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PubMed2000

RseB binding to the periplasmic domain of RseA modulates the RseA:sigmaE interaction in the cytoplasm and the availability of sigmaE.RNA polymerase.

The Escherichia coli sigmaE regulon has evolved to sense the presence of misfolded proteins in the bacterial envelope. Expression of periplasmic chaperones and folding catalysts is under the control of sigmaE RNA polymerase. The N-terminal domain of RseA sequesters sigmaE in the cytoplasmic membrane, preventing its association with core RNA polymerase. The C-terminal domain of RseA interacts with RseB, a periplasmic protein. The relative concentration of sigmaE:RseA:RseB is 2:5:1 and this ratio remains unaltered upon heat shock induction of the sigmaE regulon. Purification from crude cellular extracts yields cytoplasmic, soluble sigmaE RNA polymerase as well as membrane sequestered sigmaE.RseA and sigmaE.RseA.RseB. RseB binding to the C-terminal domain of RseA increases the affinity of RseA for sigmaE by 2- to 3-fold (Kd 50-100 nM). RseB binds also to the misfolded aggregates of MalE31, a variant of maltose binding protein that forms inclusion bodies in the periplasm. We discuss a model whereby the RseB-RiseA interaction represents a measure for misfolded polypeptides in the bacterial envelope, modulating the assembly of sigmaE RNA polymerase and the cellular heat shock response.

ATP-Binding Cassette Transporters↗
PubMed1990

CD28 ligation in T-cell activation: evidence for two signal transduction pathways.

The CD28 homodimer is thought to function as a signal transducing receptor during activation of T cells. Evidence is presented that the degree of aggregation of CD28 on the cell surface regulates two distinct CD28-associated signals. Binding of bivalent CD28 monoclonal antibody (MoAb) 9.3 upregulates lymphokine production by messenger RNA (mRNA) stabilization, without direct initiation of lymphokine mRNA transcription. This signal was not dependent on inositol phospholipid production or activation of a protein tyrosine kinase (PTK). In contrast, further crosslinking of CD28 on the cell surface rapidly induced formation of large amounts of inositol trisphosphate (InsP3) and increased cytoplasmic calcium concentration [( Ca2+]i), but did not stimulate PTK. CD28 crosslinking directly activated a subset of resting T cells, since CD25 (interleukin [IL]-2 receptor alpha chain) mRNA was rapidly induced in purified T cells, and proliferation, even without addition of exogenous IL-2, was sometimes observed. CD25 expression was detected on the cell surface of approximately 20% of CD4+ T cells. The degree of CD28 aggregation required for activation was investigated by preparing soluble 9.3 x 9.3 conjugates ranging in size from approximately 300 Kd to greater than 1,000 Kd, and comparing their function in T-cell proliferation assays with phorbol-12-myristate-13-acetate (PMA), anti-CD3, or IL-2. There was a correlation between conjugate size and proliferation with IL-2, whereas costimulation with PMA or CD3 was optimized at a lower degree of CD28 aggregation. The inositol phospholipid (InsP) generation and increase in [Ca2+]i after CD28 receptor aggregation appeared to proceed through a pathway different from the CD3/T-cell receptor (TCR) pathway since it was enhanced by pretreatment with PMA, while the InsP and [Ca2+]i signal from crosslinking CD3 was suppressed by PMA. Furthermore, the proliferation response to CD28 aggregation was resistant to inhibition by CD3 modulation. Thus, CD28 aggregation appears to trigger a phospholipase C activation pathway that differs from the CD3/TCR-linked pathway.

Animals↗
PubMed1994

The nature of multimeric forms of cymbidium ringspot tombusvirus satellite RNA.

The multimeric forms of cymbidium ringspot tombusvirus (CyRSV) satellite (sat) RNA were analysed. Attempts to amplify the putative junction region of oligomers using the polymerase chain reaction (PCR) failed, indicating the absence of such structures. SatRNA-related species having the size double than the unit length were shown to be double-stranded monomers and not single-stranded dimers. Similarly, satRNA species of a size four times the unit length were shown to be constituted by aggregates of double-stranded monomers. The absence of single-stranded CyRSV satRNA oligomers indicates that the formation of multimers is not a step in the replication of this RNA molecule.

Blotting, Northern↗
PubMed1979

[Melting and reassociation of double-stranded RNA of the encephalomyocarditis virus].

The processes of melting and reassociation of double-stranded RNA in dimethylsulfoxide were studied. The addition of a small amount of LiCl results in great results in great reduction of Tm (temperature of melting), whereas the NaCl produces the opposite effect. It is suggested, that LiCl coordinates the molecules of H2O, reducing their activity, and consequently destabilises dsRNA. Mild conditions for melting and reassociation of RNA can be created. It was found that under optimal conditions for dsRNA melting, the degree of strand separation depends on the overall concentration of RNA, irrespective of the type of RNA added to the dsRNA preparation. Reassociation of dsRNA of EMC virus proceeds much faster than that of dsRNA of a related poliovirus. Addition of poly(C) to an annealing mixture slows down the rate of reassociation of EMC dsRNA, producing no effect on the poliovirus dsRNA reassociation. It is suggested that the presence of large poly(C) and poly(G) tracts in the complementary strands of the RNA determines its anomalous fast reassociation. Upon incubation of completely separated strands of EMC dsRNA in a water solution with high ionic strength partially double-stranded aggregates are formed. The formation of aggregates is prevented by addition of poly(A), which indicates that they are produced by "zippening" of a molecule starting with poly(A):poly(U) region. The significance of homopolymeric regions for stability of dsRNA of the EMC virus as well as their role in viral multiplication are discussed.

Encephalomyocarditis virus↗
PubMed1996

Ongoing activity of RNA polymerase II precludes chromatin collapse and DNA fragmentation in Chinese hamster ovary cells.

The role of ongoing RNA synthesis in chromatin organization in Chinese hamster ovary cells was examined upon exposure to the transcription inhibitor alpha-amanitin. Treatment with alpha-amanitin led to pleomorphic nuclei with chromatin heavily condensed and with the remaining ribonucleoprotein aggregated in large compact granular masses around the margins of the nuclear periphery. Concommitant with the changes in nuclei morphology transient focal dilatation of the rough endoplasmic reticulum was observed while other cytoplasmic organelles appeared structurally unaffected. The morphological changes occurred after complete inhibition of RNA polymerase II mediated transcription. The molecular integrity of isolated DNA was monitored in parallel with the structural analysis. Fragmentation of cellular DNA occurred in a time-dependent fashion and well after the complete inhibition of RNA synthesis. Characteristic oligonucleosomesized DNA fragments of about 187 base pairs in length was produced in a cotemporal time-dependent fashion. Our findings indicate that ongoing transcription and the structural state of chromatin are very closely integrated, and provide further evidence that RNA is a structural component of the nuclear matrix, which in turn is involved in keeping chromatin physically dispersed and decondensed.

Amanitins↗
PubMed1996

A prespore-specific gene of Dictyostelium discoideum encodes the small subunit of ribonucleotide reductase.

We have isolated the gene. rnrB, that encodes the ribonucleotide reductase small subunit of Dictyostelium discoideum. The deduced amino acid sequence of rnrB exhibits about 60% sequence identity with its homologues in other eukaryotes. As demonstrated by RNA blot analysis the rnrB transcript is detected in growing cells and decreases dramatically at the onset of development. The rnrB transcript reappears after the cells have formed multicellular aggregates. To further examine the pattern of expression, we have fused the rnrB promoter and part of its coding sequence to lacZ. The transgenic strain bearing such a reporter construct expresses the fusion gene with a biphasic profile, which is indistinguishable from that of the endogenous rnrB. The multicellular aggregates of Dictyostelium are differentiated along the anterior-posterior axis. Cells in the anterior give rise to the stalk of the fruiting body while cells in the posterior are precursors of spores. Results from histochemical staining show that beta-galactosidase activity is detected exclusively in the posterior two-thirds of the aggregates. These data suggest that rnrB is expressed in prespore cells during postaggregative development and in vegetative cells.

Amino Acid Sequence↗
PubMed2006

Expression of the cadherin-11 gene is a discriminative factor between articular and growth plate chondrocytes.

OBJECTIVE: Calcification of hypertrophic chondrocytes is the final step in the differentiation of growth plates, although the precise mechanism is not known. We have established two growth plate-derived chondrocyte cell lines, MMR14 and MMR17, from p53-/- mice (Nakamata T, Aoyama T, Okamoto T, Hosaka T, Nishijo K, Nakayama T, et al. In vitro demonstration of cell-to-cell interaction in growth plate cartilage using chondrocytes established from p53-/- mice. J Bone Miner Res 2003;18:97-107). Prolonged in vitro culture produced calcified nodules in MMR14, but not in MMR17. Factors responsible for the difference in calcification between the two cell lines may also be involved in the physiological calcification in growth plate. DESIGN: Gene expression profiles of MMR14 and MMR17 were compared using a cDNA microarray to identify candidate genes involved in the calcification process. RESULTS: Forty-five genes were identified as upregulated in MMR14, including the cadherin-11 (Cdh-11) gene. The expression of Cdh-11 in MMR14 was detected in cell-cell junctions, while no expression was observed in MMR17. Primary cultured chondrocytes from growth plate (GC) also expressed the Cdh-11, and the staining of Cdh-11 was observed in the late hypertrophic zone of growth plate. Cell aggregation assays showed that chondrocytes required Ca2+ to form nodules, and knockdown of the Cdh-11 gene expression using short interfering RNA inhibited the formation of calcified nodules in MMR14. The introduction of Cdh-11 into MMR17 failed to produce calcified nodules indicating that Cdh-11 is one, but not the sole, factor responsible for the production of calcified nodules. CONCLUSION: Although the physiological role is still unclear, Cdh-11 is a discriminative factor between articular and growth plate chondrocytes.

Animals↗
PubMed1998

Rhabdomyosarcoma with rhabdoid-like features.

We describe an unusual case of a rhabdomyosarcoma (RMS) in that it had rhabdoid-like cells histologically and occurred in a female who had undergone bone marrow transplantation for chronic myelogenous leukemia. The tumor was composed of loosely cohesive cells with abundant eosinophilic cytoplasm and exhibited PAS-negative paranuclear inclusions. The tumor cells had positive vimentin, muscle-specific actin, sarcomeric actin and desmin immunoreactivity. Ultrastructurally, the tumor cells contained aggregates of thin and thick filaments. In situ hybridization did not detect human papillomavirus or cytomegalovirus DNA, or EBV DNA or RNA. The tumor fulfilled the current criteria for a diagnosis of RMS; however, it could not be further classified. The tumor appears to have a good prognosis as there has been no evidence of recurrence five years after resection. As this is the first case report, to our knowledge, of this type of tumor following bone marrow transplant, the significance of this association is not yet clear.

Abdominal Neoplasms↗
PubMed1970

Studies on ribonucleic acid and homopolyribonucleotide formation in neuronal, glial and liver nuclei.

1. Various types of nuclear preparations, with different ratios of neuronal to glial nuclei, were isolated from guinea-pig cerebral grey matter and ox cerebral grey matter and white matter. Conditions appropriate for the separate assay of RNA and poly A formation were described. Comparative rates of RNA and poly A formation were studied in cerebral and liver nuclei. 2. RNA polymerase activity per nucleus is higher in neuronal nuclei than in glial nuclei. In liver nuclei, the activity is much lower than in cerebral nuclei. The physical relationship between RNA polymerase and deoxyribonucleoprotein seems to differ in neuronal, glial and liver nuclei. 3. Poly A polymerase activity in liver nuclei is selectively activated by Mn(2+) and inhibited by GTP, CTP and UTP. On a DNA basis, the activity in an aggregate enzyme is the same as in intact nuclei. Poly A polymerase activity per nucleus is much higher in liver nuclei than in neuronal nuclei. Glial nuclei show an intermediate activity. 4. It is suggested that, in neuronal nuclei, the synthesis of RNA is more prominent than that of poly A under conditions where both polymers are formed simultaneously. This contrasts with liver nuclei, where more poly A is made than RNA. 5. In neuronal nuclei, the rate of CTP incorporation is much higher than in glial and liver nuclei. This incorporation is most probably due to poly C synthesis.

Adenine Nucleotides↗
PubMed2006

5-HT2A receptor induces ERK phosphorylation and proliferation through ADAM-17 tumor necrosis factor-alpha-converting enzyme (TACE) activation and heparin-bound epidermal growth factor-like growth factor (HB-EGF) shedding in mesangial cells.

In this study, we present multiple lines of evidence to support a critical role for heparin-bound EGF (epidermal growth factor)-like growth factor (HB-EGF) and tumor necrosis factor-alpha-converting enzyme (TACE) (ADAM17) in the transactivation of EGF receptor (EGFR), ERK phosphorylation, and cellular proliferation induced by the 5-HT(2A) receptor in renal mesangial cells. 5-hydroxy-tryptamine (5-HT) resulted in rapid activation of TACE, HB-EGF shedding, EGFR activation, ERK phosphorylation, and longer term increases in DNA content in mesangial cells. ERK phosphorylation was attenuated by 1) neutralizing EGFR antibodies and the EGFR kinase inhibitor, AG1478, 2) neutralizing HB-EGF, but not amphiregulin, antibodies, heparin, or CM197, and 3) pharmacological inhibitors of matrix-degrading metalloproteinases or TACE small interfering RNA. Exogenously administered HB-EGF stimulated ERK phosphorylation. Additionally, TACE was co-immunoprecipitated with HB-EGF. Small interfering RNA against TACE also blocked 5-HT-induced increases in ERK phosphorylation, HB-EGF shedding, and DNA content. In aggregate, this work supports a pathway map that can be depicted as follows: 5-HT --> 5-HT(2A) receptor --> TACE --> HB-EGF shedding --> EGFR --> ERK --> increased DNA content. To our knowledge, this is the first time that TACE has been implicated in 5-HT-induced EGFR transactivation or in proliferation induced by a G protein-coupled receptor in native cells in culture.

ADAM Proteins↗
PubMed1988

Ultrastructural and biochemical analysis of the stress granule in chicken embryo fibroblasts.

The ultrastructure and biochemical composition of cytoplasmic particles that form in chicken embryo fibroblasts during stress have been analyzed. We showed previously that these particles contained the small stress protein, sp 24, and antibodies specific to sp 24 were used here to identify the stress granule. In thin sections, the stress granule was a densely staining, membraneless, cytoplasmic body and appeared as a highly condensed area of cytoplasm in freeze-fracture preparations. Hypotonic swelling of cells before freeze-fracture analysis revealed a basketlike structure composed of interconnecting protein cables. No other proteins could be cross-linked to sp 24 when stress granules were treated with dithiobis-(succinimidyl propionate). High resolution autoradiographic analysis with [3H]uridine failed to identify any associated RNA synthesized in the period immediately before the stress. Thus the stress granule appears to be composed predominantly of sp 24 aggregates. Sp 24 could be purified to homogeneity from the stress granule by solubilization in 8 M urea and anion exchange chromatography.

Amino Acids↗
PubMed1983

Biosynthetic pathway of mitochondrial ATPase subunit 9 in Neurospora crassa.

Subunit 9 of mitochondrial ATPase (Su9) is synthesized in reticulocyte lysates programmed with Neurospora poly A-RNA, and in a Neurospora cell free system as a precursor with a higher apparent molecular weight than the mature protein (Mr 16,400 vs. 10,500). The RNA which directs the synthesis of Su9 precursor is associated with free polysomes. The precursor occurs as a high molecular weight aggregate in the postribosomal supernatant of reticulocyte lysates. Transfer in vitro of the precursor into isolated mitochondria is demonstrated. This process includes the correct proteolytic cleavage of the precursor to the mature form. After transfer, the protein acquires the following properties of the assembled subunit: it is resistant to added protease, it is soluble in chloroform/methanol, and it can be immunoprecipitated with antibodies to F1-ATPase. The precursor to Su9 is also detected in intact cells after pulse labeling. Processing in vivo takes place posttranslationally. It is inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). A hypothetical mechanism is discussed for the intracellular transfer of Su9. It entails synthesis on free polysomes, release of the precursor into the cytosol, recognition by a receptor on the mitochondrial surface, and transfer into the inner mitochondrial membrane, which is accompanied by proteolytic cleavage and which depends on an electrical potential across the inner mitochondrial membrane.

Adenosine Triphosphatases↗
PubMed2000

Virus-like particles assemble in plants and bacteria expressing the coat protein gene of Indian peanut clump virus.

cDNA copies of the coat protein (CP) gene of Indian peanut clump virus (IPCV)-H were introduced into cells of Nicotiana benthamiana or Escherichia coli by transformation with vectors based on pROKII or pET respectively. In both plant and bacterial cells, IPCV CP was expressed and assembled to form virus-like particles (VLP). In plant extracts, the smallest preponderant particle length was about 50 nm. Other abundant lengths were about 85 and about 120 nm. The commonest VLP length in bacterial extracts was about 30 nm. Many of the longer VLP appeared to comprise aggregates of shorter particles. The lengths of the supposed 'monomer' VLP corresponded approximately to those expected for encapsidated CP gene transcript RNA. Immunocapture RT-PCR, using primers designed to amplify the CP gene, confirmed that the VLP contained RNA encoding IPCV-H CP. The results show that encapsidation does not require the presence of the 5'-terminal untranslated sequence of the virus RNA and suggest that if there is an 'origin of assembly' motif or sequence, it lies within the CP gene. When transgenic plants expressing IPCV-H CP were inoculated with IPCV-L, a strain that is serologically distinct from IPCV-H, the virus particles that accumulated contained both types of CP.

Arachis↗
PubMed2000

Effects of birth weight and postnatal nutrition on neonatal sheep: II. Skeletal muscle growth and development.

This study investigated effects of birth weight and postnatal nutrition on growth and development of skeletal muscles in neonatal lambs. Low (L; mean +/- SD 2.289 +/- .341 kg, n = 28) and high (H; 4.840 +/- .446 kg, n = 20) birth weight male Suffolk x (Finnsheep x Dorset) lambs were individually reared on a liquid diet to grow rapidly (ad libitum fed, ADG 337 g, n = 20) or slowly (ADG 150 g, n = 20) from birth to live weights (LW) up to approximately 20 kg. At birth, weight of semitendinosus (ST) muscle in L lambs was 43% that in H lambs; aggregate weights of ST and seven other dissected muscles were similarly reduced. In ST muscle of L lambs, mass of DNA, RNA, and protein were also significantly reduced to levels 67, 60, and 34%, respectively, of those in H lambs. However, myofiber numbers of ST, tibialis caudalis, or soleus muscles did not differ between the L and H birth weight lambs and did not change during postnatal growth. During postnatal rearing, daily accretion rate of dissected muscle was lower in L than in H lambs. Accretion of muscle per kilogram of gain in empty body weight (EBW) was reduced in the slowly grown L lambs compared with their H counterparts, although the difference was less pronounced between the rapidly grown L and H lambs. Throughout the postnatal growth period, ST muscle of L lambs contained less DNA with a higher protein:DNA ratio at any given muscle weight than that of H lambs. Slowly grown lambs had heavier muscles at any given EBW than rapidly grown lambs. Content of DNA and protein:DNA ratio in ST muscle were unaffected by postnatal nutrition, but RNA content and RNA:DNA were greater and protein:RNA was lower at any given muscle weight in rapidly grown lambs. Results suggest that myofiber number in fetal sheep muscles is established before the presumed, negative effects of inadequate fetal nutrient supply on skeletal muscle growth and development become apparent. However, proliferation of myonuclei may be influenced by fetal nutrition in late pregnancy. Reduced myonuclei number in severely growth-retarded newborn lambs may limit the capacity for postnatal growth of skeletal muscles.

Animal Nutritional Physiological Phenomena↗
PubMed1998

Aggregation of the Intracellular Domain of the Human Thrombopoietin Receptor c-MPL.

Thrombopoietin (TPO) is the major cytokine which is involved in platelet production and exerts its effects via the receptor c-MPL. The yeast two-hybrid system has been used to study the aggregation of the intracellular domain of c-MPL in TPO signal transduction. First, the cDNA fragment of MPLP intracellular domain was amplified and cloned by using RT-PCR method from the total RNA of HEL cells. Then the cDNA fragment was sequenced and subcloned into two-hybrid vectors pAS2 and pGAD424, respectively, and the recombinants are named as pASMM and pGADMM. Co-transformation of these plasmids into yeast activated his3 and lacZ reporter genes, demonstrating in vivo interaction of the MPLP receptor intracellular domain itself. The aggregation may be important in TPO signal transduction.

Journal Article↗
PubMed1992

Enhanced degradation of the ferritin repressor protein during induction of ferritin messenger RNA translation.

Induction of ferritin synthesis in cultured cells by heme or iron is accompanied by degradation of the ferritin repressor protein (FRP). Intermediates in the degradative pathway apparently include FRP covalently linked in larger aggregates. The effect of iron on FRP degradation is enhanced by porphyrin precursors but is decreased by inhibitors of porphyrin synthesis, which implies that heme is an active agent. These results suggest that translational induction in this system may be caused by enhanced repressor degradation. While unique among translational regulatory systems, this process is common to a variety of other biosynthetic control mechanisms.

5-Aminolevulinate Synthetase↗
PubMed1996

Long-term ganciclovir chemotherapy for congenital duck hepatitis B virus infection in vivo: effect on intrahepatic-viral DNA, RNA, and protein expression.

Long-term antiviral chemotherapy using the nucleoside analogue ganciclovir was undertaken with the aim of eliminating hepadnaviral covalently closed circular (CCC) DNA from the livers of ducks that were congenitally infected with the duck hepatitis B virus (DHBV). Twenty-four weeks of ganciclovir therapy caused a substantial reduction in viremia, intrahepatic viral DNA replicative intermediates, and viral core proteins. Unfortunately, ganciclovir therapy did not substantially affect CCC DNA or viral RNA levels, and the treatment resulted in an increase in the intrahepatic expression of the viral envelope proteins, pre-S and S. By the completion of therapy, the viral envelope proteins had assembled into large aggregates within the cytoplasm of most hepatocytes. Viral replication in the bile duct epithelial cells and in the extrahepatic sites was likewise not affected by long-term ganciclovir therapy. In conclusion, 24 weeks of ganciclovir therapy decreased most viral replication markers within the liver, except for those of viral CCC DNA, RNA, and envelope proteins. Long-term therapeutic strategies using nucleoside analogs such as ganciclovir should be used with caution in chronic hepatitis B virus (HBV) infection. The careful monitoring of serum and hepatic markers of viral replication may therefore be important to avoid possible toxic consequences, such as the selective accumulation of viral proteins.

Animals↗
PubMed1987

Distribution by immunofluorescence of viral products and actin-containing cytoskeletal filaments in rubella virus-infected cells.

Rubella virus (RV)-host cell interactions were examined by indirect immunofluorescence staining using antibodies to viral products and cytoskeletal components as probes. The patterns of immunofluorescence observed with human convalescent sera indicated that in infected Vero cells RV-specified proteins were distributed throughout the rough endoplasmic reticulum with some possible accumulation in the region of the Golgi complex. Viral RNA synthesis, detected with anti-double stranded RNA, appeared to be confined to small, intensely stained foci irregularly distributed in the cytoplasm. When cells were infected at a higher multiplicity, these foci appeared to aggregate into linear arrays. Infection with RV had a profound effect on the organization of actin in both Vero and BHK 21 cells, as shown by anti-actin antibodies. Actin microfilaments were observed to disintegrate progressively into amorphous aggregates of apparently monomeric actin as the infection proceeded. Because of the role actin microfilaments may play in cell mitosis it is postulated that this effect may be related to the inhibition of cell division reported to be associated with the congenital rubella syndrome.

Actin Cytoskeleton↗