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Biomedical subjects

T Pan

Publications and source records attributed to T Pan.

At least 73 records · Page 4Linked to original sources
PubMed1999

Keratinocyte growth factor-induced hyperplasia of rat alveolar type II cells in vivo is resolved by differentiation into type I cells and by apoptosis.

Keratinocyte growth factor (KGF) is a potent mitogen of alveolar epithelial type II cells (AEII). AEII hyperplasia is resolved within several days following intratracheal instillation of KGF by unknown mechanism(s). AEII hyperplasia was induced in rat lungs by intrabronchial instillation of 5 mg recombinant human (rh)KGF x kg body weight(-1) or an equivalent amount of diluent. Epithelial architecture, cell proliferation, transformation of AEII into type I cells (AEI) and apoptosis were investigated by means of immunohistochemistry, stereology, double immunofluorescence microscopy, electron microscopy and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling (TUNEL) technique in lungs fixed 1, 2, 3 and 7 days after treatment. After 1 day of rhKGF instillation, an increase was observed in the nuclear antigen Ki-67, a proliferation marker detected by the antibody MIB-5-expressing surfactant protein (SP)-B, -C, -D-positive AEII. The incidence of mitosis was increased by day 2, resulting in AEII micropapillae with intense basolateral expression of the exon 6 containing isoform (v6) of CD446 (CD44v6), a marker for AEII. By day 3, monolayers of AEII exhibiting lateral CD44v6 covered 45% of the alveolar surface. After 7 days, there were numerous intermediate AEII/AEI cells characterized by a flat elongated shape, staining for SP-D, apical appearance of AEI marker Lycopersicon esculentum lectin and lateral staining for AEII marker CD44v6. Increased numbers of TUNEL-positive epithelial cells were seen at days 2-7. In conclusion, restoration of normal alveolar epithelium after instillation of recombinant human keratinocyte growth factor is accomplished by terminal differentiation and apoptosis of hyperplastic alveolar epithelial type II cells in vivo.

Animals↗
PubMed1999

Mg2+-dependent folding of a large ribozyme without kinetic traps.

The folding kinetics of the catalytic domain of Bacillus subtilis ribonuclease P is analyzed here by fluorescence and catalytic activity. The folding pathway is apparently free of kinetic traps, as indicated by a decrease in folding rates upon the addition of urea. We apply Mg2+ and urea chevron analysis to fully describe the folding and unfolding kinetics of this ribozyme. A folding scheme containing two kinetic intermediates completely accounts for the free energy, the Mg2+ Hill coefficient and the surface buried in the equilibrium transition. At saturating Mg 2+concentrations, folding is limited by a barrier that is independent of Mg2+ and urea. These results describe the first trap-free folding pathway of a large ribozyme and indicate that kinetic traps are not an obligate feature of RNA folding.

Animals↗
PubMed1999

Saccharomyces cerevisiae G1 cyclins are differentially involved in invasive and pseudohyphal growth independent of the filamentation mitogen-activated protein kinase pathway.

Several lines of evidence suggest that the morphogenetic transition from the yeast form to pseudohyphae in Saccharomyces cerevisiae may be regulated by the cyclin-dependent kinase (Cdk). To examine this hypothesis, we mutated all of the G1 cyclin genes in strains competent to form pseudohyphae. Interestingly, mutation of each G1 cyclin results in a different filamentation phenotype, varying from a significant defect in cln1/cln1 strains to enhancement of filament production in cln3/cln3 strains. cln1 cln2 double mutants are more defective in pseudohyphal development and haploid invasive growth than cln1 strains. FLO11 transcription, which correlates with the level of invasive growth, is low in cln1 cln2 mutants and high in grr1 cells (defective in proteolysis of Cln1,2), suggesting that Cln1,2/Cdks regulate the pseudohyphal transcriptional program. Epistasis analysis reveals that Cln1,2/Cdk and the filamentation MAP kinase pathway function in parallel in regulating filamentous and invasive growth. Cln1 and Cln2, but not Ste20 or Ste12, are responsible for most of the elevated FLO11 transcription in grr1 strains. Furthermore, phenotypic comparison of various filamentation mutants illustrates that cell elongation and invasion/cell-cell adhesion during filamentation are separable processes controlled by the pseudohyphal transcriptional program. Potential targets for G1 cyclin/Cdks during filamentous growth are discussed.

Cell Division↗
PubMed1998

Recognition of a pre-tRNA substrate by the Bacillus subtilis RNase P holoenzyme.

The holoenzyme of the bacterial RNase P has broader selectivity for biological substrates compared to the RNA alone (denoted P RNA) reaction. The structural basis of the substrate selectivity is investigated using a pre-tRNA substrate containing single-atom modifications by single turnover kinetics. Hydroxyl radical protection of the holoenzyme in the absence of the substrate shows that the RNase P protein binds to several regions in P RNA. The holoenzyme interacts with a subset of functional groups in the T stem-loop region of a pre-tRNA substrate previously identified to directly contact P RNA. The subtle change in structural recognition allows the holoenzyme to recognize RNA structures with only a small perturbation in an A-form helix at the corresponding position of the T stem-loop. This altered profile may permit the holoenzyme to bind non-tRNA substrates with little change in catalytic efficiency. The holoenzyme recognizes the same set of functional groups as the P RNA reaction in the region around the cleavage site and shows similar cleavage site selection compared to the P RNA reaction. These results suggest that the holoenzyme does not alter the fundamental mechanism of this enzymatic reaction. Rather, the holoenzyme significantly affects the binding affinity of an RNA substrate through additional interactions with the 5' leader [Kurz, C. A., Niranjanakumari, S., and Fierke, C. A. (1998) Biochemistry 37, 2393] and through altered recognition of the substrate structure.

Bacillus subtilis↗
PubMed1998

Interaction of structural modules in substrate binding by the ribozyme from Bacillus subtilis RNase P.

The ribozyme from bacterial ribonuclease P recognizes two structural modules in a tRNA substrate: the T stem-loop and the acceptor stem. These two modules are connected through a helical linker. The T stem-loop binds at a surface confined in a folding domain away from the active site. Substrates for the Bacillus subtilis RNase P RNA were previously selected in vitro that are shown to bind comparably well or better than a tRNA substrate. Chemical modification of P RNA-substrate complexes with dimethylsulfate and kethoxal was performed to determine how the P RNA recognizes three in vitro selected substrates. All three substrates bind at the surface known to interact with the T stem-loop of tRNA. Similar to a tRNA, the secondary structure of these substrates contains a helix around the cleavage site and a hairpin loop at the corresponding position of the T stem-loop. Unlike a tRNA, these two structural modules are connected through a non-helical linker. The two structural modules in the tRNA and in the selected substrates bind to two different domains in P RNA. The properties of substrate recognition exhibited by this ribozyme may be exploited to isolate new ribozyme-substrate pairs with interactive structural modules.

Bacillus subtilis↗
PubMed1998

Recognition of the 5' leader and the acceptor stem of a pre-tRNA substrate by the ribozyme from Bacillus subtilis RNase P.

The catalysis by the ribozyme from bacterial RNase P involves specific interactions with the structure of the tRNA substrate. Recognition of the T stem-loop by this ribozyme occurs in a groove-like structure dictated by the tertiary folding of tRNA [Loria, A., and Pan, T. (1997) Biochemistry 36, 6317]. Effects of 2'-OH --> 2'-H modifications within the acceptor stem and the 5' leader on substrate binding and catalysis are determined using a tRNAPhe substrate that is significantly cleaved at more than one site. In all but one case, the 2'-deoxy substitution has little effect on binding for cleavage at the correct and incorrect sites. Substitution of the 2'-OH group at the correct site, however, decreases the cleavage chemistry by more than 3.4 kcal/mol for cleavage at both the correct and incorrect sites. Substitutions of the 2'-OH groups at the incorrect sites have no effect for cleavage at the incorrect and correct sites. Truncation of the 5' leader results in differential effects on cleavage at different sites. These observations lead to a model in which cleavage at the correct and incorrect sites involves formation of different ribozyme-substrate complexes depending on binding of specific nucleotides in the 5' leader. Binding of the T stem-loop of tRNA and the 2'-OH group at the correct cleavage site is common for all ES complexes. An A/U-rich 5' leader significantly promotes formation of the ES complex and accelerates the cleavage chemistry over those of a C/G-rich 5' leader, but only moderately enhances cleavage at the correct site over cleavage at the incorrect sites. Since cleavage at different sites requires formation of different ES complexes, cleavage site selection can occur at the level of the ES complex and at the chemical step.

Bacillus subtilis↗
PubMed1998

A toxin homology domain in an astacin-like metalloproteinase of the jellyfish Podocoryne carnea with a dual role in digestion and development.

Metalloproteinases of the astacin family such as tolloid play major roles in animal morphogenesis. Cnidarians are thought to be evolutionary simple organisms and, therefore, a metalloproteinase from the marine hydrozoan Podocoryne carnea was analysed to evaluate the role of this conserved gene familiy at the base of animal evolution. Surprisingly, the proteinase domain of Podocornyne PMP1 is more similar to human meprin than to HMP1 from another hydrozoan, the freshwater polyp Hydra vulgaris. However, PMP1 and HMP1 both contain a small C-terminal domain with six cysteines that distinguishes them from other astacin-like molecules. Similar domains have been described only recently from sea anemone toxins specific for potassium channels. This toxin homology (Tox1) domain is clearly distinct from epidermal growth factor (EGF)-like domains or other cysteine-rich modules and terminates with the characteristic pattern CXXXCXXC with three out of six cysteines in the last eight residues of the protein. PMP1 is transiently expressed at various sites of morphogenetic activity during medusa bud development. In the adult medusa, however, expression is concentrated to the manubrium, the feeding organ, where the PMP1 gene is highly induced upon feeding. These disparate expression patterns suggest a dual role of PMP1 comparable to tolloid in development and, like astacin in the crayfish, also for food digestion. The Tox1 domain of PMP1 could serve as a toxin to keep the pray paralysed after ingestion, but as a sequence module such Tox1 domains with six cysteines are neither restricted to cnidarians nor to toxins.

Amino Acid Sequence↗
PubMed1998

Influence of the cytoskeleton on surfactant protein gene expression in cultured rat alveolar type II cells.

We have investigated the role of the cytoskeleton in surfactant protein gene expression. Cytochalasin D (CD), colchicine (Col), or nocodazole (Noco) were tested on primary cultures of adult rat alveolar type II cells. Treatment with any of the drugs did not result in dramatic cell shape changes, but ultrastructural examination revealed that the cytoplasm of cells treated with CD was markedly disorganized; cells treated with Col did not exhibit such changes. Treatment with any of the three drugs resulted in a reduction in surfactant protein (SP) mRNAs. These decreases were not the result of cell toxicity, since overall protein synthesis was unimpaired by drug treatment. Washing the cells followed by an additional 2 days of culture resulted in a reaccumulation of SP mRNAs in CD-treated cells but not in Col-treated cells. Washing of Noco-treated cultures resulted in partial recovery. SP mRNA stability was estimated in the presence or absence of cytoskeleton-disrupting drugs. Disruption of either microfilaments or microtubules significantly affected the half-lives of mRNAs for SP-A, SP-B, and SP-C. These data support a role for the cytoskeleton in the maintenance of type II cell differentiation and suggest that the role of the cytoskeleton is at least in part to stabilize SP mRNAs.

Animals↗
PubMed1998

[Spatial tuning curve recording in inferior colliculus during electrical stimulation of the cochlea].

OBJECTIVE: To explore the electrical activity mechanism of auditory center to intracochlear electrical stimulation and provide physiological foundation for spatial encoding of electrical stimulation in multiple channel cochlear implant. METHODS: The spatial tuning curves(STC) in the inferior colliculus(IC) to monopolar electrical stimulation, bipolar electrical stimulation and tone stimulation in cats were composed by using the single unit and the multi-units cluster recording method. RESULTS: The bipolar electrical stimulation excited the response in the specific region in IC, which was similar to the response induced by tone stimulation. The monopolar electrical stimulation excited an extensive response and could not provide the spatial encoding. CONCLUSION: Using the bipolar electrical stimulation, the stimulation region is the key point for improving the spatial encoding. The physiological results of spatial encoding of monopolar electrical stimulation contradicted the psychophysical conclusion and the clinical effect of CI patients. The explanation to this needs further studies.

Animals↗
PubMed1997

Transplantation of CG4 oligodendrocyte progenitor cells in the myelin-deficient rat brain results in myelination of axons and enhanced oligodendroglial markers.

Transplantation of oligodendrocyte (Ol) progenitor cells into the central nervous system is a promising approach for the treatment of myelin disorders. This approach requires providing adequate numbers of healthy cells with myelinating potential. We recently showed the successful transplantation of Ol progenitors into the myelin-deficient (md) rat brain. In the present work, CG4 cells, a cell line with properties of Ol progenitors, were labeled with fast blue and grafted into P3-P5 pups born to carrier mothers. Examination of host brains 2 weeks posttransplant indicated that CG4 cells display a much more extensive migration capacity than their wild-type counterparts. These cells synthesized myelin components. In addition, ultrastructural analysis showed myelin formation along axons of md hosts in various brain regions, including corpus callosum, cerebellum, and brainstem. Furthermore, in situ hybridization studies performed on sagittal sections revealed extensive expression of transferrin-mRNA within the md host parenchyma. The high survival and functional features displayed by CG4 cells after transplantation, together with their striking wide distribution within the host parenchyma, as assessed by the presence of myelinated fibers in mutant hosts, emphasizes the importance of using highly motile and proliferative Ol progenitor cells. Strategies to improve the condition and life span of md rat pups are currently under investigation.

Animals↗
PubMed1997

Recognition of the T stem-loop of a pre-tRNA substrate by the ribozyme from Bacillus subtilis ribonuclease P.

The ribozyme from bacterial ribonuclease P (denoted P RNA) specifically recognizes the coaxially stacked T stem-loop and the acceptor stem of a tRNA substrate. This recognition is mediated primarily through tertiary interactions. At least four 2'-OH groups in the T stem-loop region have been implicated as direct contacts with Bacillus subtilis P RNA [Pan, T., et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 12510]. Effects of six single 2'-OH --> 2'-H substitutions and two base mutants of the G19-C56 tertiary interaction in tRNA on substrate binding (Kd) and the chemical step of the reaction (k2) have been determined using a tRNA(Phe) substrate containing a 2'-deoxy residue at the cleavage site. Our results show that at least five functional groups in the T stem-loop of tRNA directly participate in P RNA binding. They include the 2'-OH groups of residues 54, 56, 61, and 62 and possibly the 4-amino group of the conserved C56. The 2'-OHs of residues 54, 61, and 62 are positioned within the same minor groove due to stacking of the reverse Hoogsteen U54-A58 pair on the G53-C61 Watson-Crick pair in the T stem. This groove is extended to the 4-amino group of C56 through the tertiary structure of tRNA. We use the term "tertiary groove" to describe alignment of functional groups through tertiary folding of an RNA. The binding also includes the 2'-OH of nucleotide C56 which is not located in this tertiary groove. Assuming additivity, these five interactions can contribute 7.4 kcal/mol or 10(5)-fold in binding but only -0.5 kcal/mol or approximately 2-fold in chemistry at 37 degrees C. The P RNA binding site for the T stem-loop includes at least the previously identified A230 as well as the A130 in B. subtilis P RNA. The Kd and k2 data from the A130G mutant of B. subtilis P RNA suggest that A130 may be proximal to residue 56 in tRNA. These results show how the highly structured T stem-loop region in a pre-tRNA substrate is bound by the B. subtilis P RNA. This is among the first examples of how a nonhelical RNA structure can be recognized by another RNA through tertiary interactions.

Bacillus subtilis↗
PubMed1997

An experimental comparative study on the characteristics of ventricular fibrillation during cardiac arrest and methoxamine administration.

The effect of a pure alpha-adrenergic agent, methoxamine on ventricular fibrillation (VF) amplitude and the relation between hemodynamic parameters and survival in a rodent cardiopulmonary resuscitation (CPR) model were studied. Our results suggested that: 1) VF amplitude decreased during untreated VF, but it increased during pericardial chest compression: 2) methoxamine significantly increased the mean aortic pressure (MAP) and coronary perfusion pressure (CPP) but not VF amplitude, and the survival also increased due to elevation of CPP; and 3) all surviving animals with successful defibrillation had a higher VF amplitude.

Adrenergic alpha-Agonists↗
PubMed1997

Effect of different drugs on end-tidal carbon dioxide during rodent CPR.

The purpose of this study was to investigate the effect of non-adrenergic agents on cardiopulmonary resuscitation (CPR) and end-tidal CO2 (ETCO2) during CPR in a rodent model. Our results suggested that: 1) coronary perfusion pressure (CPP) after drugs infusion was increased significantly by methoxamine, arginine vasopresin (AVP) and angiotension-II (ANG-II), but not by endothelin-1 (ET-1); 2) ETCO2 prior to defibrillation was decreased significantly by a pure alpha 1 adrenergic agents, methoxamine and were increased significantly by non-adrenergic agents, ANG-II and ET-1 during rodent CPR; 3) a significant positive correlation between ETCO2 and CPP was observed in AVP group, suggesting that AVP have little effect on pulmonary circulation; and 4) methoxamine, AVP and ANG-II have similar effect on resuscitability during rodent CPR.

Adrenergic alpha-Agonists↗
PubMed1997

Novel and variant ribozymes obtained through in vitro selection.

Ribozymes are RNA molecules capable of catalyzing chemical reactions. Natural ribozymes generally accelerate the rate of cleavage and ligation of specific phosphodiester bonds. In vitro selection of RNA is now being used as a powerful technique to isolate novel and variant ribozymes that carry out catalysis at phosphodiester and carbon bonds. The range of reactions catalyzed by in vitro selected ribozymes is now well beyond the scope of known natural ribozymes.

Carbon↗
PubMed1997

Intermediates and kinetic traps in the folding of a large ribozyme revealed by circular dichroism and UV absorbance spectroscopies and catalytic activity.

The folding thermodynamics and kinetics for the ribozyme from Bacillus subtilis RNase P are analyzed using circular dichroism and UV absorbance spectroscopies and catalytic activity. At 37 degrees C, the addition of Mg2+ (Kd approximately 50 microM) to the unfolded state produces an intermediate state within 1 ms which contains a comparable amount of secondary structure as the native ribozyme. The subsequent transition to the native state (Kd[Mg] approximately 0.8 mM, Hill coefficient approximately 3.5) has a half-life of hundreds of seconds as measured by circular dichroism at 278 nm and by a ribozyme activity assay. Surprisingly, the formation of the native structure is accelerated strongly by the addition of a denaturant; approximately 30-fold at 4.5 M urea. Thus, the rate-limiting step entails the disruption of a considerable number of interactions. The folding of this, and presumably other large RNAs, is slow due to the structural rearrangement of kinetically trapped species. Taken together with previous submillisecond relaxation kinetics of tRNA tertiary structure, we suggest that error-free RNA folding can be on the order of milliseconds.

Bacillus subtilis↗
PubMed1996

Multiple substrate binding sites in the ribozyme from Bacillus subtilis RNase P.

The ribozyme from Bacillus subtilis RNase P (P RNA) recognizes an RNA structure consisting of the acceptor stem and the T stem-loop of tRNA substrates. An in vitro selection experiment was carried out to obtain potential RNA substrates that may interact with the P RNA differently from the tRNA substrate. Using a P RNA-derived ribozyme that contains most, if not all, of the structural elements thought to be involved in active site formation of P RNA, but lacks the putative binding site for the T stem-loop of tRNA, a single RNA substrate was isolated after nine rounds of selection. This RNA is a competent substrate for the ribozyme used in selection as well as for the full-length P RNA. Biochemical characterization shows that this selected substrate interacts at a different site compared with the tRNA substrate. The selection experiment also identified a self-cleaving RNA seemingly different from other known ribozymes. These results indicate that a biological ribozyme can contain different binding sites for different RNA substrates. This alternate binding site model suggests a simple mechanism for evolving existing ribozymes to recognize RNA substrates of diverse structures.

Bacillus subtilis↗