Relationships between mitochondria and longevity determinants in the ascomycetePodospora anserina

The study of heteroplasmons obtained by mixing together ground mycelia, which differ by their longevities on one band and by their mitochondrial alleles [cap₁^r/cap₁^s] on the other, revealed new types of lengevities. This result showed that a given mitochondrial type can be dissociated from a given type of longevity determinant. The presence of chloramphenicol-resistant mitochondria permitted the coexistence of several types of longevity determinants within a single cytoplasm. Senescent strains can be rejuvenated when [cap₁^r] mitochondria are present in the cytoplasm.     

Repair of a genetically-caused defect in oogenesis in Drosophila melanogaster by transplantation of cytoplasm from wild-type eggs and by injection of pyrimidine nucleosides

Eggs produced by homozygous mutant rudimentary (r⁹, 154.5) females of Drosophila melanogaster die during embryogenesis, apparently because the mutant female fails to incorporate certain substances into the egg during oogenesis. These eggs can be rescued by injecting them at the preblastoderm stage with wild-type nuclei and cytoplasm or wild-type cytoplasm alone from unfertilized eggs. Some preblastoderm eggs injected with 1.5% of egg volume of cytoplasm from unfertilized wild-type eggs were able to complete both embryonic and postembryonic development and emerged as adults, whereas not a single uninjected control egg was able to complete embryonic development. The eggs of rudimentary mothers can also be rescued by injecting each egg at the blastoderm stage with 0.01 μg of pyrimidine nucleosides. The results demonstrate that a pyrimidine deficiency is the cause of abortive embryogenesis, and confirm the previous finding that the rudimentary mutation leads to pyrimidine auxotrophy.     

Probing water compartments and membrane permeability in plant cells by 1H NMR relaxation measurements

¹H NMR relaxation times (T₁ and T₂) in parenchyma tissue of apple can identify three populations of water with different relaxation characteristics. By following the uptake of Mn²⁺ ions in the tissue it is shown that the observed relaxation times originate from particular water compartments: the vacuole, the cytoplasm, and the cell wall/extracellular space.

Proton exchange between these compartments is controlled by the plasmalemma and tonoplast membranes. During the Mn²⁺ penetration experiment, conditions occur that cause the relaxation times of protons of cytoplasmic water to be much shorter than their residence time in the cytoplasm. Then the tonoplast permeability coefficient P_d for water can be calculated from the vacuolar T₁ and T₂ values to be 2.44 10^-5 m·s^-1.

     

Differential accumulation and localization of maternal poly(A)-containing RNA during early development of the ascidian,Styela

The regional distribution of poly(A)⁺ RNA was examined in sections of Styela oocytes and fertilized eggs by in situ hybridization with [³H]poly(U). The nucleus and cytoplasm of previtellogenic oocytes contain equivalent densities of [³H]poly(U) binding sites. The concentration of these sites is reduced in the cytoplasm, but not the nucleus, during vitellogenesis. Consequently, the germinal vesicle (GV) plasm of mature oocytes is characterized by an eightfold elevation in [³H]poly(U) binding activity relative to the surrounding cytoplasm. The distinctive cytoplasmic regions of the mature oocyte do not exhibit differential concentrations of [³H]poly(U) binding sites. Following fertilization which triggers GV breakdown, meiosis, and ooplasmic segregation, the high density of [³H]poly(U) binding sites characteristic of the GV plasm is conserved in the basophilic cytoplasm during its extensive migration and eventual accumulation in the animal hemisphere of the egg. The insensitivity of the [³H]poly(U) binding sites of the basophilic cytoplasm to actinomycin D suggests that they are of maternal origin. It is concluded that maternal poly(A)⁺ RNA is subject to differential accumulation in the GV plasm and its derivative ooplasm during the early development of Styela.     

Properties of a cytostatic factor from Xenopus laevis eggs.

The cytoplasm of mature eggs of Xenopus laevis was found to contain a cytostatic factor (CSF) which induces cleavage arrest at metaphase when microinjected into one blastomere of a two-cell embryo of Xenopus laevis or Rana pipiens. The Rana CSF was found to be incapable of arresting mitosis in Xenopus embryos. Both Xenopus and Rana CSF were stabilized during the transfer procedure by Ca²⁺-chelation in the donor egg. The Xenopus CSF was not present in the germinal vesicle of immature oocytes, but arose in the cytoplasm at the time of germinal vesicle breakdown and subsequently disappeared at the time of fertilization or egg activation.     

Helminthosporium maydis Race T Toxin Induces Leakage of NAD from T Cytoplasm Corn Mitochondria

The mechanism by which Helminthosporium maydis race T toxin inhibits respiration dependent on NAD⁺-linked substrates in T cytoplasm corn mitochondria was investigated. The toxin did not cause leakage of the soluble matrix enzyme malate dehydrogenase from the mitochondria or inhibit malate dehydrogenase or isocitrate dehydrogenase directly. The toxin did increase the permeability of the inner membranes of T cytoplasm, but not N cytoplasm, mitochondria to NAD⁺. Added NAD⁺ partially or fully restored toxin-inhibited electron transport in T cytoplasm mitochondria. Thiamin pyrophosphate had a similar effect when malate was the substrate. It was concluded that the inhibition of respiration of NAD⁺-linked substrates by the toxin is due to depletion of the intramitochondrial pool of NAD⁺ and other coenzymes.     

Direct injection of functional single-domain antibodies from E. coli into human cells

Intracellular proteins have a great potential as targets for therapeutic antibodies (Abs) but the plasma membrane prevents access to these antigens. Ab fragments and IgGs are selected and engineered in E. coli and this microorganism may be also an ideal vector for their intracellular delivery. In this work we demonstrate that single-domain Ab (sdAbs) can be engineered to be injected into human cells by E. coli bacteria carrying molecular syringes assembled by a type III protein secretion system (T3SS). The injected sdAbs accumulate in the cytoplasm of HeLa cells at levels ca. 10⁵–10⁶ molecules per cell and their functionality is shown by the isolation of sdAb-antigen complexes. Injection of sdAbs does not require bacterial invasion or the transfer of genetic material. These results are proof-of-principle for the capacity of E. coli bacteria to directly deliver intracellular sdAbs (intrabodies) into human cells for analytical and therapeutic purposes.     

Synthesis and secretion of hydroxyproline containing macromolecules in carrots. I. Kinetic analysis

When disks of carrot (Daucus carota) phloem parenchyma are incubated for 6 days there is a 10-fold increase in cell wall hydroxyproline due to the synthesis and secretion of hydroxyproline-containing macromolecules. The synthesis of these molecules and their secretion are demonstrated by measuring the kinetics of incorporation and of chase of ¹⁴C-proline and hydroxyproline in different fractions of the cytoplasm and the cell wall. The hydroxyproline-containing molecules which are secreted are associated with the membranous organelles of the cytoplasm. They can be fractionated into trichloroacetic acid-soluble and trichloroacetic acid-precipitated fractions. The properties of the trichloroacetic acid-soluble fraction associated with the membranous organelles are consistent with its role as a cell wall precursor.     

An increase of calcium/manganese binding sites in cell ghosts associated with the acquisition of aggregative competence in Dictyostelium discoideum.

In Dictyostelium discoideum, the formation of multicellular aggregates represents the first morphogenetic event that leads ultimately to the construction of fruiting bodies. The altered adhesive properties of the cells can be demonstrated in ghosts derived from them which consist of largely intact membranes containing a few empty vesicles and exploded mitochondria but lacking nuclei, RNA, soluble cytoplasm and ATP [4]. A cofactor requirement for the aggregation of the ghosts can be satisfied by the following divalent cations: Ca²⁺, Mn²⁺, Zn²⁺ and Cu²⁺. In this paper it is shown that associated with the acquisition of aggregative competence is a 15–20-fold increase in the ghosts of sites capable of binding either Ca²⁺ or Mn²⁺ with relatively high affinity.     

Differential distribution of poly(A)-containing RNA in the embryonic cells of Oncopeltus fasciatus: Analysis by in situ hybridization with a [3H]poly(U) probe

The regional distribution of poly(A)⁺ RNA was examined in the embryonic cells of the milkweed bug, Oncopeltus fasciatus, by in situ hybridization of histological sections with a [³H]poly(U) probe. As shown by a number of control experiments, this probe interacts specifically with poly(A) sequences preserved in the sections. Using this method, it was shown that labeling of periplasmic and vitellophage nuclei increases markedly early during syncytial blastoderm formation. At this time, label also increases in the vitellophage cytoplasm but not in the cytoplasm surrounding the blastodermal nuclei. Labeling continues to increase in the blastodermal nuclei during cellularization and germ band differentiation without a concomitant accumulation in the blastodermal cell cytoplasm. At the time of germ band invagination, the region of the most intense subcellular labeing shifts from the nucleus to the cytoplasm of the invaginated cells. This shift is not evident in the blastodermal cells which remain at the surface of the egg to become the serosa. In the serosa and the vitellophage energids, labeling then decreases as histogenesis proceeds. Significant labeling of the nuclei and cytoplasm of the invaginated germ band cells continues through germ layer formation. It is concluded that poly(A)⁺ RNA, probably synthesized de novo following oviposition, is subject to differential intracellular distribution in three types of Oncopeltus embryonic cells which may reflect cell-specific patterns of mRNA or poly(A) metabolism.     

The role of calcium in depigmentation of iris epithelial cells during cell-type conversion

During the conversion of newt iris epithelial cells into lens cells, melanosomes disappear from the cytoplasm. In this “depigmentation,” exocytosis of melanosomes is involved. The role of Ca²⁺ in this process has been the subject of this work. The intracellular Ca²⁺ concentration of cultured iris epithelial cells was increased by three methods: microinjection of 10^?3, M CaCl₂ into the cytoplasm, fusion of phospholipid vesicles containing 10^?3, M CaCl₂ with the cell membrane, and exposure to the calcium ionophore A23187. Each of these treatments caused an increase in the release of melanosomes. Further experiments suggest that cAMP stimulates exocytosis probably by liberating Ca²⁺ from intracellular stores.     

Role of Met58 in the regulation of electron/proton transfer in trihaem cytochrome PpcA from Geobacter sulfurreducens

The bacterium Gs (Geobacter sulfurreducens) is capable of oxidizing a large variety of compounds relaying electrons out of the cytoplasm and across the membranes in a process designated as extracellular electron transfer. The trihaem cytochrome PpcA is highly abundant in Gs and is most probably the reservoir of electrons destined for the outer surface. In addition to its role in electron transfer pathways, we have previously shown that this protein could perform e⁻/H⁺ energy transduction. This mechanism is achieved by selecting the specific redox states that the protein can access during the redox cycle and might be related to the formation of proton electrochemical potential gradient across the periplasmic membrane. The regulatory role of haem III in the functional mechanism of PpcA was probed by replacing Met⁵⁸, a residue that controls the solvent accessibility of haem III, with serine, aspartic acid, asparagine or lysine. The data obtained from the mutants showed that the preferred e⁻/H⁺ transfer pathway observed for PpcA is strongly dependent on the reduction potential of haem III. It is striking to note that one residue can fine tune the redox states that can be accessed by the trihaem cytochrome enough to alter the functional pathways.     

Inhibition of (3H)vitellogenin uptake by isolated amphibian oocytes injected with cytoplasm from progesterone-treated mature oocytes.

Fully grown meiotically immature (germinal vesicle stage) amphibian oocytes incorporate radioactive protein ([³H]vitellogenin) following in vitro culture. In vitro exposure of such oocytes to exogenous progesterone induces germinal vesicle breakdown and inhibits incorporation of vitellogenin. In the present studies, we have investigated the effects of cytoplasm taken from mature and immature oocytes on incorporation of vitellogenin and nuclear breakdown following microinjection of this material into immature oocytes. Vitellogenin incorporation was markedly suppressed in oocytes which underwent nuclear breakdown following injection with cytoplasm from mature oocytes. Incorporation of vitellogenin into oocytes which did not mature after injection with cytoplasm taken from mature oocytes resembled that seen in oocytes injected with immature cytoplasm. The degree of suppression of vitellogenin incorporation following cytoplasmic injections was similar to that seen in uninjected oocytes treated with progesterone. Oocytes injected with cytoplasm obtained from immature oocytes did not undergo either nuclear breakdown or changes in vitellogenin incorporation. The results suggest that cytoplasm obtained from mature oocytes contains a factor(s) which alters directly or indirectly the capacity of the oocyte cell membrane to incorporate vitellogenin. Enucleated immature oocytes also incorporated [³H]vitellogenin, and injection of such oocytes with mature, but not immature, oocyte cytoplasm suppressed vitellogenin incorporation. Suppressive effects of injected cytoplasm thus appear to be mediated through physiological changes in the recipient oocyte cytoplasm rather than the nuclear component.     

Evidence for a constitutive cytoplasmic cutinase in ungerminated conidia of Botrytis cinerea Pers.: Fr.

Cytoplasmic soluble proteins from ungerminated conidia of Botrytis cinerea exhibited cutinase activity, while cell wall binding proteins lacked this activity. Cutinase activity in proteins extracted from cell walls and cytoplasm of ungerminated conidia of Botrytis cinerea was determined using p-nitrophenyl butyrate (PNB) and TLC analysis of products derived from hydrolysis of [³H]cutin. Treatment of conidia with indoxyl acetate, a substrate indicative of non-specific esterase and cutinase activity, also gave a positive reaction in the cytoplasm of ungerminated conidia. The possible role of a putative constitutive cutinase in the cytoplasm of conidia in the early stages of infection of plants by B. cinerea is discussed.     

Mislocalization of XPF-ERCC1 Nuclease Contributes to Reduced DNA Repair in XP-F Patients

Xeroderma pigmentosum (XP) is caused by defects in the nucleotide excision repair (NER) pathway. NER removes helix-distorting DNA lesions, such as UV–induced photodimers, from the genome. Patients suffering from XP exhibit exquisite sun sensitivity, high incidence of skin cancer, and in some cases neurodegeneration. The severity of XP varies tremendously depending upon which NER gene is mutated and how severely the mutation affects DNA repair capacity. XPF-ERCC1 is a structure-specific endonuclease essential for incising the damaged strand of DNA in NER. Missense mutations in XPF can result not only in XP, but also XPF-ERCC1 (XFE) progeroid syndrome, a disease of accelerated aging. In an attempt to determine how mutations in XPF can lead to such diverse symptoms, the effects of a progeria-causing mutation (XPF^R153P) were compared to an XP–causing mutation (XPF^R799W) in vitro and in vivo. Recombinant XPF harboring either mutation was purified in a complex with ERCC1 and tested for its ability to incise a stem-loop structure in vitro. Both mutant complexes nicked the substrate indicating that neither mutation obviates catalytic activity of the nuclease. Surprisingly, differential immunostaining and fractionation of cells from an XFE progeroid patient revealed that XPF-ERCC1 is abundant in the cytoplasm. This was confirmed by fluorescent detection of XPF^R153P-YFP expressed in Xpf mutant cells. In addition, microinjection of XPF^R153P-ERCC1 into the nucleus of XPF–deficient human cells restored nucleotide excision repair of UV–induced DNA damage. Intriguingly, in all XPF mutant cell lines examined, XPF-ERCC1 was detected in the cytoplasm of a fraction of cells. This demonstrates that at least part of the DNA repair defect and symptoms associated with mutations in XPF are due to mislocalization of XPF-ERCC1 into the cytoplasm of cells, likely due to protein misfolding. Analysis of these patient cells therefore reveals a novel mechanism to potentially regulate a cell''s capacity for DNA repair: by manipulating nuclear localization of XPF-ERCC1.     

Phospholipase C delta 4 (PLCδ4) is a nuclear protein involved in cell proliferation and senescence in mesenchymal stromal stem cells

Ca²⁺ is an important second messenger, and it is involved in many cellular processes such as cell death and proliferation. The rise in intracellular Ca²⁺ levels can be due to the generation of inositol 1,4,5-trisphosphate (InsP₃), which is a product of phosphatidylinositol 4,5-bisphosphate (PIP₂) hydrolysis by phospholipases C (PLCs), that leads to Ca²⁺ release from endoplasmic reticulum by InsP₃ receptors (InsP₃R). Ca²⁺ signaling patterns can vary in different regions of the cell and increases in nuclear Ca²⁺ levels have specific biological effects that differ from those of Ca²⁺ increase in the cytoplasm. There are PLCs in the cytoplasm and nucleus, but little is known about the functions of nuclear PLCs. This work aimed to characterize phenotypically the human PLCδ4 (hPLCδ4) in mesenchymal stem cells. This nuclear isoform of PLC is present in different cell types and has a possible role in proliferative processes. In this work, hPLCδ4 was found to be mainly nuclear in human adipose-derived mesenchymal stem cells (hASC). PLCδ4 knockdown demonstrated that it is essential for hASC proliferation, without inducing cell death. An increase of cells in G1, and a reduction of cells on interphase and G2/M in knockdown cells were seen. Furthermore, PLCδ4 knockdown increased the percentage of senescent cells, p16^INK4A+ and p21^Cip1 mRNAs expression, which could explain the impaired cell proliferation. The results show that hPLCδ4 is in involved in cellular proliferation and senescence in hASC.     

Na+-ATPases of halotolerant microalgae

Na⁺ homeostasis in the cytoplasm is a common property of all organisms, irrespective of their taxonomic position. Low Na⁺ concentrations in the cytoplasm of living cells are maintained by specialized Na⁺-transporting molecular machines operating in the cell membranes. In eukaryotic cells, Na⁺-transporting ATPases of P-type play the important role in keeping the Na⁺ homeostasis. This review summarizes the authors’ investigations demonstrating the operation of the Na⁺-transporting P-type ATPases in the plasma membrane of green marine microalgae. Experiments described here provided the first evidence for the existence of the primary Na⁺-pump in plasma membranes of organisms attributed to the plant kingdom. The significance of the Na⁺-ATPases in halotolerant microalgae Dunaliella maritima and Tetraselmis viridis inhabiting saline environments is discussed.     

Extracellular Allosteric Na Binding to the Na,K-ATPase in Cardiac Myocytes

Whole-cell patch-clamp measurements of the current, I_p, produced by the Na⁺,K⁺-ATPase across the plasma membrane of rabbit cardiac myocytes show an increase in I_p over the extracellular Na⁺ concentration range 0–50 mM. This is not predicted by the classical Albers-Post scheme of the Na⁺,K⁺-ATPase mechanism, where extracellular Na⁺ should act as a competitive inhibitor of extracellular K⁺ binding, which is necessary for the stimulation of enzyme dephosphorylation and the pumping of K⁺ ions into the cytoplasm. The increase in I_p is consistent with Na⁺ binding to an extracellular allosteric site, independent of the ion transport sites, and an increase in turnover via an acceleration of the rate-determining release of K⁺ to the cytoplasm, E2(K⁺)₂ → E1 + 2K⁺. At normal physiological concentrations of extracellular Na⁺ of 140 mM, it is to be expected that binding of Na⁺ to the allosteric site would be nearly saturated. Its purpose would seem to be simply to optimize the enzyme’s ion pumping rate under its normal physiological conditions. Based on published crystal structures, a possible location of the allosteric site is within a cleft between the α- and β-subunits of the enzyme.     

Host Erythrocyte Environment Influences the Localization of Exported Protein 2, an Essential Component of the Plasmodium Translocon

Malaria parasites replicating inside red blood cells (RBCs) export a large subset of proteins into the erythrocyte cytoplasm to facilitate parasite growth and survival. PTEX, the parasite-encoded translocon, mediates protein transport across the parasitophorous vacuolar membrane (PVM) in Plasmodium falciparum-infected erythrocytes. Proteins exported into the erythrocyte cytoplasm have been localized to membranous structures, such as Maurer''s clefts, small vesicles, and a tubovesicular network. Comparable studies of protein trafficking in Plasmodium vivax-infected reticulocytes are limited. With Plasmodium yoelii-infected reticulocytes, we identified exported protein 2 (Exp2) in a proteomic screen of proteins putatively transported across the PVM. Immunofluorescence studies showed that P. yoelii Exp2 (PyExp2) was primarily localized to the PVM. Unexpectedly, PyExp2 was also associated with distinct, membrane-bound vesicles in the reticulocyte cytoplasm. This is in contrast to P. falciparum in mature RBCs, where P. falciparum Exp2 (PfExp2) is exclusively localized to the PVM. Two P. yoelii-exported proteins, PY04481 (encoded by a pyst-a gene) and PY06203 (PypAg-1), partially colocalized with these PyExp2-positive vesicles. Further analysis revealed that with P. yoelii, Plasmodium berghei, and P. falciparum, cytoplasmic Exp2-positive vesicles were primarily observed in CD71⁺ reticulocytes versus mature RBCs. In transgenic P. yoelii 17X parasites, the association of hemagglutinin-tagged PyExp2 with the PVM and cytoplasmic vesicles was retained, but the pyexp2 gene was refractory to deletion. These data suggest that the localization of Exp2 in mouse and human RBCs can be influenced by the host cell environment. Exp2 may function at multiple points in the pathway by which parasites traffic proteins into and through the reticulocyte cytoplasm.