C-terminal region amino acid substitutions contribute to catalytic differences between murine class alpha glutathione transferases mGSTA1-1 and mGSTA2-2 toward anti-diol epoxide isomers of benzo[c]phenanthrene

The molecular basis for catalytic differences between structurally closely related murine class alpha glutathione (GSH) transferases mGSTA1-1 and mGSTA2-2 in the GSH conjugation of anti-diol epoxide isomers of benzo[c]phenanthrene (anti-B[c]PDE) was investigated. GSH conjugation of both (-)- and (+)-enantiomers of anti-B[c]PDE was observed in the presence of mGSTA1-1 (60 and 40% GSH conjugation, respectively), whereas mGSTA2-2 exhibited a preference for the (-)-anti-isomer (>97%). In addition, the specific activity of mGSTA2-2 toward the (-)-anti-B[c]PDE isomer was relatively higher than that of mGSTA1-1. The amino acid sequences of mGSTA1-1 and mGSTA2-2 differ at 10 positions that are distributed in three sections. Section I contains amino acid residues in positions 65 and 95; section II contains residues in positions 157, 162, and 169, and section III contains residues in positions 207, 213, 218, 221, and 222. Enzyme activity measurements with chimeras of mGSTA1-1 and mGSTA2-2 revealed that amino acid substitutions in section III account for their differential enantioselectivity and catalytic activity toward anti-B[c]PDE. Site-directed mutagenesis of amino acid residues in section III of mGSTA2-2 with corresponding residues of mGSTA1-1 followed by activity measurements of the wild type and mutated enzymes indicates that leucine 207 and phenylalanine 221 may be critical for the high catalytic activity of mGSTA2-2 toward (-)-anti-B[c]PDE. Molecular modeling studies demonstrated that the active site of mGSTA1-1 accommodates both enantiomers of anti-B[c]PDE, whereas the (-)-anti-isomer interacts more favorably with active site residues in mGSTA2-2. The results of this study clearly indicate that amino acid substitutions in the C-terminal region contribute to catalytic differences between mGSTA1-1 and mGSTA2-2 with respect to anti-B[c]PDE.     

OsGRF4 controls grain shape,panicle length and seed shattering in rice

Traits such as grain shape, panicle length and seed shattering, play important roles in grain yield and harvest. In this study, the cloning and functional analysis of PANICLE TRAITS 2 (PT2), a novel gene from the Indica rice Chuandali (CDL), is reported. PT2 is synonymous with Growth‐Regulating Factor 4 (OsGRF4), which encodes a growth‐regulating factor that positively regulates grain shape and panicle length and negatively regulates seed shattering. Higher expression of OsGRF4 is correlated with larger grain, longer panicle and lower seed shattering. A unique OsGRF4 mutation, which occurs at the OsmiRNA396 target site of OsGRF4, seems to be associated with high levels of OsGRF4 expression, and results in phenotypic difference. Further research showed that OsGRF4 regulated two cytokinin dehydrogenase precursor genes (CKX5 and CKX1) resulting in increased cytokinin levels, which might affect the panicle traits. High storage capacity and moderate seed shattering of OsGRF4 may be useful in high‐yield breeding and mechanized harvesting of rice. Our findings provide additional insight into the molecular basis of panicle growth.     

Crystal structure determination of the β-cyclodextrin-p-aminobenzoic acid inclusion complex from powder X-ray diffraction data

The 1:1 inclusion complex of β-cyclodextrin and p-aminobenzoic acid was prepared and characterized by TG-DTA. The crystal structure of the complex was solved directly from powder X-ray diffraction data using the direct space approach and refined using Rietveld refinement techniques. The complex crystallizes in monoclinic P2₁ space group, with unit cell parameters a = 20.7890 ?, b = 10.2084 ?, c = 15.1091 ?, β = 110.825°, V = 2997 ?³. The amino group is located at the wide side of the β-cyclodextrin cavity, forming hydrogen bonds with β-cyclodextrin, and the carboxyl group is located at the narrow side. The crystallographic data obtained from powder diffraction data were compared with the single crystallographic data, and the result shows that solving crystal structure of cyclodextrins inclusion complexes of such complexity is accessible to powder diffractionists to some extent.     

Trehalose synthase converts glycogen to trehalose

Trehalose (alpha,alpha-1,1-glucosyl-glucose) is essential for the growth of mycobacteria, and these organisms have three different pathways that can produce trehalose. One pathway involves the enzyme described in the present study, trehalose synthase (TreS), which interconverts trehalose and maltose. We show that TreS from Mycobacterium smegmatis, as well as recombinant TreS produced in Escherichia coli, has amylase activity in addition to the maltose <--> trehalose interconverting activity (referred to as MTase). Both activities were present in the enzyme purified to apparent homogeneity from extracts of Mycobacterium smegmatis, and also in the recombinant enzyme produced in E. coli from either the M. smegmatis or the Mycobacterium tuberculosis gene. Furthermore, when either purified or recombinant TreS was chromatographed on a Sephacryl S-200 column, both MTase and amylase activities were present in the same fractions across the peak, and the ratio of these two activities remained constant in these fractions. In addition, crystals of TreS also contained both amylase and MTase activities. TreS produced both radioactive maltose and radioactive trehalose when incubated with [(3)H]glycogen, and also converted maltooligosaccharides, such as maltoheptaose, to both maltose and trehalose. The amylase activity was stimulated by addition of Ca(2+), but this cation inhibited the MTase activity. In addition, MTase activity, but not amylase activity, was strongly inhibited, and in a competitive manner, by validoxylamine. On the other hand, amylase, but not MTase activity, was inhibited by the known transition-state amylase inhibitor, acarbose, suggesting the possibility of two different active sites. Our data suggest that TreS represents another pathway for the production of trehalose from glycogen, involving maltose as an intermediate. In addition, the wild-type organism or mutants blocked in other trehalose biosynthetic pathways, but still having active TreS, accumulate 10- to 20-fold more glycogen when grown in high concentrations (> or = 2% or more) of trehalose, but not in glucose or other sugars. Furthermore, trehalose mutants that are missing TreS do not accumulate glycogen in high concentrations of trehalose or other sugars. These data indicate that trehalose and TreS are both involved in the production of glycogen, and that the metabolism of trehalose and glycogen is interconnected.     

The angiosomes of the head and neck: anatomic study and clinical applications

The angiosome concept was introduced over a decade ago by Taylor and Palmer, whereby the body was considered to be composed anatomically of multiple three-dimensional composite blocks of tissue supplied by particular source arteries. Since then, detailed studies of the forearm and leg have been examined by Taylor and his coworkers. This study focuses on another region--the head and neck. Six fresh head and neck cadaver specimens were examined after infusion with a radio-opaque lead oxide mixture and correlated with over 24 previous body studies. The vascular anatomy of the skin, superficial musculoaponeurotic system (SMAS), muscles, brain, dura, and bone was examined. Each layer was painstakingly removed, photographed, labeled, and mapped to the respective arteries and veins. A radiologic subtraction technique was used to allow successive layers to be compared. This information was then scanned into a computer, analyzed, color coded, and labeled, thereby producing a three-dimensional study of the head and neck region to identify the respective angiosomes. As in previous detailed examinations of the leg and forearm, the angiosomes were found to be connected usually within tissues, such as muscle, skin, specialized organs or glands, rather than between the tissues. The muscles usually had vessels of two or more angiosomes supplying them and fell into three major groups based on the similarity of their arterial supply. In some areas, the midline anastomoses were rich, especially in the integument of the scalp, forehead, and lips. In other regions, the midline vascular connections were poor, especially in the tongue and palate. No fewer than 13 angiosomes of the head and neck, supplied by the branches of the external carotid, internal carotid, and subclavian arteries, have been defined, mapping their three-dimensional territories in the skin, the deep soft tissues, and the bones. Although most angiosomes spanned between skin and bone, three territories, those of the vertebral, lingual, and ascending pharyngeal vessels, were confined to the deep tissues without cutaneous representation. Finally, this study provides additional data for the surgeon to help plan safer incisions and better reconstructive flap procedures. It also gives information that may help explain the etiology and treatment of head and neck arteriovenous vascular malformations.     

球毛壳菌(Chaetomium globosum)NK102 纤维素酶基因及其表达条件

[目的]生物质的利用是当前生物技术研究的一个热点.本小组分离到一株高效降解纤维素球毛壳菌(Chaetomium globosum)NK102,本文拟探索研究此菌的纤维素酶表达系统并寻找影响酶基因表达的关键因素.[方法]通过对NK102测序,本文界定了球毛壳菌NK102的主要纤维素酶编码基因,使用数字基因表达谱升级版(RNA-Seq)的方法得到纤维素酶基因的表达差异,然后观察了营养、物理条件下纤维素酶基因表达和酶活性变化的情况.[结果]发现随着培养时间的延长,纤维素酶基因整体上表达量升高.在所选基因中,外切葡聚糖酶、纤维二糖脱氢酶和内切葡聚糖酶基因(cbh1,cdh和egl1)的表达量最高.糖代谢的负调控因子ACE I和CreA的随时间表达量均降低,而Hap2/3/5复合体的表达量反而升高.之后检测了不同碳源培养基对纤维素酶基因表达量和酶活性的影响,发现葡萄糖为强阻遏因子,纤维二糖为其诱导物,而山梨醇没有影响.特别是,我们发现光照也影响纤维素酶基因的表达,黑暗条件明显抑制酶基因的表达.[结论]转录组学的方法可以初步探索纤维素酶表达的规律,酶基因的表达受到营养、物理条件的影响.本研究为揭示球毛壳菌降解纤维素分子机理和阐释生物质糖代谢途径提供了有用参考.     

Differences in genotypes of Helicobacter pylori from different human populations

DNA motifs at several informative loci in more than 500 strains of Helicobacter pylori from five continents were studied by PCR and sequencing to gain insights into the evolution of this gastric pathogen. Five types of deletion, insertion, and substitution motifs were found at the right end of the H. pylori cag pathogenicity island. Of the three most common motifs, type I predominated in Spaniards, native Peruvians, and Guatemalan Ladinos (mixed Amerindian-European ancestry) and also in native Africans and U.S. residents; type II predominated among Japanese and Chinese; and type III predominated in Indians from Calcutta. Sequences in the cagA gene and in vacAm1 type alleles of the vacuolating cytotoxin gene (vacA) of strains from native Peruvians were also more like those from Spaniards than those from Asians. These indications of relatedness of Latin American and Spanish strains, despite the closer genetic relatedness of Amerindian and Asian people themselves, lead us to suggest that H. pylori may have been brought to the New World by European conquerors and colonists about 500 years ago. This thinking, in turn, suggests that H. pylori infection might have become widespread in people quite recently in human evolution.     

Human liver alkaline phosphatase, purification and partial sequencing: homology with the placental isozyme

Human liver alkaline phosphatase (AP) has been purified to homogeneity. The enzyme has a molecular weight of 150,000 in its native state and consists of two identical subunits of Mr 75,000. After treatment with endoglycosidase F the molecular weight is reduced to 50,000 indicating a high degree of glycosylation. The amino-terminal sequence up to 22 residues was found to be Leu-Val-Pro-Glu-Lys-Glu-Lys-Asp-Pro-Lys-Tyr-(Ala)-Arg-Asp-Gln-Ala-Gln-?- Thr-Leu-Lys-Tyr. The amino-terminal portions of human and bovine liver AP are identical. The amino termini of the human liver and human placental AP isozymes have appreciable homology. Conformationally the amino termini are very similar.     

Distal NF-kB binding motif functions as an enhancer for nontypeable H. influenzae-induced DEFB4 regulation in epithelial cells

Among the antimicrobial molecules produced by epithelial cells, DEFB4 is inducible in response to proinflammatory signals such as cytokines and bacterial molecules. Nontypeable Haemophilus influenzae (NTHi) is an important human pathogen that exacerbates chronic obstructive pulmonary disease in adult and causes otitis media and sinusitis in children. Previously, we have demonstrated that DEFB4 effectively kills NTHi and is induced by NTHi via TLR2 signaling. The 5′-flanking region of DEFB4 contains several NF-κB binding motifs, but their NTHi-specific activity remains unclear. In this study, we aimed to elucidate molecular mechanism involved in DEFB4 regulation, focusing on the role of the distal NF-κB binding motif of DEFB4 responding to NTHi. Here, we show that the human middle ear epithelial cells up-regulate DEFB4 expression in response to NTHi via NF-κB activation mediated by IκKα/β−IκBα signaling. Deletion of the distal NF-κB binding motif led to a significant reduction in NTHi-induced DEFB4 up-regulation. A heterologous construct containing the distal NF-κB binding motif was found to increase the promoter activity in response to NTHi, indicating a NTHi-responding enhancer activity of the distal NF-κB binding motif. Furthermore, electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that the p65 domain of NF-κB binds to the distal NF-κB binding motif in response to NTHi. Taken together, our results suggest that NTHi-induced binding of p65 NF-κB to the distal NF-κB binding motif of DEFB4 enhances NTHi-induced DEFB4 regulation in epithelial cells.     

Molecular characterization of avian-like H1N1 swine influenza a viruses isolated in Eastern China, 2011

Currently, three predominant subtypes of influenza virus are prevalent in pig populations worldwide: H1N1, H3N2, and H1N2. European avian-like H1N1 viruses, which were initially detected in European pig populations in 1979, have been circulating in pigs in eastern China since 2007. In this study, six influenza A viruses were isolated from 60 swine lung samples collected from January to April 2011 in eastern China. Based on whole genome sequencing, molecular characteristics of two isolates were determined. Phylogenetic analysis showed the eight genes of the two isolates were closely related to those of the avian-like H1N1 viruses circulating in pig populations, especially similar to those found in China. Four potential glycosylation sites were observed at positions 13, 26, 198, 277 in the HA1 proteins of the two isolates. Due to the presence of a stop codon at codon 12, the isolates contained truncated PB1-F2 proteins. In this study, the isolates contained 591Q, 627E and 701N in the polymerase subunit PB2, which had been shown to be determinants of virulence and host adaptation. The isolates also had a D rather than E at position 92 of the NS1, a marker of mammalian adaptation. Both isolates contained the GPKV motif at the PDZ ligand domain of the 3′ end of the NS1, a characteristic marker of the European avian-like swine viruses since about 1999, which is distinct from those of avian, human and classical swine viruses. The M2 proteins of the isolates have the mutation (S31N), a characteristic marker of the European avian-like swine viruses since about 1987, which may confer resistance to amantadine and rimantadine antivirals. Our findings further emphasize the importance of surveillance on the genetic diversity of influenza A viruses in pigs, and raise more concerns about the occurrence of cross-species transmission events.