Epistasis effects of COMT and MTHFR on inter-individual differences in mental health: Under the inverted U-shaped prefrontal dopamine model

Higher cognitive performance, maintenance of mental health and psychological well-being require adequate prefrontal cortex (PFC) function. “Inverted U-shaped” dopamine model indicates optimal PFC dopamine level is important to attain its function while high or low levels have adverse effects. Catechol-O-methyltransferase (COMT) and methylenetetrahydrofolate reductase (MTHFR) may be involved in this complex non-linear PFC dopamine regulation. We addressed whether genetic variation reflecting COMT and MTHFR activities can explain the inter-individual mental health differences in healthy Japanese men (n = 188). The mental health was measured by Mental Health Inventory (MHI)-5 score. The rs4633–rs4818–rs4680 haplotypes were used to represent the multilevel COMT activities, while for MTHFR, the functional single polymorphism, rs1801133 (C677T), was used. We examined the effectiveness of haplotype-based association analysis of COMT on mental health together with studying its interaction with MTHFR-C677T. As a result, the relation between activity-ranked COMT genotype and MHI-5 score showed a tendency to fit into an “inverted U-shaped” quadratic curve (P = 0.054). This curvilinear correlation was significant in the subjects with MTHFR-CC (P < 0.001), but not with MTHFR T-allele carriers (P = 0.793). Our pilot study implies a potential influence of COMT and MTHFR genotypic combination on normal variation of mental health.     

High-throughput antibody generation using multiplexed immunization and immunogen array analysis

In this work, the authors developed a new screening approach using multiplexed immunization and immunogen array analysis to improve the efficiency of antibody screening for high-throughput antibody generation. The immunogen array is based on a 96-well format in which different immunogens and negative as well as positive controls are immobilized in each well, thus making it possible to screen hundreds of antibody candidates simultaneously. To demonstrate this approach, a model of 4 mixed immunogens immunization was employed. In total, 675 antibody candidates were screened before and after established antibody hybridomas in parallel with immunogen arrays and enzyme-linked immunosorbent assay. The signal intensity, specificity, and cross-reactivity of produced antibody candidates were analyzed using a hierarchical cluster algorithm to track the characteristics of antibody candidates during antibody generation, which might reduce the number of false-positive and false-negative binding of antibodies. Moreover, 4 monoclonal antibodies that were produced successfully recognized their corresponding target antigens.     

Metformin-like effects of Quei Fu Di Huang Wan, a Chinese herbal mixture, on streptozotocin-induced diabetic rat.

Effect on plasma glucose concentration of Quei Fu Di Huang Wan (Quei Fu DHW), the herbal mixture widely used to treat diabetic disorder in Chinese traditional medicine, was investigated in diabetic rats deficient in insulin. Changes of plasma glucose in streptozotocin-induced diabetic rats (STZ-diabetic rats) receiving repeated oral administration of Quei Fu DHW were determined. Also, the mRNA level (by Northern blotting) and protein level (by Western blotting) of phosphoenolpyruvate carboxykinase (PEPCK) in liver from STZ-diabetic rats were measured to compare differences between groups receiving repeated oral administration of Quei Fu DHW, metformin, and two active herbs (Zou Guei or Fuzei) at effective dosages. In STZ-diabetic rats, acute oral administration of Quei Fu DHW decreased the plasma glucose level significantly in a dose-dependent manner from 5 mg/kg to 26.0 mg/kg. Similar treatment with Quei Fu DHW also brought on a plasma glucose-lowering effect in normal rats, although the effectiveness was not as significant as in STZ-diabetic rats. Repeated oral treatment of Quei Fu DHW at 26 mg/kg every 8 h, three times daily for 3 days, produced a plasma glucose-lowering activity similar to that of metformin-treatment in STZ-diabetic rats. Oral administration of Zou Guei (Cinnamomi Cortex) or Fuzei (Aconiti Tuber), the individual constituent of Quei Fu DHW, at the dose of 50 mg/kg into STZ-diabetic rats for 3 days normalized hyperglycemia. Similar to the repeated treatment with Quei Fu DHW, Fuzei at the effective dose reversed the elevated mRNA and protein levels of PEPCK in liver from STZ-diabetic rats. This is consistent with findings that metformin restored the increased gene expression of PEPCK in liver from STZ-diabetic rats. However, the gene expression of PEPCK in STZ-diabetic rats was not influenced by similar treatment with Zou Guei. The present study found that oral administration of Quei Fu DHW could decrease hepatic gluconeogenesis in a way similar to metformin in lowering plasma glucose in diabetic rats lacking insulin. Thus, this preparation may be a helpful adjuvant for the treatment of diabetic disorders in clinical practice.     

Hyphantria cunea ferritin heavy chain homologue: cDNA sequence and mRNA expression

We have sequenced a cDNA clone encoding a 26-kDa ferritin subunit, which was heavy chain homologue (HCH), in fall webworm, Hyphantria cunea. The HCH cDNA was obtained from the screening of a cDNA library using a PCR product. H. cunea ferritin is composed of 221 amino acid residues and their calculated mass is 26,160 Da. The protein contains the conserved motifs for the ferroxidase center typical for heavy chains of vertebrate ferritin. The iron-responsive element sequence with a predicted stem-loop structure is present in the 5'-untranslated region of ferritin HCH mRNA. The sequence alignment of ferritin HCH shows 68.9 and 68.7% identity with Galleria mellonella HCH (26 kDa ferritin) and Manduca sexta HCH, respectively. While G type insect ferritin vertebrate light chain homologue (LCH) is distantly related to H. cunea ferritin HCH (17.2-20.8%), the Northern blot analysis revealed that H. cunea ferritin HCH was ubiquitously expressed in various tissues and all developmental stages. The ferritin expression of midgut is more responsive to iron-fed, compared to fat body in H. cunea.     

Tumor necrosis factor-alpha stimulates gastric epithelial cell proliferation

TNF-alpha is a cytokine produced during gastric mucosal injury. We examined whether TNF-alpha could promote mucosal repair by stimulation of epithelial cell proliferation and explored further the underlying mechanisms in a rat gastric mucosal epithelial cell line (RGM-1). TNF-alpha treatment (1-10 ng/ml) for 12 or 24 h significantly increased cell proliferation but did not induce apoptosis in RGM-1 cells. TNF-alpha treatment significantly increased cytosolic phospholipase A(2) and cyclooxygenase-2 (COX-2) protein expression and PGE(2) level but did not affect the protein levels of EGF, basic fibroblast growth factor, and COX-1 in RGM-1 cells. The mRNA of TNF receptor (TNF-R) 2 but not of TNF-R1 was also increased. Dexamethasone dose dependently inhibited the stimulatory effect of TNF-alpha on cell proliferation, which was associated with a significant decrease in cellular COX-2 expression and PGE(2) level. A selective COX-2 inhibitor 3-(3-fluorophenyl)-4-[4-(methylsulfonyl)phenyl]-5,5-dimethyl-(5)H-furan-2-one (DFU) by itself had no effect on basal cell proliferation but significantly reduced the stimulatory effect of TNF-alpha on RMG-1 cells. Combination of dexamethasone and DFU did not produce an additive effect. PGE(2) significantly reversed the depressive action of dexamethasone on cell proliferation. These results suggest that TNF-alpha plays a regulatory role in epithelial cell repair in the gastric mucosa via the TNF-alpha receptor and activation of the arachidonic acid/PG pathway.     

Photosynthetic characteristics and tolerance to photo-oxidation of transgenic rice expressing C4 photosynthesis enzymes

The photosynthetic characteristics of four transgenic rice lines over-expressing rice NADP-malic enzyme (ME), and maize phosphoenolpyruvate carboxylase (PC), pyruvate,orthophosphate dikinase (PK), and PC+PK (CK) were investigated using outdoor-grown plants. Relative to untransformed wild-type (WT) rice, PC transgenic rice exhibited high PC activity (25-fold increase) and enhanced activity of carbonic anhydrase (more than two-fold increase), while the activity of ribulose-bisphosphate carboxylase/oxygenase (Rubisco) and its kinetic property were not significantly altered. The PC transgenic plants also showed a higher light intensity for saturation of photosynthesis, higher photosynthetic CO₂ uptake rate and carboxylation efficiency, and slightly reduced CO₂ compensation point. In addition, chlorophyll a fluorescence analysis indicates that PC transgenic plants are more tolerant to photo-oxidative stress, due to a higher capacity to quench excess light energy via photochemical and non-photochemical means. Furthermore, PC and CK transgenic rice produced 22–24% more grains than WT plants. Taken together, these results suggest that expression of maize C₄ photosynthesis enzymes in rice, a C₃ plant, can improve its photosynthetic capacity with enhanced tolerance to photo-oxidation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Computational studies on polynitropurines as potential high energy density materials

As part of a search for high energy density materials (HEDMs), a series of purine derivatives with nitro groups were designed computationally. The relationship between the structures and the performances of these polynitropurines was studied. Density functional theory (DFT) at the B3LYP/6-311G** level was employed to evaluate the heats of formation (HOFs) of the polynitropurines by designing an isodesmic reaction method. Results indicated that the HOFs were influenced by the number and positions of substituent groups. Detonation properties were evaluated using the Kamlet–Jacobs equations, based on the theoretical densities and heats of formation of the polynitropurines. The relative stabilities of the polynitropurines were studied via the pyrolysis mechanism and the UB3LYP/6-311G** method. Homolysis of the ring–NO₂ bond is predicted to be the initial step in the thermal decomposition of these purine derivatives. Considering their detonation properties and relative stabilities, the tetranitropurine (D1) derivatives may be regarded as potential candidates for practical HEDCs. These results may provide useful information for further investigations.     

High glucose concentration induces endothelial cell proliferation by regulating cyclin‐D2‐related miR‐98

Cyclin D2 is involved in the pathology of vascular complications of type 2 diabetes mellitus (T2DM). This study investigated the role of cyclin‐D2‐regulated miRNAs in endothelial cell proliferation of T2DM. Results showed that higher glucose concentration (4.5 g/l) significantly promoted the proliferation of rat aortic endothelial cells (RAOECs), and significantly increased the expression of cyclin D2 and phosphorylation of retinoblastoma 1 (p‐RB1) in RAOECs compared with those under low glucose concentration. The cyclin D2‐3′ untranslated region is targeted by miR‐98, as demonstrated by miRNA analysis software. Western blot also confirmed that cyclin D2 and p‐RB1 expression was regulated by miR‐98. The results indicated that miR‐98 treatment can induce RAOEC apoptosis. The suppression of RAOEC growth by miR‐98 might be related to regulation of Bcl‐2, Bax and Caspase 9 expression. Furthermore, the expression levels of miR‐98 decreased in 4.5 g/l glucose‐treated cells compared with those treated by low glucose concentration. Similarly, the expression of miR‐98 significantly decreased in aortas of established streptozotocin (STZ)‐induced diabetic rat model compared with that in control rats; but cyclin D2 and p‐RB1 levels remarkably increased in aortas of STZ‐induced diabetic rats compared with those in healthy control rats. In conclusion, this study demonstrated that high glucose concentration induces cyclin D2 up‐regulation and miR‐98 down‐regulation in the RAOECs. By regulating cyclin D2, miR‐98 can inhibit human endothelial cell growth, thereby providing novel therapeutic targets for vascular complication of T2DM.     

Outlier Analysis Defines Zinc Finger Gene Family DNA Methylation in Tumors and Saliva of Head and Neck Cancer Patients

Head and Neck Squamous Cell Carcinoma (HNSCC) is the fifth most common cancer, annually affecting over half a million people worldwide. Presently, there are no accepted biomarkers for clinical detection and surveillance of HNSCC. In this work, a comprehensive genome-wide analysis of epigenetic alterations in primary HNSCC tumors was employed in conjunction with cancer-specific outlier statistics to define novel biomarker genes which are differentially methylated in HNSCC. The 37 identified biomarker candidates were top-scoring outlier genes with prominent differential methylation in tumors, but with no signal in normal tissues. These putative candidates were validated in independent HNSCC cohorts from our institution and TCGA (The Cancer Genome Atlas). Using the top candidates, ZNF14, ZNF160, and ZNF420, an assay was developed for detection of HNSCC cancer in primary tissue and saliva samples with 100% specificity when compared to normal control samples. Given the high detection specificity, the analysis of ZNF DNA methylation in combination with other DNA methylation biomarkers may be useful in the clinical setting for HNSCC detection and surveillance, particularly in high-risk patients. Several additional candidates identified through this work can be further investigated toward future development of a multi-gene panel of biomarkers for the surveillance and detection of HNSCC.     

The complete amino-acid sequence of the proteinase inhibitor B from the root of the arrowhead (Sagittaria sagittifolia L.)

After reduction and alkylation of the disulfide bonds of the proteinase inhibitor B from the root of the arrowhead (Sagittaria sagittifolia L.) followed by CNBr cleavage three peptide fragments with 68, 62 and 11 amino-acid residues could be separated on DEAE-Sepharose CL-6B. The peptides or the inhibitor itself were further specifically cleaved either by trypsin or by the mixture of (CH3)2SO/HCl/HBr at the arginyl- and the tryptophyl-peptide bond, respectively. The complete amino-acid sequences of the peptides were determined by manual solid phase DABITC/PITC double coupling micro-method and the primary structure of the arrowhead inhibitor B consisting of 141 amino-acid residues was then elucidated. Twenty pairs of amino-acid residues are repeated in the molecule of this inhibitor, three of these pairs even occur three times. The possible locations of the reactive sites are discussed. On the basis of sequence comparisons between this inhibitor and all other serine proteinase inhibitors the arrowhead inhibitor may belong to a new family.