Elucidating Drug-Like Compounds and Potential Mechanisms of Corn Silk (Stigma Maydis) against Obesity: A Network Pharmacology Study

Corn silk (Stigma Maydis) has been utilized as an important herb against obesity by Chinese, Korean, and Native Americans, but its phytochemicals and mechanisms(s) against obesity have not been deciphered completely. This study aimed to identify promising bioactive constituents and mechanism of action(s) of corn silk (CS) against obesity via network pharmacology. The compounds from CS were identified using Gas Chromatography Mass Spectrometry (GC-MS) and were confirmed ultimately by Lipinski’s rule via SwissADME. The relationships of the compound-targets or obesity-related targets were confirmed by public bioinformatics. The signaling pathways related to obesity, protein-protein interaction (PPI), and signaling pathways-targets-bioactives (STB) were constructed, visualized, and analyzed by RPackage. Lastly, Molecular Docking Test (MDT) was performed to validate affinity between ligand(s) and protein(s) on key signaling pathway(s). We identified a total of 36 compounds from CS via GC-MS, all accepted by Lipinski’s rule. The number of 36 compounds linked to 154 targets, 85 among 154 targets related directly to obesity-targets (3028 targets). Of the final 85 targets, we showed that the PPI network (79 edges, 357 edges), 12 signaling pathways on a bubble chart, and STB network (67 edges, 239 edges) are considered as therapeutic components. The MDT confirmed that two key activators (β-Amyrone, β-Stigmasterol) bound most stably to PPARA, PPARD, PPARG, FABP3, FABP4, and NR1H3 on the PPAR signaling pathway, also, three key inhibitors (Neotocopherol, Xanthosine, and β-Amyrone) bound most tightly to AKT1, IL6, FGF2, and PHLPP1 on the PI3K-Akt signaling pathway. Overall, we provided promising key signaling pathways, targets, and bioactives of CS against obesity, suggesting crucial pharmacological evidence for further clinical testing.     

Structure-guided affinity maturation of a single-chain variable fragment antibody against the Fu-bc epitope of the dengue virus envelope protein

Dengue is one of the most dominant arthropod-borne viral diseases, infecting at least 390 million people every year throughout the world. Despite this, there is no effective treatment against dengue, and the only available vaccine has already been withdrawn owing to the significant adverse effects. Therefore, passive immunotherapy using monoclonal antibodies is now being sought as a therapeutic option. To date, many dengue monoclonal antibodies have been identified, most of which are serotype-specific, and only a few of which are cross-reactive. Furthermore, antibodies that cross-react within serotypes are weakly neutralizing and frequently induce antibody-dependent enhancement, which promotes viral entry and replication. Therefore, broadly neutralizing antibodies with no risk of antibody-dependent enhancement are required for the treatment of dengue. Here, we developed a single-chain variable fragment (scFv) antibody from an anti-fusion loop E53 antibody (PDB: 2IGF). We introduced previously predicted favorable complementarity-determining region (CDR) mutations into the gene encoding the scFv antibody for affinity maturation, and the resultant variants were tested in vitro against the highly conserved fusion and bc epitope of the dengue virus envelope protein. We show some of these scFv variants with two to three substitution mutations in three different CDRs possess affinity constants (K_D) ranging from 20 to 200 nM. The scFv-mutant15, containing D31L, Y105W, and S227W substitutions, showed the lowest affinity constant, (K_D = 24 ± 7 nM), approximately 100-fold lower than its parental construct. We propose that the scFv-derivative antibody may be a good candidate for the development of an effective and safe immunotherapy.     

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.     

Metabolic regulation analysis of <Emphasis Type="Italic">icd</Emphasis>-gene knockout <Emphasis Type="Italic">Escherichia coli</Emphasis> based on 2D electrophoresis with MALDI-TOF mass spectrometry and enzyme activity measurements

An integrated study of cell growth characteristics, enzyme activities and protein expression patterns was carried out to investigate how the central metabolism of Escherichia coli changes upon knockout of the isocitrate dehydrogenase (ICDH) gene (icd) in the tricarboxylic acid cycle. Deletion of the icd gene led to reduced specific growth rate and reduced specific glucose consumption rate. The reduced specific growth rate in the icd mutant was due mainly to the lower intracellular ATP/ADP ratio as well as to the lower NADPH/NADP⁺ ratio compared with those in the parent strain. However, the specific carbon dioxide evolution rate was found to be higher in the icd mutant strain compared to the parent E. coli. This may be due to the higher activity of 6-phosphogluconate dehydrogenase, phosphoenol pyruvate carboxykinase and NADP⁺-dependent malic enzymes. The glyoxylate pathway was also utilized, as evidenced by the significant upregulation of isocitrate lyase and malate synthase activity in the icd mutant E. coli. The appearance of the glyoxylate pathway caused lower acetate production. Of 21 proteins showing altered expression levels, 17 were successfully identified with the aid of MALDI-TOF mass spectrometry. The results showed that the abolition of ICDH activity significantly affected the respiratory system and electron transport chain, as evidenced by the significant downregulation of proteins encoded by the genes nuoE, nuoH, cydA and cyoA in icd mutant E. coli compared to the parent.     

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.     

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.