Escherichia coli adenylate kinase dynamics: comparison of elastic network model modes with mode-coupling (15)N-NMR relaxation data

The dynamics of adenylate kinase of Escherichia coli (AKeco) and its complex with the inhibitor AP(5)A, are characterized by correlating the theoretical results obtained with the Gaussian Network Model (GNM) and the anisotropic network model (ANM) with the order parameters and correlation times obtained with Slowly Relaxing Local Structure (SRLS) analysis of (15)N-NMR relaxation data. The AMPbd and LID domains of AKeco execute in solution large amplitude motions associated with the catalytic reaction Mg(+2)*ATP + AMP --> Mg(+2)*ADP + ADP. Two sets of correlation times and order parameters were determined by NMR/SRLS for AKeco, attributed to slow (nanoseconds) motions with correlation time tau( perpendicular) and low order parameters, and fast (picoseconds) motions with correlation time tau( parallel) and high order parameters. The structural connotation of these patterns is examined herein by subjecting AKeco and AKeco*AP(5)A to GNM analysis, which yields the dynamic spectrum in terms of slow and fast modes. The low/high NMR order parameters correlate with the slow/fast modes of the backbone elucidated with GNM. Likewise, tau( parallel) and tau( perpendicular) are associated with fast and slow GNM modes, respectively. Catalysis-related domain motion of AMPbd and LID in AKeco, occurring per NMR with correlation time tau( perpendicular), is associated with the first and second collective slow (global) GNM modes. The ANM-predicted deformations of the unliganded enzyme conform to the functional reconfiguration induced by ligand-binding, indicating the structural disposition (or potential) of the enzyme to bind its substrates. It is shown that NMR/SRLS and GNM/ANM analyses can be advantageously synthesized to provide insights into the molecular mechanisms that control biological function.     

Temporal and spatial variation of forest biomass in relation to stand dynamics in a mature,lowland tropical rainforest,Malaysia

To clarify consistency in the size of carbon pool of a lowland tropical rainforest, we calculated changes in above-ground biomass in the Pasoh Forest Reserve, Peninsular Malaysia. We estimated the total above-ground biomass of a mature stand using tree census data obtained in a 6-ha plot every 2years from 1994 to 1998. The total above-ground biomass decreased consistently from 1994 (431Mgha^–1) to 1998 (403Mgha^–1) (1Mg=10³ kg). These are much lower than that in 1973 for a 0.2ha portion of the same area, suggesting that the the total above-ground biomass reduction might have been consistent in recent decades. This trend contrasted with a major trend for neotropical forests. During 1994–1998, the forest gained 23.0 and 0.88Mgha^–1 of the total above-ground biomass by tree growth and recruitment, respectively, and lost 51.9Mgha^–1 by mortality. Overall, the biomass decreased by 28.4Mgha^–1 (i.e. 7.10Mgha^–1·year^–1), which is almost equivalent to losing a 76-cm-diameter living tree per hectare per year. Analysis of positive and negative components of biomass change revealed that deaths of large trees dominated the total above-ground biomass decrease. The forest biomass also varied spatially, with the total above-ground biomass density ranging 212–655Mgha^–1 on a 0.2-ha basis (n= 30 subplots, 1998) and 365–440Mgha^–1 on a 1ha basis. A large decrease of the total above-ground biomass density (>50Mg per ha per 2years) in several 0.2-ha subplots contributed to the overall decrease in the 6-ha total above-ground biomass. In the present study, we discuss the association between forest dynamics and biomass fluctuation, and the implication for carbon cycling in mature forests with emphasis on forest monitoring and assessments of soil and decomposition systems.     

Requirement of a relatively high threshold level of Mg(2+) for cell growth of a rhizoplane bacterium, Sphingomonas yanoikuyae EC-S001

Mg(2+) is one of the essential elements for bacterial cell growth. The presence of the magnesium cation (Mg(2+)) in various concentrations often affects cell growth restoration in plant-associating bacteria. This study attempted to determine whether Mg(2+) levels in Sphingomonas yanoikuyae EC-S001 affected cell growth restoration in the host plant and what the threshold level is. S. yanoikuyae EC-S001, isolated from the rhizoplane of spinach seedlings grown from surface-sterilized seeds under aseptic conditions, displayed uniform dispersion and attachment throughout the rhizoplane and phylloplane of the host seedlings. S. yanoikuyae EC-S001 did not grow in potato-dextrose broth medium but grew well in an aqueous extract of spinach leaves. Chemical investigation of the growth factor in the spinach leaf extract led to identification of the active principle as the magnesium cation. A concentration of ca. 0.10 mM Mg(2+) or more allowed S. yanoikuyae EC-S001 to grow in potato-dextrose broth medium. Some saprophytic and/or diazotrophic bacteria used in our experiment were found to have diverse threshold levels for their Mg(2+) requirements. For example, Burkholderia cepacia EC-K014, originally isolated from the rhizoplane of a Melastoma sp., could grow even in Mg(2+)-free Hoagland's no. 2 medium with saccharose and glutamine (HSG medium) and requires a trace level of Mg(2+) for its growth. In contrast, S. yanoikuyae EC-S001, together with Bacillus subtilis IFO12113, showed the most drastic restoring responses to subsequent addition of 0.98 mM Mg(2+) to Mg(2+)-free HSG medium. Our studies concluded that Mg(2+) is more than just the essential trace element needed for cell growth restoration in S. yanoikuyae EC-S001 and that certain nonculturable bacteria may require a higher concentration of Mg(2+) or another specific essential element for their growth.     

High-efficiency bypass of DNA damage by human DNA polymerase Q

Endogenous DNA damage arises frequently, particularly apurinic (AP) sites. These must be dealt with by cells in order to avoid genotoxic effects. DNA polymerase theta; is a newly identified enzyme encoded by the human POLQ gene. We find that POLQ has an exceptional ability to bypass an AP site, inserting A with 22% of the efficiency of a normal template, and continuing extension as avidly as with a normally paired base. POLQ preferentially incorporates A opposite an AP site and strongly disfavors C. On nondamaged templates, POLQ makes frequent errors, incorporating G or T opposite T about 1% of the time. This very low fidelity distinguishes POLQ from other A-family polymerases. POLQ has three sequence insertions between conserved motifs in its catalytic site. One insert of approximately 22 residues into the tip of the polymerase thumb subdomain is predicted to confer considerable flexibility and additional DNA contacts to affect enzyme fidelity. POLQ is the only known enzyme that efficiently carries out both the insertion and extension steps for bypass of AP sites, commonly formed as endogenous genomic lesions.     

Interesting electrophysiological findings in a patient with coincidental right ventricular outflow tract and atrioventricular nodal reentrant tachycardia

Tachycardia induced tachycardias are not common in clinical practice, and it is believed that most cases of double tachycardia are coincidental. The existence of two different tachycardias in the same patient almost always poses problems in the electrophysiology laboratory. However, in rare instances, the emergence of a second tachycardia can actually provide invaluable information about the first one. In this report, we describe a 30-year-old woman who presented with palpitations. Electrophysiological study revealed that atrial programmed stimulation at baseline induced right ventricular outflow tract (RVOT) tachycardia and supraventricular tachycardia. The study also showed that each of the tachycardias was able to induce the other. A short run of RVOT tachycardia during supraventricular tachycardia was able to entrain the latter. This finding provided important information about the nature of the supraventricular tachycardia, which proved to be atrioventricular nodal reentrant tachycardia. Both of these tachycardias were successfully ablated, and the patient's palpitations disappeared.     

Mechanism of action of low recurrence of gastritis caused by Helicobacter pylori with the type II urease B gene

Background. Low recurrence of gastritis is seen in patients infected with Helicobacter pylori carrying the type II urease B gene, compared with H. pylori carrying types I and III. The underlying mechanism has been studied in terms of the urease activity and interleukin (IL)‐8 production capacity of different strains of H. pylori. Materials and Methods. Forty‐five patients infected with different strains of H. pylori (type I; 15, type II; 15 and type III; 15) were enrolled in the study. H. pylori was isolated from gastric mucosa and cultured in the presence of urea at pH 5.5 to evaluate urease activity. The capacity of different strains of H. pylori to induce IL‐8 mRNA and IL‐8 from a human gastric cancer cell line and human peripheral blood mononuclear cells was evaluated. Results. The urease activity of type II H. pylori[523 ± 228 µg of ammonia/dl/10⁸ colony‐forming units (CFU)/ml] was significantly lower than that of type I (1355 ± 1369 µg of ammonia/dl/10⁸ CFU/ml) and type III (1442 ± 2229 µg of ammonia/dl/10⁸ CFU/ml) (p < .05). Gastric cancer cells cocultured with type II H. pylori produced lower levels of IL‐8 mRNA compared with type I and type III H. pylori. The levels of IL‐8 were also significantly lower in cultures induced by type II H. pylori compared with those induced by type I and type III H. pylori. Peripheral blood mononuclear cells also produced lower levels of IL‐8 when cocultured with type II compared with type I H. pylori. Conclusions. These results indicate that both the lower level of urease activity and the low IL‐8‐inducing capacity of type II H. pylori might underlie the lower recurrence rate of gastritis caused by type II H. pylori.     

Fluorescence resonance energy transfer analysis of cytochromes P450 2C2 and 2E1 molecular interactions in living cells

The molecular organization of microsomal cytochromes P450 (P450s) and formation of complexes with P450 reductase have been studied previously with isolated proteins and in reconstituted systems. Although these studies demonstrated that some P450s oligomerize in vitro, neither oligomerization nor interactions of P450 with P450 reductase have been studied in living cells. Here we have used fluorescence resonance energy transfer (FRET) to study P450 oligomerization and binding to P450 reductase in live transfected cells. Cytochrome P450 2C2, but not P450 2E1, forms homo-oligomeric structures, and this self-association is mediated by the signal-anchor sequence. Because P450 2C2, in contrast to P450 2E1, is directly retained in the endoplasmic reticulum (ER), these results could suggest that oligomerization may prevent transport from the ER. However, P450 2C1 signal-anchor sequence chimera defective in ER retention also formed oligomers, and chimera containing the cytoplasmic domain of P450 2C2, which is directly retained in the ER, did not exhibit self-oligomerization, which indicates that oligomerization is not correlated with direct retention. By using FRET, we have also detected binding of P450 2C2 and P450 2E1 to P450 reductase. In contrast to self-oligomerization, the catalytic domain can mediate an interaction of P450 2C2 with P450 reductase. These results suggest that microsomal P450s may differ in their quaternary structure but that these differences do not detectably affect interaction with the reductase or transport from the ER.     

Streptococcus pneumoniae hyaluronate lyase contains two non-cooperative independent folding/unfolding structural domains: characterization of functional domain and inhibitors of enzyme

Hyaluronate lyase contributes directly to bacterial invasion by degrading hyaluronan, the major component of host extracellular matrix of connective tissues. Streptococcus pneumoniae hyaluronate lyase (SpnHL) is built from two structural domains that interact through interface residues, in addition to being connected by a peptide linker. For the first time we demonstrate that the N- and C-terminal domains of SpnHL fold/unfold independent of each other suggesting the absence of any significant cooperative interactions between them. The C-terminal domain of SpnHL is less stable than the N-terminal domain against thermal and guanidine hydrochloride denaturation. The intact N-terminal domain was purified after limited proteolysis of SpnHL under conditions where only the C-terminal domain was unfolded. Isolated N-terminal domain of SpnHL had similar thermal stability as when present in the native enzyme and was found to be enzymatically active demonstrating that it is capable of carrying out enzymatic reaction on its own. Functional studies demonstrated that guanidine hydrochloride, guanidine isothiocyanate, l-arginine methyl ester, and l-arginine inhibit the enzymatic activity of SpnHL at very low concentrations. This provides a lead for new chemical entities that can be exploited for designing effective inhibitors of SpnHL.     

Hsp70 family member, mot-2/mthsp70/GRP75, binds to the cytoplasmic sequestration domain of the p53 protein

Hsp70 family member mot-2/mthsp70/GRP75/PBP74 was shown to bind to the tumor suppressor protein p53. In this study, by in vivo coimmunoprecipitation of mot-2 with p53 and its deletion mutants, the mot-2 binding site of p53 was mapped to its C-terminal amino acid residues 312-352, a region of p53 that includes its cytoplasmic sequestration domain. These data demonstrate that cytoplasmic sequestration and inactivation of p53 by mot-2 occurs by its binding to the cytoplasmic sequestration domain. Therefore, perturbation of mot-p53 interactions can be employed to abrogate cytoplasmic retention of wild-type p53 in tumors.     

Cohesin defects lead to premature sister chromatid separation,kinetochore dysfunction,and spindle-assembly checkpoint activation

Scc1/Mcd1 is a component of the cohesin complex that plays an essential role in sister chromatid cohesion in eukaryote cells. Knockout experiments of this gene have been described in budding yeast, fission yeast, and chicken cells, but no study has been reported on human Scc1 thus far. In this study, we found that an N-terminally truncated human Scc1 shows a dominant-negative effect, and we examined the phenotypes of human cells defective in Scc1 function. Scc1 defects led to failure of sister chromatid cohesion in both interphase and mitotic cells. Interestingly, four chromatids derived from two homologues occupied four distinct territories in the nucleus in chromosome painting experiments. In mitotic Scc1-defective cells, chromatids were disjoined with normal condensation, and the spindle-assembly checkpoint was activated. We also found that, although the disjoined kinetochore (half-kinetochore) in Scc1-defective cells contains CENP-A, -B, -C, and -E normally, it apparently does not establish the kinetochore-microtubule association. These results indicate that Scc1 is essential for the association of kinetochores with microtubules.