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T Pan

Publications and source records attributed to T Pan.

At least 19 recordsLinked to original sources
PubMed2001

Modular construction of a tertiary RNA structure: the specificity domain of the Bacillus subtilis RNase P RNA.

The structure of the specificity domain (S-domain) of the Bacillus subtilis RNase P RNA has been proposed to be composed of a core and a buttress module, analogous to the bipartite structure of the P4-P6 domain of the Tetrahymena group I ribozyme. The core module is the functional unit of the S-domain and contains the binding site for the T stem-loop of a tRNA. The buttress module provides structural stability to the core module and consists of a GA3 tetraloop and its receptor. To explicitly test the hypothesis that modular construction can describe the structure of the S-domain and is a useful RNA design strategy, we analyzed the equilibrium folding and substrate binding of three classes of S-domain mutants. Addition or deletion of a base pair in the helical linker region between the modules only modestly destabilizes the tertiary structure. tRNA binding selectivity is affected in one but not in two other mutants of this class. Elimination of the GA3 tetraloop-receptor interactions significantly destabilizes the core module and results in the loss of tRNA binding selectivity. Replacing the buttress module with that of a homologous RNase P RNA maintains the tRNA binding selectivity. Overall, we have observed that the linker regions between the two modules can tolerate moderate structural changes and that the buttress modules can be shuffled between homologous S-domains. These results suggest that it is feasible to design an RNA using a buttress module to stabilize a functional module.

Bacillus subtilis↗
PubMed2001

Novel differences between two human prion strains revealed by two-dimensional gel electrophoresis.

The phenotype of human sporadic prion diseases is affected by patient genotype at codon 129 of the prion protein (PrP) gene, the site of a common methionine/valine polymorphism, and by the type of the scrapie PrP (PrP(Sc)), which likely reflects the prion strain. However, two distinct disease phenotypes, identified as sporadic Creutzfeldt-Jakob disease (M/M2 sCJD) and sporadic fatal insomnia (sFI), share methionine homozygosity at codon 129 and PrP(Sc) type 2. One-dimensional gel electrophoresis and immunoblotting reveal no difference between the M/M2 sCJD and sFI species of PrP(Sc) in gel mobility and glycoform ratio. In contrast, the two-dimensional immunoblot demonstrates that in M/M2 sCJD the full-length PrP(Sc) form is overrepresented and carries glycans that are different from those present in the PrP(Sc) of sFI. Because the altered glycans are detectable only in the PrP(Sc) and not in the normal or cellular PrP (PrP(C)), they are likely to result from preferential conversion to PrP(Sc) of rare PrP(C) glycoforms. This is the first evidence that a qualitative difference in glycans contributes to prion diversity.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2001

Modular construction for function of a ribonucleoprotein enzyme: the catalytic domain of Bacillus subtilis RNase P complexed with B. subtilis RNase P protein.

The bacterial RNase P holoenzyme catalyzes the formation of the mature 5'-end of tRNAs and is composed of an RNA and a protein subunit. Among the two folding domains of the RNase P RNA, the catalytic domain (C-domain) contains the active site of this ribozyme. We investigated specific binding of the Bacillus subtilis C-domain with the B.subtilis RNase P protein and examined the catalytic activity of this C-domain-P protein complex. The C-domain forms a specific complex with the P protein with a binding constant of approximately 0.1 microM. The C-domain-P protein complex and the holoenzyme are equally efficient in cleaving single-stranded RNA (approximately 0.9 min(-1) at pH 7.8) and substrates with a hairpin-loop 3' to the cleavage site (approximately 40 min(-1)). The holoenzyme reaction is much more efficient with a pre-tRNA substrate, binding at least 100-fold better and cleaving 10-500 times more efficiently. These results demonstrate that the RNase P holoenzyme is functionally constructed in three parts. The catalytic domain alone contains the active site, but has little specificity and affinity for most substrates. The specificity and affinity for the substrate is generated by either the specificity domain of RNase P RNA binding to a T stem-loop-like hairpin or RNase P protein binding to a single-stranded RNA. This modular construction may be exploited to obtain RNase P-based ribonucleoprotein complexes with altered substrate specificity.

Bacillus subtilis↗
PubMed2001

The thermodynamic origin of the stability of a thermophilic ribozyme.

Understanding the mechanism of thermodynamic stability of an RNA structure has significant implications for the function and design of RNA. We investigated the equilibrium folding of a thermophilic ribozyme and its mesophilic homologue by using hydroxyl radical protection, small-angle x-ray scattering, and circular dichroism. Both RNAs require Mg(2+) to fold to their native structures that are very similar. The stability is measured as a function of Mg(2+) and urea concentrations at different temperatures. The enhanced stability of the thermophilic ribozyme primarily is derived from a tremendous increase in the amount of structure formed in the ultimate folding transition. This increase in structure formation and cooperativity arises because the penultimate and the ultimate folding transitions in the mesophilic ribozyme become linked into a single transition in the folding of the thermophilic ribozyme. Therefore, the starting point, or reference state, for the transition to the native, functional thermophilic ribozyme is significantly less structured. The shift in the reference state, and the resulting increase in folding cooperativity, is likely due to the stabilization of selected native interactions that only form in the ultimate transition. This mechanism of using a less structured intermediate and increased cooperativity to achieve higher functional stability for tertiary RNAs is fundamentally different from that commonly proposed to explain the increased stability of thermophilic proteins.

Base Sequence↗
PubMed2001

Differential expression of cellular prion protein in mouse brain as detected with multiple anti-PrP monoclonal antibodies.

The normal cellular prion protein (PrP(C)) plays an essential role in the development of prion diseases. Indirect evidence has suggested that different PrP(C) glycoforms may be expressed in different brain regions and perform distinct functions. However, due to a lack of monoclonal antibodies (Mabs) that are specific for mouse PrP(C), the expression of PrP(C) in the mouse brain has not been studied in great detail. We used Mabs specific for either the N-terminus or the C-terminus of the mouse PrP(C) to study its expression in the mouse brain by immunoblotting and immunohistochemistry. Immunoblotting studies demonstrated that the expression of PrP(C) differed quantitatively as well as qualitatively in different regions of the brain. The anti-C-terminus Mabs reacted with all three molecular weight bands of PrP(C); the anti-N-terminus Mabs only reacted with the 39-42 kDa PrP(C). The results from immunohistochemical staining revealed the spatial distribution of PrP(C) in the mouse brain, which were consistent with that from immunoblotting. Although expression of PrP(C) has been reported to be required for long-term survival of Purkinje cells, we were unable to detect PrP(C) in the Purkinje cell layer in the cerebellum with multiple anti-PrP Mabs. Our findings suggest that PrP(C) variants, i.e. various glycoforms and truncated forms, might be specifically expressed in different regions of mouse brain and might have different functions.

Animals↗
PubMed2001

Altering the intermediate in the equilibrium folding of unmodified yeast tRNAPhe with monovalent and divalent cations.

The isothermal equilibrium folding of the unmodified yeast tRNA(Phe) is studied as a function of Na(+), Mg(2+), and urea concentration with hydroxyl radical protection, circular dichroism, and diethyl pyrocarbonate (DEPC) modification. These assays indicate that this tRNA folds in Na(+) alone. Similar to folding in Mg(2+), folding in Na(+) can be described by two transitions, unfolded-to-intermediate-to-native. The I-to-N transition has a Na(+) midpoint of approximately 0.5 M and a Hill constant of approximately 4. Unexpectedly, the urea m-value, the dependence of free energy on urea concentration, for the I-to-N transition is significantly smaller in Na(+) than in Mg(2+), 0.4 versus 1.7 kcal mol(-1) M(-1), indicating that more structure is formed in the Mg(2+)-induced transition. DEPC modification indicates that the I state in Na(+)-induced folding contains all four helices of tRNA and the I-to-N transition primarily corresponds to the formation of the tertiary structure. In contrast, the intermediate in Mg(2+)-induced folding contains only three helices, and the I-to-N transition corresponds to the formation of the acceptor stem plus tertiary structure. The cation dependence of the intermediates arises from the differences in the stability of the acceptor stem and the tertiary structure. The acceptor stem is stable at a lower Na(+) concentration than required for the tertiary structure formation. The relative stability is reversed in Mg(2+) so that the acceptor stem and the tertiary structure form simultaneously in the I-to-N transition. These results demonstrate that formation of the RNA secondary structure can be independent or coupled to the formation of the tertiary structure depending on their relative stability in monovalent and divalent ions.

Adenosine↗
PubMed2001

Normal cellular prion protein is preferentially expressed on subpopulations of murine hemopoietic cells.

We studied the expression of normal cellular prion protein (PrP(C)) in mouse lymphoid tissues with newly developed mAbs to PrP(C). Most of the mature T and B cells in the peripheral lymphoid organs do not express PrP(C). In contrast, most thymocytes are PrP(C+). In the bone marrow, erythroid cells and maturing granulocytes are PrP(C+). Approximately 50% of the cells in the region of small lymphocytes and progenitor cells also express PrP(C). Most of these PrP(C+) cells are CD43(+), but B220(-), surface IgM(-) (sIgM(-)), and IL-7R(-), a phenotype that belongs to cells not yet committed to the B cell lineage. Another small group of the PrP(C+) cell are B220(+), and some of these are also sIgM(+). The majority of the B220(+) cells, however, are PrP(C-). Therefore, PrP(C) is preferentially expressed in early bone marrow progenitor cells and subsets of maturing B cells. Supporting this interpretation is our observation that stimulation of bone marrow cells in vitro with PMA results in a decrease in the number of PrP(C+)B220(-) cells with a corresponding increase of sIgM(+)B220(high) mature B cells. This result suggests that the PrP(C+)B220(-) cells are potential progenitors. Furthermore, in the bone marrow of Rag-1(-/-) mice, there are an increased number of PrP(C+)B220(-) cells, and most of the developmentally arrested pro-B cells in these mice are PrP(C+). Collectively, these results suggest that PrP(C) is expressed preferentially in immature T cells in the thymus and early progenitor cells in the bone marrow, and the expression of PrP(C) is regulated during hemopoietic differentiation.

Animals↗
PubMed2001

Peak protein expression of IL-2 and IFN-gamma correlate with the peak rejection episode in a spontaneously tolerant model of rat liver transplantation.

Spontaneous orthotopic liver transplant (OLT) tolerance occurs uniformly between the inbred rat strains of DA (MHC haplotype RT1(a)) into PVG (RT1(c)) despite a fully allogeneic barrier. Animals transplanted in this combination do, however, undergo a rejection episode which appears to be self-limiting. In order to clarify this further we under took in situ measurements of the cytokines IL-2, IFN-gamma and TNF-alpha prior to, during and post rejection episode. The cytokine protein product was examined via immunoblotting assays and mRNA levels by RT-PCR. Comparisons were also made for syngeneic transplant combinations over the same time period. Peak protein expression of IL-2 and, to a lesser extent, IFN-gamma, occurred during the rejection episode between days 10 and 14. IFN-gamma was still present in syngeneic OLT on day 10 but was only present in allogeneic OLT on day 14. IL-2 was only detectable in allogeneic OLT on days 10 and 14. Transient increases in TNF-alpha occurred in allogeneic and syngeneic OLT with TNF-alpha levels falling by the peak rejection episode. Immunoblotting also confirmed the ability of hepatocytes to produce each of the cytokines studied. mRNA levels, by contrast, were maximal at days 1 and 10 for IL-2 and day 3 for IFN-gamma in allogeneic OLT when compared with syngeneic and non-transplanted controls. Earlier increases in IL-2 and IFN-gamma mRNA and time of peak protein expression do not correlate in this model. We therefore conclude that the measurement of peak mRNA levels alone are not enough to evaluate the rejection process especially since it is the cytokine protein products which have potential biological activity.

Animals↗
PubMed2001

Absence of protease-resistant prion protein in the cerebrospinal fluid of Creutzfeldt-Jakob disease.

Creutzfeldt-Jakob disease (CJD), believed to be caused by a protease-resistant isoform of prion protein (PrP(Sc)), usually manifests itself as a clinically distinctive age-related dementia because of its rapid progression, occasionally accompanied by cerebellar ataxia. Recently, a variant CJD (vCJD) has been described, which has prominent early psychiatric symptoms and an earlier age of death. Although cerebrospinal fluid (CSF) is part of the extracellular fluid of the central nervous system (CNS), the bulk of its proteins are derived from the plasma and there is increasing concern about possible transmission of prion disease by blood. As investigation of CSF has played a significant role in the diagnosis and management of several neurological diseases, it was decided to characterize PrP present in the CSF of CJD individuals. Significant variation was observed in the level of PrP in the CSF from both non-CJD and CJD (including vCJD) patients, and the detected PrP forms are protease-sensitive. Using a conformation-dependent immunoassay, it was further demonstrated that the PrP detected in the CSF from CJD patients was broadly similar in conformation to that found in non-CJD patients. Taken together, the results of this study fail to demonstrate any correlation between the presence of protease-resistant PrP isoform (PrP(Sc)) in the CSF and disease manifestation.

Creutzfeldt-Jakob Syndrome↗
PubMed2001

Induction of HO-1 and NOS in doppel-expressing mice devoid of PrP: implications for doppel function.

Ectopic expression of the doppel (Dpl) protein, a homologue of the prion protein (PrP), was recently associated with cerebellar Purkinje cell degeneration observed in two aging prion protein knock-out (Prnp(0/0)) mouse lines. We investigated the possible role of Dpl in oxidative metabolism. Two Prnp(0/0) mouse lines of similar genetic background were studied. One line expresses Dpl in the brain and displays Dpl-associated cerebellar abnormalities. The other has no elevated expression of Dpl and no cerebellar abnormalities. We observed a correlation between Dpl expression and the induction of both heme oxygenase 1 (HO-1) and nitric oxide synthase systems (nNOS and iNOS). These responses are suggestive of increased oxidative stress in the brains of the Dpl-expressing Prnp(0/0) mice. No induction was observed with Hsp-60, indicating a specific response by the HO/NOS system. We proposed that Dpl expression exacerbates oxidative damage that is antagonistic to the protective function of wild-type PrP.

Animals↗
PubMed2001

The Bacillus subtilis RNase P holoenzyme contains two RNase P RNA and two RNase P protein subunits.

Ribonuclease P (RNase P) catalyzes the 5' maturation of precursor tRNA transcripts and, in bacteria, is composed of a catalytic RNA and a protein. We investigated the oligomerization state and the shape of the RNA alone and the holoenzyme of Bacillus subtilis RNase P in the absence of substrate by synchrotron small-angle X-ray scattering and affinity retention. The B. subtilis RNase P RNA alone is a monomer; however, the scattering profile changes upon the addition of monovalent ions, possibly suggesting different interdomain angles. To our surprise, the X-ray scattering data combined with the affinity retention results indicate that the holoenzyme contains two RNase P RNA and two RNase P protein molecules. We propose a structural model of the holoenzyme with a symmetrical arrangement of the two RNA subunits, consistent with the X-ray scattering results. This (P RNA)2(P protein)2 complex likely binds substrate differently than the conventional (P RNA)1(P protein)1 complex; therefore, the function of the B. subtilis RNase P holoenzyme may be more diverse than previously thought. These revisions to our knowledge of the RNase P holoenzyme suggest a more versatile role for proteins in ribonucleoprotein complexes.

Avidin↗
PubMed2001

Remyelination of the adult demyelinated mouse brain by grafted oligodendrocyte progenitors and the effect of B-104 cografts.

The 4e transgenic mouse is characterized by overexpression of the PLP gene. Heterozygous littermates containing three PLP gene copies develop and myelinate normally. However, a progressive CNS demyelination begins at 3-4 months of age. Despite focal demyelination, these animals survive for one year with hind limb paralysis. We used this CNS demyelination model to determine if grafts of CG4 oligodendrocyte progenitors would survive and myelinate the adult CNS. Either CG4 cells, or co-grafts of CG4/B 104 cells 11:1 ratio respectively) were performed. Grafted cells survived and migrated in the normal and transgenic brain. Non-treated transgenic animals revealed extensive lack of myelin. Three months post-transplant hosts with CG4 or co-transplants displayed a near normal myelin pattern. Double immunofluorescence for neurofilament and myelin basic protein revealed the presence of many naked axons in non-grafted transgenic animals. Those grafted with progenitor CG4 cells or cografts displayed a clear increase in remyelination. This data provides a new direction for the development of cell replacement therapies in demyelinating diseases.

Animals↗
PubMed2001

Increased levels of oxidative stress markers detected in the brains of mice devoid of prion protein.

Although minor abnormalities have been reported in prion protein (PrP) knock-out (Prnp-/-) mice, the normal physiological function of PrP, the causative agent implicated in transmissible spongiform encephalopathies (TSE), remains unresolved. Since there are increasing correlations between oxidative stress and amyloidoses, we decided to investigate whether PrP plays a role in oxidative modulation. We found higher levels of oxidative damage to proteins and lipids in the brain lysates of Prnp-/- as compared to wild-type (WT) mice of the same genetic background. These two indicators, protein oxidation and lipid peroxidation, are hallmarks of cellular oxidative damage. Elevated levels of ubiquitin-protein conjugates were also observed in Prnp-/- mice, a probable consequence of cellular attempts to remove the damaged proteins as indicated by increased proteasome activity. Taken together, these findings are indicative of a role for PrP in oxidative homeostasis in vivo.

Aldehydes↗
PubMed2001

Aberrant metal binding by prion protein in human prion disease.

Human prion diseases are characterized by the conversion of the normal prion protein (PrP(C)) into a pathogenic isomer (PrP(Sc)). Distinct PrP(Sc) conformers are associated with different subtypes of prion diseases. PrP(C) binds copper and has antioxidation activity. Changes in metal-ion occupancy can lead to significant decline of the antioxidation activity and changes in conformation of the protein. We studied the trace element status of brains from patients with sporadic Creutzfeldt-Jakob disease (sCJD). We found a decrease of up to 50% of copper and an increase in manganese of approximately 10-fold in the brain tissues from sCJD subjects. We have also studied the metal occupancy of PrP in sCJD patients. We observed striking elevation of manganese and, to a lesser extent, of zinc accompanied by significant reduction of copper bound to purified PrP in all sCJD variants, determined by the PrP genotype and PrP(Sc) type, combined. Both zinc and manganese were undetectable in PrP(C) preparations from controls. Copper and manganese changes were pronounced in sCJD subjects homozygous for methionine at codon 129 and carrying PrP(Sc) type-1. Anti-oxidation activity of purified PrP was dramatically reduced by up to 85% in the sCJD variants, and correlated with increased in oxidative stress markers in sCJD brains. These results suggest that altered metal-ion occupancy of PrP plays a pivotal role in the pathogenesis of prion diseases. Since the metal changes differed in each sCJD variants, they may contribute to the diversity of PrP(Sc) and disease phenotype in sCJD. Finally, this study also presented two potential approaches in the diagnosis of CJD; the significant increase in brain manganese makes it potentially detectable by MRI, and the binding of manganese by PrP in sCJD might represent a novel diagnostic marker.

Antioxidants↗
PubMed2001

The basic helix-loop-helix transcription factor Cph2 regulates hyphal development in Candida albicans partly via TEC1.

Candida albicans undergoes a morphogenetic switch from budding yeast to hyphal growth form in response to a variety of stimuli and growth conditions. Multiple signaling pathways, including a Cph1-mediated mitogen-activated protein kinase pathway and an Efg1-mediated cyclic AMP/protein kinase A pathway, regulate the transition. Here we report the identification of a basic helix-loop-helix transcription factor of the Myc subfamily (Cph2) by its ability to promote pseudohyphal growth in Saccharomyces cerevisiae. Like sterol response element binding protein 1, Cph2 has a Tyr instead of a conserved Arg in the basic DNA binding region. Cph2 regulates hyphal development in C. albicans, as cph2/cph2 mutant strains show medium-specific impairment in hyphal development and in the induction of hypha-specific genes. However, many hypha-specific genes do not have potential Cph2 binding sites in their upstream regions. Interestingly, upstream sequences of all known hypha-specific genes are found to contain potential binding sites for Tec1, a regulator of hyphal development. Northern analysis shows that TEC1 transcription is highest in the medium in which cph2/cph2 displays a defect in hyphal development, and Cph2 is necessary for this transcriptional induction of TEC1. In vitro gel mobility shift experiments show that Cph2 directly binds to the two sterol regulatory element 1-like elements upstream of TEC1. Furthermore, the ectopic expression of TEC1 suppresses the defect of cph2/cph2 in hyphal development. Therefore, the function of Cph2 in hyphal transcription is mediated, in part, through Tec1. We further show that this function of Cph2 is independent of the Cph1- and Efg1-mediated pathways.

Amino Acid Sequence↗
PubMed2001

Lung fibroblasts improve differentiation of rat type II cells in primary culture.

Epithelial-mesenchymal interactions mediate prenatal lung morphogenesis and differentiation, yet little is known about their effects in the adult. In this study we have examined the influence of cocultured lung fibroblasts on rat alveolar type II cell differentiation in primary culture. Type II cells that were co-cultured with lung fibroblasts showed significant increases in messenger RNA (mRNA) levels of surfactant protein (SP)-A, SP-B, SP-C, and SP-D. Metabolic labeling and immunohistochemistry demonstrated that these mRNAs were translated and processed. Addition of 10(-7) M dexamethasone (DEX) to cocultures antagonized the effects of the fibroblasts on SP-A and SP-C, but significantly augmented the effects on SP-B; expression of SP-D was unaffected. Coculture of type II cells with lung fibroblasts also increased acetate incorporation into phospholipids 10-fold, which was antagonized by DEX. Keratinocyte growth factor (KGF) mimicked the effects of lung fibroblasts on SP gene expression, but KGF neutralizing antibodies only partially reduced the effects of lung fibroblasts. KGF increased acetate incorporation into surfactant phospholipids, and the addition of DEX augmented this response. Together, our observations suggest that epithelial--mesenchymal interactions affect type II cell differentiation in the adult lung, and that these effects are partially mediated by KGF.

Animals↗
PubMed2001

Rat alveolar type II cells inhibit lung fibroblast proliferation in vitro.

Fibroblasts stimulate alveolar type II epithelial cell differentiation and proliferation in vitro and during lung development. However, little is known about the effects of adult type II cells on fibroblasts. We investigated the effect of adult rat type II cells on proliferation of adult human lung fibroblasts. Fibroblasts were suspended within rat tail collagen which was gelled on a floating polycarbonate filter, and type II cells were cultured on Matrigel. In this coculture system, alveolar type II cells inhibited fibroblast proliferation and indomethacin blocked the inhibitory effect on fibroblast growth. Prostaglandin (PG) E2, the major PG secreted by type II cells, inhibited fibroblast proliferation and was increased during the period of inhibition of fibroblast proliferation. Incubation with arachidonate showed that most of the PGE2 in the coculture system was produced by the fibroblasts. In addition, we found that rat type II cells also inhibited rat fibroblasts and that inhibition of fibroblast growth by type II cells could be stimulated by keratinocyte growth factor. We conclude that in this coculture system, type II cells inhibit fibroblast proliferation by secreting a factor(s) that stimulates PGE2 production by fibroblasts, and that PGE2 directly inhibits fibroblast proliferation.

Adult↗