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PubMed1975

Protein migration into nuclei. II. Frog oocyte nuclei accumulate a class of microinjected oocyte nuclear proteins and exclude a class of microinjected oocyte cytoplasmic proteins.

Nuclear contents or cytoplasm from Xenopus oocytes labeled with (35-S)methionine or (3-H)proline (donor oocytes) were reinjected into unlabeled oocytes (recipient oocytes). The radioactivity injected as nuclear contents was found to enter and accumulate in the recipient oocyte nucleus. In contrast, the radioactivity injected as cytoplasm was found to enter but not to accumulate in the recipient oocyte nucleus. Sodium dodecyl sulfate (SDS) gel electrophoresis of the nucleus and cytoplasm of donor oocytes revealed the existence of three classes of labeled proteins in these oocytes: those proteins found predominantly in the nucleus (N proteins), those found predominantly in the cytoplasm (C proteins), and those found in both the nucleus and cytoplasm at similar concentrations (B proteins). SDS gel electrophoresis of the nucleus and cytoplasm of recipient oocytes showed that N proteins entered and accumulated in the nucleus but that B proteins partitioned about equally between the nucleus and cytoplasm. A similar analysis of oocytes injected with labeled cytoplasm showed that C proteins did not enter the nucleus but again B proteins partitioned about equally between the nucleus and cytoplasm.

Animals↗
PubMed1998

Disulfide bond formation in the Escherichia coli cytoplasm: an in vivo role reversal for the thioredoxins.

Cytoplasmic proteins do not generally contain structural disulfide bonds, although certain cytoplasmic enzymes form such bonds as part of their catalytic cycles. The disulfide bonds in these latter enzymes are reduced in Escherichia coli by two systems; the thioredoxin pathway and the glutathione/glutaredoxin pathway. However, structural disulfide bonds can form in proteins in the cytoplasm when the gene (trxB) for the enzyme thioredoxin reductase is inactivated by mutation. This disulfide bond formation can be detected by assessing the state of the normally periplasmic enzyme alkaline phosphatase (AP) when it is localized to the cytoplasm. Here we show that the formation of disulfide bonds in cytoplasmic AP in the trxB mutant is dependent on the presence of two thioredoxins in the cell, thioredoxins 1 and 2, the products of the genes trxA and trxC, respectively. Our evidence supports a model in which the oxidized forms of these thioredoxins directly catalyze disulfide bond formation in cytoplasmic AP, a reversal of their normal role. In addition, we show that the recently discovered thioredoxin 2 can perform many of the roles of thioredoxin 1 in vivo, and thus is able to reduce certain essential cytoplasmic enzymes. Our results suggest that the three most effective cytoplasmic disulfide-reducing proteins are thioredoxin 1, thioredoxin 2 and glutaredoxin 1; expression of any one of these is sufficient to support aerobic growth. Our results help to explain how the reducing environment in the cytoplasm is maintained so that disulfide bonds do not normally occur.

Alkaline Phosphatase↗
PubMed1995

Genetics of male sterility in gynodioecious Plantago coronopus. I. Cytoplasmic variation.

Inheritance of male sterility was studied in the gynodioecious species Plantago coronopus using five plants and their descendants from an area of approximately 50 m2 in each of six locations. The crosses were planned to test for cytoplasmic inheritance of male sterility. In four locations significant differences between reciprocal crosses were observed. The progenies of these reciprocal crosses were used in a crossing scheme designed to test whether these reciprocal differences were caused by different cytoplasmic types between the plants. In all four locations, the existence of at least two cytoplasmic types could be shown. Moreover, the results of the crosses between locations showed that the same two cytoplasmic types were present in all four locations. We therefore argue that there is only limited cytoplasmic variation in P. coronopus. In each cytoplasmic type a series of intermediate sex forms occurred. A marked difference in restoration level existed between the two cytoplasmic types. Plants with cytoplasmic type 2 hardly segregated male steriles, in contrast to plants with cytoplasmic type 1.

Crosses, Genetic↗
PubMed2004

Cytoplasmic droplets are normal structures of human sperm but are not well preserved by routine procedures for assessing sperm morphology.

BACKGROUND: There is a discrepancy between the use of terminology employed by clinicians and basic scientists concerning the cytoplasmic droplets of sperm. Most clinicians consider their presence on sperm to be indicative of abnormal sperm, whereas basic scientists consider them to be attributes of normal sperm. METHODS: The presence of cytoplasmic droplets on human sperm was examined using conventional air-dried, fixed and stained sperm smears and in living and fixed wet preparations. RESULTS: Cytoplasmic droplets were found on the majority of motile sperm and in fixed preparations but only half of them were found in air-dried smears. There was no relationship between the presence of abnormally large cytoplasmic droplets, indicative of abnormal sperm, and the droplets found on living cells. CONCLUSION: The term 'cytoplasmic droplet' is confusingly used to describe two different sperm structures: large amounts of retained, excessive cytoplasmic remnants, that survive the air-drying procedure and are observed on abnormal sperm in conventionally stained sperm smears, and osmotically sensitive vesicles that are present on normal living sperm. A plea is made to retain the term 'cytoplasmic droplet' for the latter structure of normal sperm and to use the term 'excess residual cytoplasm' to describe the abnormally retained cytoplasm observed on abnormal sperm in smears.

Air↗
PubMed1992

Distribution of the microtubule-dependent motors cytoplasmic dynein and kinesin in rat testis.

To examine the possible role of microtubule-based transport in testicular function, we used immunofluorescent techniques to study the presence and localization of the microtubule mechanoenzymes cytoplasmic dynein (a slow-growing end-directed motor) and kinesin (a fast-growing end-directed motor) within rat testis. Cytoplasmic dynein immunofluorescence was observed in Sertoli cells during all stages of spermatogenesis, with a peak in apical cytoplasm during stages IX-XIV. Cytoplasmic dynein immunofluorescence was also localized within Sertoli cells to steps 9-14 (stages IX-XIV) germ cell-associated ectoplasmic specializations. In germ cells, cytoplasmic dynein immunofluorescence was observed in manchettes of steps 15-17 (stages I-IV) spermatids, and small, hollow circular structures were seen in the cytoplasm of step 17 and step 18 spermatids during stages V and VI. Kinesin immunofluorescence was observed in manchettes of steps 10-18 spermatids (stages X-VI). The stage-dependent apical Sertoli cell cytoplasmic dynein immunofluorescence, in conjunction with the previously reported orientation of Sertoli cell microtubules (slow-growing ends toward the lumen) and peak secretion of androgen-binding protein and transferrin, is consistent with the hypothesis that cytoplasmic dynein is involved in Sertoli cell protein transport and secretion. Further, the localization of cytoplasmic dynein and kinesin to manchettes is consistent with current hypotheses concerning manchette function.

Animals↗
PubMed2007

Antineutrophil cytoplasmic autoantibody-associated small-vessel vasculitis.

PURPOSE OF REVIEW: This review focuses on recent advances in the diagnosis, pathogenesis and treatment of antineutrophil cytoplasmic autoantibody-associated small-vessel vasculitis. RECENT FINDINGS: Antineutrophil cytoplasmic autoantibodies are closely associated with Wegener's granulomatosis and microscopic polyangiitis. Within the Churg-Strauss syndrome, antineutrophil cytoplasmic autoantibodies, mostly directed towards myeloperoxidase, characterize patients with glomerulonephritis and small-vessel vasculitis. There is increasing evidence that myeloperoxidase-antineutrophil cytoplasmic autoantibodies are directly involved in the pathogenesis of necrotizing vasculitis. This is less clear for proteinase 3-antineutrophil cytoplasmic autoantibodies, markers for Wegener's granulomatosis. With respect to proteinase 3-antineutrophil cytoplasmic autoantibodies, complementary proteinase 3, a peptide translated from the antisense DNA strand of proteinase 3 and homologous to several microbial peptides, may be involved in induction of proteinase 3-antineutrophil cytoplasmic autoantibodies. Currently, various controlled trials have been initiated. Methotrexate has been shown to be effective for induction of remission in locoregional Wegener's granulomatosis. Other trials are underway. SUMMARY: Apart from its diagnostic potential, antineutrophil cytoplasmic autoantibodies, particularly myeloperoxidase-antineutrophil cytoplasmic autoantibodies, are directly involved in the pathogenesis of the associated vasculitides. New treatment modalities, supposedly more efficacious and less toxic than daily oral cyclophosphamide, are being tested in randomized controlled trials.

Animals↗
PubMed1991

Rev is necessary for translation but not cytoplasmic accumulation of HIV-1 vif, vpr, and env/vpu 2 RNAs.

The effect of Rev on cytoplasmic accumulation of the singly spliced human immunodeficiency virus type 1 (HIV-1) vif, vpr, and env/vpu RNAs was examined by using a quantitative RNA polymerase chain reaction (PCR) analysis following transfection of complete proviral molecular clones into lymphoid cells. Previously published studies using subgenomic env constructs in nonlymphoid cell types concluded that Rev was necessary for cytoplasmic accumulation of high levels of unspliced env RNA and that, by analogy, Rev must be necessary for the cytoplasmic accumulation of all HIV-1 RNAs that contain the Rev-responsive element (RRE). We confirm those results in COS cells. Unexpectedly, in lymphoid cells, we find that although Rev acts somewhat to increase the cytoplasmic level of full-length HIV-1 RNA, Rev has little or no effect on cytoplasmic accumulation of singly spliced HIV-1 RNAs. However, Env protein expression was greatly reduced in the absence of Rev. Analysis of the cytoplasmic RNA revealed that in the absence of Rev or the RRE, the cytoplasmic vif, vpr, and env/vpu 2 RNAs were not associated with polysomes but with a complex of 40S-80S in size. Consequently, efficient expression of the Vif, Vpr, Vpu, and Env proteins from these RNAs is dependent on Rev. These results exclude a mechanism whereby the sole function of Rev is simply to export RNAs from nucleus to cytoplasm. We discuss other models to take into account the dependence on Rev for efficient translation of cytoplasmic HIV-1 RNAs.

Base Sequence↗
PubMed1975

Characterization of cytoplasmic DNA of mouse-myeloma cells.

Cytoplasmic DNA from mouse myeloma cells comprised between 1% and 2% of the total cellular DNA. Detergent-prepared cytoplasmic lysate consisted mainly of 8-S and 22-S species. While these DNA species were present in the 13000 times g pellet of the detergent-prepared cytoplasmic lysate, only the light DNA species was present in the 13000 times g supernatant fraction. In neutral CsCl gradients the DNA of both cytoplasmic fractions had a buoyant density of 1700 g/cm3, which is identical to that of nuclear DNA. The similarity between the cytoplasmic and nuclear DNA was also demonstrated by analysis on alkaline CsCl gradients. A small proportion of closed-circular DNA, presumably of mitochondrial origin, was demonstrated only in cytoplasmic fraction obtained from mechanically disrupted cells and not in detergent-prepared cytoplasmic lysate. It was found that poly (A)-containing mRNA and 28-S ribosomal RNA hybridized to about the same extent to the cytoplasmic DNA as compared to nuclear DNA. The results indicate that most of the cytoplasmic DNA in myeloma cells is similar to nuclear DNA and does not consist of mitochondrial DNA.

Animals↗
PubMed1982

Mitochondrial modifications associated with the cytoplasmic male sterility in faba beans.

Isolated mitochondria of faba beans carrying two different determinisms of the cytoplasmic male sterility (cytoplasms 447 and 350) have been compared to fertile lines. 1. In addition to the major mitochondrial DNA, five small DNA species (in the range of 1000-2000 base pairs) were detected by agarose gel electrophoresis in the four cytoplasms. An additional small DNA species was found specifically in the cytoplasm 350. After endonuclease restriction of the mitochondrial DNA, the patterns obtained for both male-sterile cytoplasms were identical to each other but distinct by two to four fragments from the patterns obtained for male-fertile cytoplasms. 2. [35S]Methionine labeling in situ of the mitochondrial protein synthesis revealed an additional polypeptide (Mr = 25000) detected only in the two male-sterile cytoplasms. 3. The male-sterile cytoplasm 350 showed a decrease of the respiratory state 3 of oxygen uptake during oxidation of NADH or malate + pyruvate. This decrease is thought to reflect a smaller capacity of the respiratory chain. These specific mitochondrial modifications support the hypothesis of a mitochondrial localization of the cytoplasmic male sterility determinant in faba beans.

Cell Nucleus↗
PubMed2004

Salt-inducible kinase-1 represses cAMP response element-binding protein activity both in the nucleus and in the cytoplasm.

Salt-inducible kinase-1 (SIK1) is phosphorylated at Ser577 by protein kinase A in adrenocorticotropic hormone-stimulated Y1 cells, and the phospho-SIK1 translocates from the nucleus to the cytoplasm. The phospho-SIK1 is dephosphorylated in the cytoplasm and re-enters the nucleus several hours later. By using green-fluorescent protein-tagged SIK1 fragments, we found that a peptide region (586-612) was responsible for the nuclear localization of SIK1. The region was named the 'RK-rich region' because of its Arg- and Lys-rich nature. SIK1s mutated in the RK-rich region were localized mainly in the cytoplasm. Because SIK1 represses cAMP-response element (CRE)-mediated transcription of steroidogenic genes, the mutants were examined for their effect on transcription. To our surprise, the cytoplasmic mutants strongly repressed the CRE-binding protein (CREB) activity, the extent of repression being similar to that of SIK1(S577A), a mutant localized exclusively in the nucleus. Several chimeras were constructed from SIK1 and from its isoform SIK2, which was localized mainly in the cytoplasm, and they were examined for intracellular localization as well as CREB-repression activity. A SIK1-derived chimera, where the RK-rich region had been replaced with the corresponding region of SIK2, was found in the cytoplasm, its CREB-modulating activity being similar to that of wild-type SIK1. On the other hand, a SIK2-derived chimera with the RK-rich region of SIK1 was localized in both the nucleus and the cytoplasm, and had a CREB-repressing activity similar to that of the wild-type SIK2. Green fluorescent protein-fused transducer of regulated CREB activity 2 (TORC2), a CREB-specific co-activator, was localized in the cytoplasm and nucleus of Y1 cells, and, after treatment with adrenocorticotropic hormone, cytoplasmic TORC2 entered the nucleus, activating CREB. The SIK1 mutants, having a strong CRE-repressing activity, completely inhibited the adrenocorticotropic hormone-induced nuclear entry of green fluorescent protein-fused TORC2. This suggests that SIK1 may regulate the intracellular movement of TORC2, and as a result modulates the CREB-dependent transcription activity. Together, these results indicate that the RK-rich region of SIK1 is important for determining the nuclear localization and attenuating CREB-repressing activity, but the degree of the nuclear localization of SIK1 itself does not necessarily reflect the degree of SIK1-mediated CREB repression.

3T3-L1 Cells↗
PubMed1990

Kinematics of cytoplasmic deformation in neutrophils during active motion.

A procedure is proposed to measure the cytoplasmic deformation in active motile neutrophils in the form of cytoplasmic strains and strain rates. Three neighboring microspheres in a local region of the cytoplasm serve as markers for local motion. Their positions are tracked by means of a high resolution light microscope and serve to compute nonlinear measures of strains and strain rates together with the principal strains and principal directions. Active neutrophils exhibit large cytoplasmic strains both during periodic pseudopod projections and during continuous locomotion in a polarized shape. The cytoplasmic motion is often synchronized with the whole cell deformation. The local cytoplasmic strains exceed the strains estimated for the whole cell and are not reversible except in some cases of single pseudopod projections. Large strains are observed both in attached and freely suspended cells. Strain rates are relatively constant but show an increase during the pseudopod retraction phase. Local cytoplasmic strains in neutrophils are inhomogeneous and reach large values during passage of the contraction rings. Neutrophils rendered passive by treatment with cytochalasin or EDTA show a random motion of microspheres with much smaller displacements. These observations suggest that the cytoplasm of active neutrophil exhibits large cytoplasmic strains and strain rates in the absence of an external stress resulting in a high degree of intracellular mixing. The proposed technique may be applied to a wide range of problems in cell biology.

Biomechanical Phenomena↗
PubMed1990

Localization of enterobacterial common antigen immunoreactivity in the ribosomal cytoplasm of Escherichia coli cells cryosubstituted and embedded at low temperature.

The application of two on-section immunogold labeling techniques, the Lowicryl K4M (progressive lowering of temperature) procedure and the cryosection technique of Tokuyasu, in a previous work to study the topology of enterobacterial common antigen (ECA) biosynthesis revealed the presence of label on the outer membrane and in areas associated with the inner side of the cytoplasmic membrane. However, labeling was also observed in the ribosomal cytoplasm. The question of whether the cytoplasmic label was a result of ECA displacement during the more slowly acting aldehyde fixation or whether cytoplasmic ECA precursors are true constituents of the ribosomal cytoplasm could not be resolved from these results. In the study described here, cells of the same Escherichia coli F470 strain were reinvestigated by comparison of the progressive lowering of temperature and improved cryosubstitution-low-temperature embedment techniques. The latter procedure, applied directly to nonpretreated and noncentrifuged cells, led to superior ultrastructural preservation of the cytoplasmic organization, with little opportunity for cytoplasmic antigen displacement after the primary cryofixation step; the label distribution obtained supports the conclusion that N-acetylmannosaminuronic acid (ManNAcA)-containing ECA precursors are real constituents of the ribosomal cytoplasm. Results from tunicamycin inhibition studies of ECA biogenesis in the E. coli mutant 2465 suggested that even the ECA precursor UDP-ManNAcA alone or a chemically unidentified product(s) generated from accumulated ManNAcA residues may react with the monoclonal antibody used, leading to weak but clearly positive cytoplasmic labeling. The relatively intense labeling obtained with cells grown in the absence of the drug can be explained by the reactivity of further ManNAcA-containing ECA precursors with the monoclonal antibody used.

Antigens, Bacterial↗
PubMed2007

UL20 protein functions precede and are required for the UL11 functions of herpes simplex virus type 1 cytoplasmic virion envelopment.

Egress of herpes simplex virus type 1 (HSV-1) from the nucleus of the infected cell to extracellular spaces involves a number of distinct steps, including primary envelopment by budding into the perinuclear space, de-envelopment into the cytoplasm, cytoplasmic reenvelopment, and translocation of enveloped virions to extracellular spaces. UL20/gK-null viruses are blocked in cytoplasmic virion envelopment and egress, as indicated by an accumulation of unenveloped or partially enveloped capsids in the cytoplasm. Similarly, UL11-null mutants accumulate unenveloped capsids in the cytoplasm. To assess whether UL11 and UL20/gK function independently or synergistically in cytoplasmic envelopment, recombinant viruses having either the UL20 or UL11 gene deleted were generated. In addition, a recombinant virus containing a deletion of both UL20 and UL11 genes was constructed using the HSV-1(F) genome cloned into a bacterial artificial chromosome. Ultrastructural examination of virus-infected cells showed that both UL20- and UL11-null viruses accumulated unenveloped capsids in the cytoplasm. However, the morphology and distribution of the accumulated capsids appeared to be distinct, with the UL11-null virions forming aggregates of capsids having diffuse tegument-derived material and the UL20-null virus producing individual capsids in close juxtaposition to cytoplasmic membranes. The UL20/UL11 double-null virions appeared morphologically similar to the UL20-null viruses. Experiments on the kinetics of viral replication revealed that the UL20/UL11 double-null virus replicated in a manner similar to the UL20-null virus. Additional experiments revealed that transiently expressed UL11 localized to the trans-Golgi network (TGN) independently of either gK or UL20. Furthermore, virus infection with the UL11/UL20 double-null virus did not alter the TGN localization of transiently expressed UL11 or UL20 proteins, indicating that these proteins did not interact. Taken together, these results show that the intracellular transport and TGN localization of UL11 is independent of UL20/gK functions, and that UL20/gK are required and function prior to UL11 protein in virion cytoplasmic envelopment.

Animals↗
PubMed1974

Simian virus 40-host cell interactions. II. Cytoplasmic and nucleolar accumulation of simian virus 40 virion protein.

We have used immunofluorescence in parallel with transmission and scanning electron microscopy to characterize the unusual cytoplasmic and nucleolar accumulation of Simian virus 40 (SV40) virion protein (C antigen) at restrictive temperatures (39 to 41 C) in monkey cells infected with a temperature-sensitive mutant of SV40 defective in virion assembly, tsB11. Cytoplasmic and nucleolar accumulation of C antigen did not occur in wild-type-infected cells at any temperature. Wild-type- and tsBll-infected cells were not distinguishable at 33 C by immunofluorescence or electron microscopy. Temperature-shift experiments using metabolic inhibitors of DNA (cytosine arabinonucleoside, 20 mug/ml), RNA (actinomycin D, 5 mug/ml), and protein synthesis (cycloheximide, 2 x 10(-4) to 10 x 10(-4) M) were used to investigate the requirements for ongoing DNA, RNA, and protein synthesis in the distribution of virion protein between the nucleus, nucleolus, and cytoplasm. The transport of C antigen from the nucleolus and cytoplasm into the nucleus was complete after a temperature shift-down (41 and 39 to 33 C). Limited virus particle formation occurred after the shift-down in the presence of actinomycin D and cycloheximide, indicating some of the 39 to 41 C synthesized virion protein could be used for capsid assembly at 33 C in the absence of further virion protein synthesis. Nucleolar and cytoplasmic accumulations of C antigen occurred in the absence of drugs after a shift-up (33 to 39 C and 41 C) indicating a continuous requirement for the tsB11 mutant function. Furthermore, the virion protein synthesized at 33 C remained confined to the nucleus when the cells were shifted to 39 and 41 C in the presence of actinomycin D or cycloheximide. In the presence of cytosine arabinonucleoside, however, the virion protein accumulated in large aggregates in the nucleus and nucleolus after the shift-up, but did not migrate into the cytoplasm as it did in drug-free tsB11-infected control cells. Colchicine (10(-3) M) had no effect on the abnormal accumulation of C antigen during shift-up or shift-down experiments suggesting that microtubular transport plays little if any role in the abnormal transport of tsB11 virion protein from cytoplasm to nucleus. Although virus particles were never observed by electron microscopy and V antigen was not detected by immunofluorescence at 39 or 41 C in tsB11-infected cells, dense amorphous accumulations were formed in the nucleoli and cytoplasm. We suggest that the tsB11 function is continuously required for the normal transport of SV40 virion protein between the cytoplasm, nucleolus, and nucleus and for the assembly of capsids and virions. Several possible mechanisms for the altered tsB11 function or protein are discussed. One of the virion proteins may also be involved in some presently undetermined nucleolar function during SV40 productive infection.

Animals↗
PubMed1991

Origin of unenveloped capsids in the cytoplasm of cells infected with herpes simplex virus 1.

In cells infected with herpes simplex viruses the capsids acquire an envelope at the nuclear membrane and are usually found in the cytoplasm in structures bound by membranes. Infected cells also accumulate unenveloped capsids alone or juxtaposed to cytoplasmic membranes. The juxtaposed capsids have been variously interpreted as either undergoing terminal deenvelopment resulting from fusion of the envelope with the membrane of the cytoplasmic vesicles or undergoing sequential envelopment and deenvelopment as capsids transit the cytoplasm into the extracellular space. Recent reports have shown that (i) wild-type virus attaches to but does not penetrate cells expressing glycoprotein D (G. Campadelli-Fiume, M. Arsenakis, F. Farabegoli, and B. Roizman, J. Virol. 62:159-167, 1988) and that (ii) a mutation in glycoprotein D enables the mutant virus to productively infect cells expressing the wild-type glycoprotein (G. Campadelli-Fiume, S. Qi, E. Avitabile, L. Foa-Tomasi, R. Brandimarti, and B. Roizman, J. Virol. 64:6070-6079, 1990). If the unenveloped capsids in the cytoplasm result from fusion of the cytoplasmic membranes with the envelopes of viruses transiting the cytoplasm, cells infected with virus carrying the mutation in glycoprotein D should contain many more unenveloped capsids in the cytoplasm inasmuch as there would be little or no restriction in the fusion of the envelope with cytoplasmic membranes. Comparison of thin sections of baby hamster kidney cells infected with wild-type and mutant viruses indicated that this was the case. Moreover, in contrast to the wild-type parent, the mutant virus was not released efficiently from infected cells. The conclusion that the unenveloped capsids are arrested forms of deenveloped capsids is supported by the observation that the unenveloped capsids were unstable in that they exhibited partially extruded DNA.

Animals↗
PubMed2003

Sequestration of p53 in the cytoplasm by adenovirus type 12 E1B 55-kilodalton oncoprotein is required for inhibition of p53-mediated apoptosis.

The adenovirus E1B 55-kDa protein is a potent inhibitor of p53-mediated transactivation and apoptosis. The proposed mechanisms include tethering the E1B repression domain to p53-responsive promoters via direct E1B-p53 interaction. Cytoplasmic sequestration of p53 by the 55-kDa protein would impose additional inhibition on p53-mediated effects. To investigate further the role of cytoplasmic sequestration of p53 in its inhibition by the E1B 55-kDa protein we systematically examined domains in both the Ad12 55-kDa protein and p53 that underpin their colocalization in the cytoplasmic body and show that the N-terminal transactivation domain (TAD) of p53 is essential for retaining p53 in the cytoplasmic body. Deletion of amino acids 11 to 27 or even point mutation L22Q/W23S abolished the localization of p53 to the cytoplasmic body, whereas other parts of TAD and the C-terminal domain of p53 are dispensable. This cytoplasmic body is distinct from aggresome associated with overexpression of some proteins, since it neither altered vimentin intermediate filaments nor associated with centrosome or ubiquitin. Formation of this structure is sensitive to mutation of the Ad12 55-kDa protein. Strikingly, mutation S476/477A near the C terminus of the Ad12 55-kDa protein eliminated the formation of the cytoplasmic body. The equivalent residues in the Ad5 55-kDa protein were shown to be critical for its ability to inhibit p53. Indeed, Ad12 55-kDa mutants that cannot form a cytoplasmic body can no longer inhibit p53-mediated effects. Conversely, the Ad12 55-kDa protein does not suppress p53 mutant L22Q/W23S-mediated apoptosis. Finally, we show that E1B can still sequester p53 that contains the mitochondrial import sequence, thereby potentially preventing the localization of p53 to mitochondria. Thus, cytoplasmic sequestration of p53 by the E1B 55-kDa protein plays an important role in restricting p53 activities.

Adenovirus E1B Proteins↗
PubMed1989

Regulation of the nuclear genes encoding the cytoplasmic and mitochondrial leucyl-tRNA synthetases of Neurospora crassa.

We show that the nuclear genes for the cytoplasmic and mitochondrial leucyl-tRNA synthetase (LeuRS) of Neurospora crassa are distinct in their encoded proteins, codon usage, mRNA levels, and regulation. The 4.2-kilobase-pair region representing the structural gene for cytoplasmic LeuRS and flanking regions has been sequenced. The positions of the 5' and 3' ends of mRNA and of a single 62-base-pair intron have been mapped. The methionine-initiated open reading frame encoded a protein of 1,123 amino acids and displayed a strong codon bias. Although cytoplasmic LeuRS shares with mitochondrial LeuRS some general features common to most aminoacyl-tRNA synthetases, there is little amino acid sequence similarity between them, mRNA levels for cytoplasmic LeuRS were much higher than those for mitochondrial LeuRS. This observation and the strong codon bias in the cytoplasmic LeuRS gene may contribute to a greater abundance of cytoplasmic LeuRS than mitochondrial LeuRS. The genes for cytoplasmic and mitochondrial LeuRS are regulated independently. The cytoplasmic LeuRS gene is regulated by the cross-pathway control system in N. crassa, which is analogous to general amino acid control in Saccharomyces cerevisiae. The cytoplasmic LeuRS mRNA levels are induced by amino acid starvation resulting from the addition of aminotriazole. Part of this increase is due to utilization of new transcription start sites. In contrast, the mitochondrial LeuRS gene is not induced by amino acid limitation. However, the mitochondrial LeuRS mRNA levels did increase dramatically upon inhibition of mitochondrial protein synthesis by chloramphenicol or ethidium bromide or in the temperature-sensitive strain leu-5 carrying a mutation in the mitochondrial LeuRS structural gene.

Amino Acid Sequence↗
PubMedDate not supplied

Mammary mucinous carcinoma with marked cytoplasmic hyalinization. A report of 2 cases with emphasis on fine needle aspiration cytologic findings.

BACKGROUND: Cytoplasmic eosinophilia in breast tumors is due mainly to abundance of secretory granules, lysosomes and/or mitochondria. Diffuse cytoplasmic hyalinization caused by intermediate filaments has not been described before. CASES: Two cases of pure mammary mucinous carcinoma occurred with marked cytoplasmic hyalinization among 556 wide excision/mastectomy specimens of mammary ductal carcinoma, either in situ or invasive, encountered at Pamela Youde Nethersole Eastern Hospital, Hong Kong, during the period from 1994 to the end of 2001. Preoperative fine needle aspiration cytology was available in 1 case, showing cohesive clusters and micropapillae of tumor cells in abundant background mucin. The tumor cells had low mitotic activity and possessed round to oval nuclei, solitary macronucleoli; ample, eosinophilic cytoplasm and discrete cell borders. Cytoplasmic granularity, intracytoplasmic vacuoles or "cometlike" cells were not found. Histologic examination of the surgical specimens in both cases revealed pure mucinous carcinoma with diffuse and marked cytoplasmic hyalinization. There was no evidence of peritumoral lymphovascular permeation or regional lymph node metastasis. Ultrastructural examination showed minimal secretory activity, with abundant, loose aggregates of intermediate filaments in the cytoplasm. There was a relative paucity of other organelles. CONCLUSION: Diffuse cytoplasmic hyalinization is a peculiar morphologic change in mammary ductal carcinoma and apparently unique to pure mucinous carcinoma. Recognition of this phenomenon may help to avoid misdiagnosis of other types of ductal carcinoma with cytoplasmic eosinophilia, such as apocrine carcinoma and ductal carcinoma with neuroendocrine differentiation, which sometimes are also associated with pools of extracellular mucin.

Adenocarcinoma, Mucinous↗