel electrophoresis has allowed the assignment of fragment number and molecular weight to EcoRI, SalI and PstI restriction fragments of mitochondrial DNA from B37 normal (N) and B37 T, C and S male sterile cytoplasmic types of maize. A minimum complexity of 450-475 kb has been established. Hybridization of cloned EcoRI fragments to restriction digests of total mitochondrial DNA suggests that at least 80% of the genome is composed of unique sequences. Restriction fragments of identical size in N, T, C and S contain similar sequence information as evidenced by their hybridization behavior.—The total SalI digest and the larger PstI fragments representing 80% of the total complexity were used to calculate the fraction of shared fragments of each pairwise combination of cytoplasmic types. The C type mtDNA is most closely allied with the other mtDNAs and shares 67% of fragments with S, 65% with N, and 60% with T. The S type mtDNA is quite divergent from N (53% shared fragments) and T (56% shared fragments). N and T share 59% of the fragments. These results are discussed in terms of the origin of mtDNA diversity in maize.     

Anatomy of herpes simplex virus DNA VII. alpha-RNA is homologous to noncontiguous sites in both the L and S components of viral DNA.

Previous reports from this laboratory (Honess and Roizman, 1974) have operationally defined alpha polypeptides as the viral proteins that are synthesized first in HEp-2 cells treated with cycloheximide from the time of infection with herpes simplex virus type 1 until the withdrawal of the drug 12 to 15 h after infection. It has also been shown that the viral RNA (designated alpha RNA) that accumulates in the cytoplasm during cycloheximide treatment and on polyribosomes immediately upon withdrawal of the drug is homologous to 10 to 12% of viral DNA, whereas the viral RNA accumulating in the cytoplasm of untreated cells at 8 to 14 h after infection is homologous to 43% of viral DNA (Kozak and Roizman, 1974). In the present study, alpha RNA and cytoplasmic RNA extracted from untreated cells 8 h after infection were each hybridized in liquid to in vitro labeled restriction endonuclease fragments generated by cleavage of herpes simplex virus type 1 DNA with Hsu I, with Bgl II, and with both enzymes simultaneously. The data show that only a subset of the fragments hybridized to alpha RNA, and these are scattered within both the L and S components of the DNA. There are at least five noncontiguous regions in the DNA homologous to alpha RNA; two of these are located partially within the reiterated sequences in the S component. All fragments tested hybridized more extensively with 8-h cytoplasmic RNA than with alpha RNA. Four adjacent fragments, corresponding to 30% of the DNA and mapping within the L component, hybridized exclusively with the cytoplasmic RNA extracted from cells 8 h after infection.     

Subunit Cell Wall of Sulfolobus acidocaldarius

The cell wall of Sulfolobus acidocaldarius has been isolated. Cells were mechanically disrupted with a French press, and the cytoplasmic membrane was removed by extracting cell-envelope fragments with Triton X-100. The Triton-insoluble cell wall material retained the characteristic subunit structure when examined in the electron microscope. Isolated cell wall fragments formed in open sheets that were easily separated from cytoplasmic contamination. Chemical studies showed that the Triton-insoluble cell wall fragments consisted of lipoprotein with small amounts of carbohydrate and hexosamine. The amino acid composition indicated a highly charged hydrophobic cell surface. The presence of diaminopimelic acid with only traces of muramic acid indicates that the cell envelope does not have a rigid peptidoglycan layer. The results of chemical analyses and electron microscopy suggest a wall-membrane interaction stabilizing the cell envelope. The chemical and physical properties of this type of cell envelope would appear to form the basis for a new major division of bacteria with the definitive characteristics of a morphologically distinct subunit cell wall devoid of peptidoglycan.     

Novel cytosolic binding partners of the neural cell adhesion molecule: mapping the binding domains of PLC gamma, LANP, TOAD-64, syndapin, PP1, and PP2A

The neural cell adhesion molecule (NCAM) is implicated in important functions during development and maintenance of the nervous system. Two of the three major isoforms, NCAM 140 and NCAM 180, are transmembrane glycoproteins with large cytoplasmic domains of different length. The purpose of this study was to identify novel intracellular binding partners of NCAM 140 and NCAM 180. We expressed both cytoplasmic domains, as well as cytoplasmic fragments of NCAM, as fusion proteins in Escherichia coli and used them for ligand affinity chromatography or glutathione S-transferase (GST) pull-down assays. By peptide mass fingerprinting Western blot analysis, or both, we identified PLCgamma, LANP, syndapin, PP1, and PP2A as binding partners for both NCAM 140 and NCAM 180, whereas TOAD-64 was identified as a NCAM 180-specific interacting protein. Furthermore, we were able to show that binding of these novel binding proteins, as well as the previously described interaction partners ROK alpha (rho A binding kinase alpha) and alpha- and beta-tubulin, bind to specific cytosolic sequences of NCAM. For this purpose, we performed GST pull-down experiments using cytosolic fragments of NCAM as GST-fusion proteins and cytosolic- or cytoskeleton-enriched protein fractions of rat brain.     

Properties of dispersed, highly repeated DNA within and near the hamster CAD gene.

Dispersed, highly repeated DNA sequences were found within and near the Syrian hamster gene coding for the multifunctional protein CAD. Most of the repeated sequences were homologous to each other and had similar properties. They hybridized to many cytoplasmic polyadenylated RNAs and to 7S and 4.5S cytoplasmic non-polyadenylated RNAs. Cloned DNA fragments containing repeated sequences were transcribed in vitro by RNA polymerase III. The repeated sequences from Syrian hamsters share many properties with the Alu family of repetitive DNA from humans. The hamster sequences were homologous to total repetitive human DNA but only very weakly homologous to two cloned members of the human Alu family.     

Expression of the gene encoding the adenovirus DNA terminal protein precursor in productively infected and transformed cells.

The major product of in vitro translation of early RNA prepared from H5ts125-infected cells and selected by hybridization to adenoviral DNA fragments spanning the region from 14.7 to 31.5 map units had been shown to be identical to the 87-kilodalton terminal protein precursor. A 72- to 75-kilodalton polypeptide whose rRNA can be selected by DNA from this same region and made in the presence of anisomycin was indistinguishable from the 72-kilodalton single-stranded DNA-binding protein encoded by the region from 60.1 to 66.6 map units. The accumulation of cytoplasmic RNA sequences complementary to these l-strand genes under various conditions of infection and in certain lines of transformed cells has been investigated by solution hybridization of cytoplasmic RNA to the separated strands of restriction endonuclease fragments of adenoviral DNA. During the early phase, RNA sequences complementary to the region from 11.6 to 36.7 map units were present at a concentration of 10 to 60 copies per cell, regardless of the nature of the block used to inhibit viral DNA synthesis. By 24 h after infection in the absence of any such block, sequences complementary to the regions from 11.6 to 18.2 map units (IVa2) and from 18.6 to 36.7 map units (E2B) accumulated to concentrations of 4,800 and 280 copies per cell, respectively. The ratio of cytoplasmic E2A RNA sequences to E2B RNA sequences remained close to 10:1 throughout the time period investigated. Of the transformed cell lines which retained E2B DNA sequences that were examined, only the T2C4 line expressed these sequences in cytoplasmic RNA. The implications of these observations for regulation of expression of the adenoviral early l-strand genes are discussed.