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A new search for restorer cytoplasm: the restorer cytoplasm for the gene ms10 most probably does not exist in maize.

Since the suggestion concerning the hypothesis of the existence of restorer cytoplasms for some of the known and currently available male sterile (ms) genes in maize, a relatively limited amount of research effort has been made in order to test the hypothesis. Considering the importance of such a phenomenon, we designed a large, two-part experiment to test the idea more seriously. In the first part, 50 randomly chosen, medium-late open-pollinated (OP) varieties of maize from the Zemun Polje (ZP) collection were tested for the presence of the restorer cytoplasm in some currently known ms-genes in maize (ms1, ms2, ms3, ms4, ms5, ms6, ms7, ms8, ms9, ms11, ms12, ms13, ms17, ms22, ms23, and ms24). In the second part, the whole ZP collection of maize germplasm (more than 4,000 entries) was tested for the presence of the restorer cytoplasm for the gene ms10. After the first basic screening of OP varieties, more than 70 nonsegregating "candidates" were identified; however, after additional screening of the collection and the direct testing with respective homozygous ms-testers, all of them showed segregation, indicating that the restorer cytoplasm does not exist, especially the gene ms10. While performing this experiment, we discovered almost a hundred sources of male sterile cytoplasm, which were distinguished by their overwhelming frequency of male sterile plants in segregating test progenies.

Fertility↗

Small cytoplasmic RNAs and their location within the cytoplasm.

These studies were designed to establish the location of various species of small RNAs within the subcellular cytoplasmic compartments. Four cytoplasmic RNA-containing compartments were examined: (A) cytoskeleton-bound polyribosomal ribonucleoprotein (RNP) complexes, (B) soluble-phase polyribosomal RNP complexes, (C) cytoskeleton-bound free RNP complexes, (D) soluble-phase free RNP complexes. The presence of the small cytoplasmic RNA (scRNA) population and histone H4 and actin mRNAs in each compartment was examined to determine their spatial distribution within the cytoplasm. The 7S signal recognition RNA and the 5S and 5.8S rRNAs were distributed among all four compartments, while 4S tRNAs were localized largely in fraction D. Fraction C contained a group of seven abundant scRNAs, of approximately 105-348 nucleotides in length, which were localized almost entirely within the cytoskeleton-bound free RNP compartment. Actin mRNAs were localized in fraction A, the actively translating cytoskeleton-bound compartment. Actin mRNAs were localized in fraction A, the actively translating cytoskeleton-bound compartment. Following cytochalasin B treatment, actin mRNAs were released into the soluble phase, implicating a dependence on the integrity of actin filaments in its binding. Such treatment also released several of the scRNAs from their cytoskeleton-bound location. In contrast histone H4 mRNAs were much more widely dispersed, being present in all four cytoskeletal compartments. Approximately 60% of the H4 mRNAs, however, were localized within the soluble-phase polyribosomes in fraction B. Cytochalasin B treatment released only the small portion of untranslated histone H4 mRNA associated with the cytoskeleton in fraction C, suggesting that the binding of these H4 mRNAs was dependent in some manner upon the integrity of actin filaments.

Cytoplasm↗

Mitochondrial import of a cytoplasmic lysine-tRNA in yeast is mediated by cooperation of cytoplasmic and mitochondrial lysyl-tRNA synthetases.

Cytoplasmic tRNA(Lys)CUU is the only nuclear-encoded tRNA of Saccharomyces cerevisiae found to be associated with mitochondria. Selective import of this tRNA into isolated organelles requires cytoplasmic factors. Here we identify two of these factors as the cytoplasmic and mitochondrial lysyl-tRNA synthetases. The cytoplasmic enzyme is obligatory for in vitro import of the deacylated, but not of the aminoacylated tRNA. We thus infer that it is needed for aminoacylation of the tRNA, which is a prerequisite for its import. The mitochondrial synthetase, which cannot aminoacylate tRN(Lys)CUU, is required for import of both aminoacylated and deacylated forms. Its depletion leads to a total arrest of tRNA import, in vitro and in vivo. The mitochondrial lysyl-tRNA synthetase is able to form specific and stable RNP complexes with the amino-acylated tRNA. Furthermore, an N-terminal truncated form of the synthetase which cannot be targeted into mitochondria is unable to direct the import of the tRNA. We therefore hypothesize that the cytosolic precursor form of the mitochondrial synthetase has a carrier function for translocation of the tRNA across the mitochondrial membranes. However, cooperation of the two synthetases is not sufficient to direct tRNA import, suggesting the need of additional factor(s).

Acylation↗

Cytoplasmic retention of HIV-1 regulatory protein Vpr by protein-protein interaction with a novel human cytoplasmic protein VprBP.

Vpr is an HIV-1 auxiliary regulatory protein packaged in the virion. It has been shown to enhance the nuclear transport of the HIV-1 pre-integration complex, activate transcription of cellular and viral promoters, and arrest the cell cycle at the G2/M check-point. We previously identified a cellular protein of 180 kDa (RIP) that interacted with HIV-1 Vpr specifically. We now rename this cellular protein as Vpr-binding protein, or VprBP. In this report, we describe the cloning of the VprBP cDNA that encodes 1507 aa residues and is identical to the previously cloned cDNA KIAA0800. We demonstrate that Vpr specifically interacts with recombinantly expressed VprBP in vitro as well as in vivo. Furthermore, Vpr interacts with the cellular endogenous VprBP in the context of the HIV-1 life cycle. Mutational analysis of VprBP suggests that the Vpr binding domain is located within the C-terminal half of VprBP, which has a Pro-rich domain and several Phe-x-x-Phe repeats. Subcellular fractionation studies show that both the endogenous VprBP and the adenovirus-expressed VprBP are distributed predominantly in the cytoplasmic fraction. Consistent with previous reports, the adenovirus-expressed Vpr is distributed in both the cytoplasmic and the nuclear fractions. However, when VprBP and Vpr are expressed together, Vpr is found almost exclusively in the cytoplasm. Expression of VprBP does not affect the nuclear transport of the adenoviral nuclear protein, pTP. VprBP expressed in insect cells also blocks the nuclear transport of a Vpr-GFP fusion protein, and VprBP mutants incapable of interacting with Vpr fail to block Vpr-GFP nuclear transport. We hypothesize that Vpr interaction with VprBP may cause changes in the host cell cytoplasm that affect HIV-1 pathogenesis as well as HIV-1 replication.

Amino Acid Sequence↗

Probing the dark state tertiary structure in the cytoplasmic domain of rhodopsin: proximities between amino acids deduced from spontaneous disulfide bond formation between Cys316 and engineered cysteines in cytoplasmic loop 1.

A dark state tertiary structure in the cytoplasmic domain of rhodopsin is presumed to be the key to the restriction of binding of transducin and rhodopsin kinase to rhodopsin. Upon light-activation, this tertiary structure undergoes a conformational change to form a new structure, which is recognized by the above proteins and signal transduction is initiated. In this and the following paper in this issue [Cai, K., Klein-Seetharaman, J., Altenbach, C., Hubbell, W. L., and Khorana, H. G. (2001) Biochemistry 40, 12479-12485], we probe the dark state cytoplasmic domain structure in rhodopsin by investigating proximity between amino acids in different regions of the cytoplasmic face. The approach uses engineered pairs of cysteines at predetermined positions, which are tested for spontaneous formation of disulfide bonds between them, indicative of proximity between the original amino acids. Focusing here on proximity between the native cysteine at position 316 and engineered cysteines at amino acid positions 55-75 in the cytoplasmic sequence connecting helices I-II, disulfide bond formation was studied under strictly defined conditions and plotted as a function of the position of the variable cysteines. An absolute maximum was observed for position 65 with two additional relative maxima for cysteines at positions 61 and 68. The observed disulfide bond formation rates correlate well with proximity of these residues found in the crystal structure of rhodopsin in the dark. Modeling of the engineered cysteines in the crystal structure indicates that small but significant motions are required for productive disulfide bond formation. During these motions, secondary structure elements are retained as indicated by the lack of disulfide bond formation in cysteines that do not face toward Cys316 in the crystal structure model. Such motions may be important in light-induced conformational changes.

Amino Acid Sequence↗

Isoform-specific 3'-untranslated sequences sort alpha-cardiac and beta-cytoplasmic actin messenger RNAs to different cytoplasmic compartments.

We demonstrate that in differentiating myoblasts, the mRNAs encoding two actin isoforms, beta-cytoplasmic, and alpha-cardiac, can occupy different cytoplasmic compartments within the same cytoplasm. beta-actin mRNA is localized to the leading lamellae and alpha-actin mRNA is associated with a perinuclear compartment. This was revealed by co-hybridizing, in situ, fluorochrome-conjugated oligonucleotide probes specific for each isoform. To address the mechanism of isoform-specific mRNA localization, molecular chimeras were constructed by insertion of actin sequences between the Lac Z coding region and SV-40 3'UTR in a reporter plasmid. These constructs were transiently expressed in a mixed culture of embryonic fibroblasts, myoblasts and myotubes, beta-galactosidase activity within transfectants was revealed by a brief incubation with its substrate (X-gal). Since the blue-insoluble reaction product co-localized with the specific mRNAs expressed from each construct, it was used as a bioassay for mRNA localization. Transfectants were scored as either perinuclear, peripheral or nonlocalized with respect to the distribution of the blue product. The percentage of transfectants within those categories was quantitated as a function of the various constructs. This analysis revealed that for each actin mRNA its 3'UTR is necessary and sufficient to direct reporter transcripts to its appropriate compartment; beta-actin peripheral and alpha-actin perinuclear. In contrast, sequences from the 5'UTR through the coding region of either actin gene did not localize the blue product. Therefore, 3'UTR sequences play a key role in modulating the distribution of actin mRNAs in muscle cells. We propose that the mechanism of mRNA localization facilitates actin isoform sorting in the cytoplasm.

Actins↗

Nuclear-cytoplasmic relationships in human cells in tissue culture. III. Auto-radiographic study of interrelation of nuclear and cytoplasmic ribonucleic acid.

The movement of ribonucleic acid (RNA) from nucleus to cytoplasm has been studied, by autoradiographic techniques, in cells of the human amnion grown in tissue culture. Cells were exposed to cytidine-H(3) for 1 hour after which time only the RNA of the nuclei was labelled. After this 1 hour exposure the cells were placed in a medium containing an excess amount of unlabelled cytidine. Periodically, cells from this medium were fixed. Autoradiographs showed that there was a progressive movement of the label from nucleus to cytoplasm, such that after 24 hours essentially all the label was in the RNA of the cytoplasm. A study of the incorporation of the cytidine-H(3) in deoxyribonucleic acid (DNA), in the same population of cells at the same times, indicated that the presence of excess amounts of unlabelled cytidine almost instantaneously inhibited further utilization of cytidine-H(3). It is concluded that RNA moves from nucleus to cytoplasm as a complex polynucleotide structure.

Cell Nucleus↗

Nucleo-cytoplasmic interaction during macronuclear differentiation in ciliate protists: genetic basis for cytoplasmic control of SerH expression during macronuclear development in Tetrahymena thermophila.

A novel class of mutations affecting the developmental expression of SerH cell surface antigen genes of Tetrahymena thermophila is described. Unlike previous categories of mutation, the four independently isolated mutations of this class act through the cytoplasm to affect SerH genes during macronuclear development. That is, macronuclei which develop under the influence of mutant cytoplasm do not subsequently express H, most likely because the developmental processing of SerH genes is affected. The cytoplasmic effect is specific for the SerH locus and is independent of which SerH allele is present. In place of H, hitherto unknown antigens are expressed. Expression of SerH can be rescued during development either by wild-type cytoplasm exchanged between conjugants or by the homozygous wild-type genotype. The mutations segregate independently of the SerH genes and identify one, possibly two, bistable genes. Possible models to explain these results are discussed.

Alleles↗

Mitogenic effect of cytoplasmic membranes and a cytoplasmic fraction of Staphylococcus aureus L-forms on human peripheral blood lymphocytes.

The cytoplasmic membranes and a cytoplasmic fraction of Staphylococcus aureus L-forms increased the incorporation of [3H]thymidine by human lymphocytes in the presence of fetal bovine serum. Both fractions stimulated cord blood lymphocytes as well as adult peripheral lymphocytes, suggesting the possibility that the observed effect was not due to an antigen-specific reaction, but to an immunologically nonspecific action. The membrane mitogen(s) was resistant to trypsin, although it was partially solubilized by trypsin treatment. The mitogen(s) could not be extracted with a chloroform-methanol mixture (2:1, v/v), although the chloroform-methanol soluble fraction was strongly mitogenic to murine splenocytes. Human serum which was added to the assay system in place of fetal bovine serum definitely suppressed the mitogenic effect of both cytoplasmic membranes and the cytoplasmic fraction, especially the latter.

Cytoplasm↗

Analysis of cytoplasmic and surface antigens in childhood T-cell acute lymphoblastic leukaemias: clinical relevance of cytoplasmic TCR beta chain expression.

Expression of surface and cytoplasmic antigens on the blasts from 42 cases of childhood T-cell acute lymphoblastic leukaemia (T-ALL) were analysed. All with childhood T-ALL, except for one case expressing cytoplasmic TCR delta chain, were classified on the basis of differential expression of cytoplasmic CD3 (cCD3), TCR beta chain (cTCR beta) and surface CD3 (sCD3) into the following three groups: group I (cCD3+, cTCR beta-, sCD3-), eight cases (19.5%); group II (cCD3+, cTCR beta +, sCD3-), 23 cases (56.1%); group III (cCD3+, cTCR beta +/-, sCD3+), 10 cases (24.4%). Each group defines the stepwise maturational stage of the CD3/TCR complex along the intrathymic T-cell differentiation. Group I had the lowest initial WBC count among the three groups (P < 0.05) and showed significantly (P < 0.05) a higher event-free survival (0.75) than those of group II (0.33). There was no significant difference in both the initial WBC count and the event-free survival between groups II and III. Thus, the absence of cTCR beta in sCD3-negative T-ALL appears to be a good prognostic factor, suggesting that this classification provides a useful tool to predict the prognosis of childhood T-ALL. This is the first report, to our knowledge, studying the relationship between the expression of cytoplasmic CD3/TCR antigens and the clinical features in T-ALL.

Adolescent↗

Atypical, cytoplasmic and perinuclear anti-neutrophil cytoplasmic antibodies in patients with inflammatory bowel disease.

Atypical, cytoplasmic and perinuclear anti-neutrophil cytoplasmic antibodies (x-, c- and pANCA, respectively) are associated with a variety of inflammatory diseases, including inflammatory bowel disease (IBD). Anti-neutrophil cytoplasmic antibodies are more common in patients with ulcerative colitis (UC) than in patients with Crohn's disease (CD). Most publications only refer to p- and cANCA in relation to IBD. We have prospectively evaluated the reactivity of sera from 58 patients with IBD and 10 healthy controls against human neutrophils with emphasis on the distinction of the ANCA types. The sera were incubated with ethanol- and formaldehyde-fixed granulocytes to differentiate between c-, p- and xANCA. The results showed that 10 of 24 patients with UC were positive for ANCA, whereas only one of 34 patients with CD was ANCA positive. These results correspond to a sensitivity of 42%, a specificity of 97%, a negative predictive value of 91% and a positive predictive value of 75% in UC. Of the 11 ANCA-positive sera, two showed a cytoplasmic staining pattern, three showed a perinuclear and six an atypical staining pattern. The disease activity was not correlated to either the ANCA titre or to the presence of ANCA in the serum. In conclusion, ANCA are of limited value in differentiating between UC and CD. Because the majority of ANCA in patients with IBD are xANCA, these ANCA should be explored by not only incubating on ethanol-fixed granulocytes, but also on formaldehyde-fixed granulocytes.

Adolescent↗

Serum antineutrophil cytoplasmic autoantibodies in inflammatory bowel disease are mainly associated with ulcerative colitis. A correlation study between perinuclear antineutrophil cytoplasmic autoantibodies and clinical parameters, medical, and surgical treatment.

Perinuclear antineutrophil cytoplasmic antibodies have recently been demonstrated in the sera of patients with inflammatory bowel disease. Three hundred and sixty six sera obtained from 120 patients with ulcerative colitis, 105 patients suffering from Crohn's disease and 49 non-inflammatory bowel disease controls were tested in two laboratories, using an indirect immunofluorescence assay. In addition, a fixed-neutrophil enzyme linked immunoadsorbent assay (ELISA) was evaluated in one of the two laboratories. The results in the immunofluorescence test showed a high degree of correlation between the two laboratories (Kappa coefficient = 0.8). Ninety five of the 120 (79%) ulcerative colitis patients had a positive test whereas only 14 of the 105 (13%) patients with Crohn's disease were positive. Sera from four patients suffering from primary sclerosing cholangitis were positive as well as four of the 45 control sera (9%). The sensitivity of the perinuclear antineutrophil cytoplasmic antibody immunofluorescence test for the diagnosis of ulcerative colitis was 0.75 with a specificity of 0.88 and a positive predictive value of 0.88 (all sera). In the ELISA technique 37 of 94 ulcerative colitis sera and one of the 68 Crohn's disease sera were positive. In the control group only one of the patients suffering from primary sclerosing cholangitis reacted positively (32 non-inflammatory bowel disease sera tested). The ELISA technique had a high specificity (0.97), but a low sensitivity (0.39). There was no relation of perinuclear antineutrophil cytoplasmic antibodies in ulcerative colitis patients or in Crohn's disease patients with disease activity, duration of illness, localisation, extent of disease, previous bowel operations or medical treatment. The clinical significance of perinuclear antineutrophil cytoplasmic antibody positive and negative subsets in both groups of patients thus remains unexplained. Our study confirms that determination of serum antineutrophil cytoplasmatic antibodies in patients with inflammatory bowel disease may differentiate ulcerative colitis from Crohn's disease. Further immunological studies are needed to explain the absence of these antibodies in a subset of ulcerative colitis patients and their role in the pathogenesis of the disease.

Adult↗

Cytoplasmic induction of changes in the ultrastructure of the Acetabularia nucleus and perinuclear cytoplasm.

The ultrasturcture of the cell nucleus and perinuclear cytoplasm in Acetabularia is quite different in young and old cells. When a nucleus of an old cell was implanted into the cytoplasm of a young cell the nucleus assumed the morphology typical of a young cell within less than 10 days. The cytoplasm of an old cell was able to induce the reverse change in an implanted nucleus from a young cell. This cytoplasmic induction of nuclear ageing appeared to proceed more rapidly than rejuvenation.

Acetabularia↗

[Effects of pH and ATP on the cytoplasm of Amoeba proteus: relation between cytoplasm motility and actin polymerization].

The effect of pH and ATP was studied on isolated cytoplasm of Amoeba proteus. These two parameters were shown to influence both the motility and the organization of actin filaments in the isolated cytoplasm. Furthermore, our results demonstrate that there is a relationship between the motility and the polymeric state of actin. When the isolated cytoplasm is non-motile, actin is highly polymerized into long filaments arranged parallel in bundles. When this cytoplasm is motile, however, actin can either be weakly polymerized, i.e. observed as few short filaments, or can be polymerized in long branched filaments forming a loose network.

Actins↗

[Ultrastructural-morphometric studies on the rat liver in the postnatal period. 1. Changes in the extrahepatocyte volume, the hepatocyte nuclei the cytoplasm and and the nucleus-cytoplasm ratio].

Quantitative changes in the volume densities of extrahepatocyte spaces, nuclei of hepatocytes, cytoplasm and of the nucleus-cytoplasm-ratio were studied in male Wistar-rats aged 0 (birth), 1, 2, 3, 4, 5, 6, 7, 14, 21, 28 days and 2, 3, 4, 5 and 6 months. The ratio of hepatocyte/nonhepatocyte fractions is 40.3 : 59.7 at birth and it increases to 86.6 : 13.4 by the 6th month. We observed 2 phases of proliferations of hepatocytes in the first 4 days, while the single volume of hepatocytes reaches lower levels. After the 4th day the single volume of hepatocytes increases because of the augmentation of volume density of cytoplasm. According to these changes the nucleus-cytoplasm-ratio will be reduced to 0.077 by the 5th month.

Animals↗

[Alveolar hemorrhage, skin eruption, and gastrointestinal bleeding accompanied by crescentric glomerulonephritis in a patient with cytoplasmic anti-neutrophil cytoplasmic antibody].

A 69-year-old woman was admitted to our hospital because of hemoptysis, skin eruption, and gastrointestinal bleeding. A chest X-ray film revealed infiltrates in the left upper lung field. Analysis of bronchoalveolar lavage fluid revealed alveolar hemorrhage. Examination of skin biopsy specimens demonstrated leukocytoclastic vasculitis. Fiberoptic examination of the colon showed diffuse reddish and edematous mucosa. Histological examination of biopsy specimens from the sigmoid colon showed necrotizing vasculitis. The serum creatinine level was high: 2.2 mg/dl. Examination of transcutaneous renal biopsy specimens revealed crescentric glomerulonephritis. A test for perinuclear anti-neutrophil cytoplasmic antibody was negative, but a test for cytoplasmic anti-neutrophil cytoplasmic antibody was positive. The latter strongly suggests a diagnosis of Wegener's granulomatosis, although the association is not absolute. We diagnosed pulmonary-renal syndrome associated with anti-neutrophil cytoplasmic antibody. The findings in this case were compatible with microscopic polyangiitis.

Aged↗

Relative Contribution of the Cell Wall, Cytoplasmic Membrane, and Cytoplasm to the Gram-Positive Characteristic of Bacillus megaterium.

Bartholomew, J. W. (University of Southern California, Los Angeles), and Thomas Cromwell. Relative contribution of the cell wall, cytoplasmic membrane, and cytoplasm to the gram-positive characteristic of Bacillus megaterium. J. Bacteriol. 90:643-647. 1965.-A comparison of the roles of the cell wall, cytoplasmic membrane, and cytoplasmic components revealed that the intact cell wall was the dominant contributor to the gram-positive state. Protoplasts of Bacillus megaterium were confirmed as being gram-negative, as reported by Gerhardt et al. The "gram-positive protoplast" report of Amano et al. was shown to be a laboratory-produced artifact, resulting from the comparison of smears made from saline suspensions of Escherichia coli cells with smears made from formalin-sucrose suspensions of B. megaterium protoplasts.

Journal Article↗

The Chédiak-Higashi syndrome; the nature of the giant neutrophil granules and their interactions with cytoplasm and foreign particulates. I. Progressive enlargement of the massive inclusions in mature neutrophils. II. Manifestations of cytoplasmic injury and sequestration. III. Interactions between giant organelles and foreign particulates.

Defective bactericidal functioning of polymorphonuclear leukocytes (PMNs) from patients with the Chédiak-Higashi syndrome (CHS) has been related in previous reports to a failure of the giant granules characteristics of the disorder to participate in degranulation after uptake of foreign particulates by neutrophils. However, the reason massive CHS inclusions do not fuse with and discharge their contents into phagocytic vacuoles has not been defined. The problem is particularly puzzling because it has been postulated that the hugh organelles in CHS neutophils originate by fusion of small azurophilic granules in promyelocytes and myelocytes. The present series of investigations into the cytopathology of the CHS has employed electron microscopy and ultrastructural cytochemistry to characterize the progressive enlargement of the hugh bodies in mature PMNs, their interaction with cytoplasmic constituents resulting in various manifestations of cell injury, and their response to foreign particulates. Each study clarifies abnormal features of the giant organelles essential to the understanding of their role in the defective bactericidal function of CHS neutrophils. The first report demonstrates that most of the hugh inclusions in PMNs are not primary lysosomes. The interaction and fusion of giant azurophilic granules with each other, with normal-sized primary and secondary granules, and with cytoplasmic components converts the massive primary granules into huge secondary lysosomes. Transformation to secondary hysosomes represents a critical alteration in the state of the giant granules that underlies their damaging influence on the cytoplasm and loss of reactivity wtih phagocytic vacuoles.

Adolescent↗