Characterization of a novel gene for strain typing reveals substructuring of Aspergillus fumigatus across North America

Fifty-five epidemiologically linked Aspergillus fumigatus isolates obtained from six nosocomial outbreaks of invasive aspergillosis were subtyped by sequencing the polymorphic region of the gene encoding a putative cell surface protein, Afu3g08990 (denoted as CSP). Comparative sequence analysis showed that genetic diversity was generated in the coding region of this gene by both tandem repeats and point mutations. Each unique sequence in an outbreak cluster was assigned an arbitrary number or CSP sequence type. The CSP typing method was able to identify "clonal" and genotypically distinct A. fumigatus isolates, and the results of this method were concordant with those of another discriminatory genotyping technique, the Afut1 restriction fragment length polymorphism typing method. The novel single-locus sequence typing (CSP typing) strategy appears to be a simple, rapid, discriminatory tool that can be readily shared across laboratories. In addition, we found that A. fumigatus isolates substructured into multiple clades; interestingly, one clade consisted of isolates predominantly representing invasive clinical isolates recovered from cardiac transplant patients from two different outbreak situations. We also found that the A. fumigatus isolate Af293, whose genome has been sequenced, possesses a CSP gene structure that is substantially different from those of the other A. fumigatus strains studied here, highlighting the need for further taxonomic study.     

Ecological application of wavelet analysis in the scaling of spatial distribution patterns of Ceratoides ewersmanniana

Ecological experiments are usually conducted on small scales, but the ecological and environmental issues are usually on large scales. Hence, there is a clear need of scaling. Namely, when we deal with patterns and processes on larger scales, a special connection needs to be established on the small scales that we are familiar with. Here we presented a wavelet analysis method that could build relationships between spatial distribution patterns on different scales. With this method, we also studied how spatial heterogeneity and distribution patterns changed with the scale. We investigated the distribution and the habitat of C. ewersmanniana in two plots (200 m × 200 m; the distance between these two plots is 15 km) at Mosuowan desert. The results demonstrated that spatial heterogeneity and distribution patterns were incorporated into larger scales when the wavelet scale varied from one (5 m) to four (20 m). However, if the wavelet scale was above five (25 m), the spatial distribution patterns varied placidly, the oscillation frequency of landforms stabilized at 110 m, and the dynamic quantity period of C. ewersmanniana stabilized at 115–125 m. We also identified signal mutation points with wavelet analysis and verified the heterogeneity degree of local space with position variance. We found that position variance decomposed the distribution patterns on large scales into small sampling plots, and the position with the largest variance also had the strongest heterogeneity. In a word, the wavelet analysis method could scale-up spatial distribution patterns and habitat heterogeneity. With this method and other methods derived from this one, such as wavelet scale, wavelet variance, position variance and extremely direct-viewing graphs, wavelet analysis could be widely applied in solving the scaling problem in ecological and environmental studies.     

Identification of CD46 binding sites within the adenovirus serotype 35 fiber knob

Species B human adenoviruses (Ads) are often associated with fatal illnesses in immunocompromised individuals. Recently, species B Ads, most of which use the ubiquitously expressed complement regulatory protein CD46 as a primary attachment receptor, have gained interest for use as gene therapy vectors. In this study, we focused on species B Ad serotype 35 (Ad35), whose trimeric fiber knob domain binds to three CD46 molecules with a K_D (equilibrium dissociation constant) of 15.5 nM. To study the Ad35 knob-CD46 interaction, we generated an expression library of Ad35 knobs with random mutations and screened it for CD46 binding. We identified four critical residues (Phe242, Arg279, Ser282, and Glu302) which, when mutated, ablated Ad35 knob binding to CD46 without affecting knob trimerization. The functional importance of the identified residues was validated in surface plasmon resonance and competition binding studies. To model the Ad35 knob-CD46 interaction, we resolved the Ad35 knob structure at 2-Å resolution by X-ray crystallography and overlaid it onto the existing structure for Ad11-CD46 interaction. According to our model, all identified Ad35 residues are in regions that interact with CD46, whereby one CD46 molecule binds between two knob monomers. This mode of interaction might have potential consequences for CD46 signaling and intracellular trafficking of Ad35. Our findings are also fundamental for better characterization of species B Ads and design of antiviral drugs, as well as for application of species B Ads as in vivo and in vitro gene transfer vectors.     

Isolation and Molecular Characterization of a Poliovirus Type 1 Mutant That Replicates in the Spinal Cords of Mice

The Mahoney strain of poliovirus type 1 (OM) is generally unable to cause paralysis in mice. We isolated a mouse-adapted mutant, PV1/OM-SA (SA), from the spinal cord of a mouse that had been intracerebrally inoculated with OM. SA showed mouse neurovirulence only with intraspinal inoculation, and the infected mice developed a flaccid paralysis, which was indistinguishable from that observed in poliovirus-sensitive transgenic mice inoculated with OM. SA antigens were detected in neurons of the spinal cords of the infected mice. Nucleotide (nt) sequence analysis revealed 9 nt changes on the SA genome, resulting in three amino acid (a.a.) substitutions, i.e., one each in the capsid proteins VP4 and VP1 and in the noncapsid protein 2C. To identify the key mutation site(s) for the mouse neurovirulence, virus recombinants between OM and SA were constructed by using infectious cDNA clones of these two viruses and tested for their mouse neurovirulence after inoculation via an intraspinal route. The results indicated that a mutation at nt 928 (replacement of A with G), resulting in a substitution of Met for Ile at a.a. 62 within VP4, was responsible for conferring the mouse neurovirulence phenotype of the mutant SA. The mutation in VP4 may render the virus accessible to a molecule that acts as a virus receptor and is located on the surfaces of neurons of the mouse spinal cord. This molecule appears not to be expressed in the mouse brain.     

长白山白眉蝮蛇蛇毒磷脂酶A2的分离和初步表征

东北长白山白眉蝮蛇（ＡｇｋｉｓｔｒｏｄｏｎｂｌｏｍｈｏｆｆｉＵｓｕｒｅｎｓｉｓ）蛇毒经ＤＥＡＥＳｅｐｈａｄｅｘＡ５０离子交换层析柱,连续３步ＳｅｐｈａｄｅｘＧ７５凝胶过滤柱得到了磷脂酶Ａ２（ＰＬＡ２）的纯品。ＳＤＳ聚丙烯酰胺凝胶电泳（ＳＤＳＰＡＧＥ）以及基质辅助激光解析电离飞行时质谱（ＭＡＬＤＩ／ＴＯＦ／ＭＳ）表征为单一蛋白,其准确分子量为（１４．００８±０．００７）ｋＤ。最适ｐＨ范围８．０～９．０,最适的反应温度为４５℃。在溶液中有多聚体的存在。     

Jittery, a Mutator distant relative with a paradoxical mobile behavior: excision without reinsertion

The unstable mutation bz-m039 arose in a maize (Zea mays) stock that originated from a plant infected with barley stripe mosaic virus. The instability of the mutation is caused by a 3.9-kb mobile element that has been named Jittery (Jit). Jit has terminal inverted repeats (TIRs) of 181 bp, causes a 9-bp direct duplication of the target site, and appears to excise autonomously. It is predicted to encode a single 709-amino acid protein, JITA, which is distantly related to the MURA transposase protein of the Mutator system but is more closely related to the MURA protein of Mutator-like elements (MULEs) from Arabidopsis thaliana and rice (Oryza sativa). Like MULEs, Jit resembles Mutator in the length of the element's TIRs, the size of the target site duplication, and in the makeup of its transposase but differs from the autonomous element Mutator-Don Robertson in that it encodes a single protein. Jit also differs from Mutator elements in the high frequency with which it excises to produce germinal revertants and in its copy number in the maize genome: Jit-like TIRs are present at low copy number in all maize lines and teosinte accessions examined, and JITA sequences occur in only a few maize inbreds. However, Jit cannot be considered a bona fide transposon in its present host line because it does not leave footprints upon excision and does not reinsert in the genome. These unusual mobile element properties are discussed in light of the structure and gene organization of Jit and related elements.     

The Ku protein complex interacts with YY1, is up-regulated in human heart failure, and represses alpha myosin heavy-chain gene expression

Human heart failure is accompanied by repression of genes such as alpha myosin heavy chain (alphaMyHC) and SERCA2A and the induction of fetal genes such as betaMyHC and atrial natriuretic factor. It seems likely that changes in MyHC isoforms contribute to the poor contractility seen in heart failure, because small changes in isoform composition can have a major effect on the contractility of cardiac myocytes and the heart. Our laboratory has recently shown that YY1 protein levels are increased in human heart failure and that YY1 represses the activity of the human alphaMyHC promoter. We have now identified a region of the alphaMyHC promoter that binds a factor whose expression is increased sixfold in failing human hearts. Through peptide mass spectrometry, we identified this binding activity to be a heterodimer of Ku70 and Ku80. Expression of Ku represses the human alphaMyHC promoter in neonatal rat ventricular myocytes. Moreover, overexpression of Ku70/80 decreases alphaMyHC mRNA expression and increases skeletal alpha-actin. Interestingly, YY1 interacts with Ku70 and Ku80 in HeLa cells. Together, YY1, Ku70, and Ku80 repress the alphaMyHC promoter to an extent that is greater than that with YY1 or Ku70/80 alone. Our results suggest that Ku is an important factor in the repression of the human alphaMyHC promoter during heart failure.     

Study on the communities of endophytic fungi and endophytic actinomycetes from rice and their antipathogenic activities in vitro

The populations of endophytic fungi and actinomycetes from four rice cultivars in the Panyu district (Site 1) and Wushan district (Site 2) in Guangdong province, South China, were studied. The preponderant endophytic fungi and actinomycetes isolated belonged to Fusarium and Streptomyces respectively. The incidence of Streptomycetes griseofuscus ranged from 36.1 to 69% out of all the different rice cultivars from the two sites. It is the commonest population of endophytic actinomycetes, and constituted the greatest part of all the antagonistic communities. The distributions of endophytic fungi and actinomycetes in roots and leaves were different, endophytic fungi from leaves were diverse, some were organ-specific. More diverse endophytic actinomycetes were isolated from roots than from leaves. The endophytic fungi isolated from rice in Site 2 were more diverse than that in Site 1. The diversity of the endophytic actinomycetes, however, was less than that in Site 1. Acid soil in Site 2 is ideal for the growth and colonization of fungi while the alkaline soil in Site 1 is better for the growth and colonization of actinomycetes. The results suggested that differences in the chemical composition of soil could influence the endophytic microbial communities of rice plants. The endophytic fungi and actinomycetes isolated from poor-growing seedlings and susceptible rice cultivars were more abundant than that the disease-resistant counterparts. In the dual culture and activity detection of the metabolites, 41.2% of all the isolated endophytic fungi showed antagonism to rice pathogens. Fifty percent of all the isolated endophytic actinomycetes were antagonistic to those pathogens. The percentage of Streptomyces griseofuscus and hygroscopicus reached 55.4 and 21.4% of all the active actinomycetes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Novel Robinow syndrome causing mutations in the proximal region of the frizzled-like domain of ROR2 are retained in the endoplasmic reticulum

ROR2 is a member of the cell surface receptor tyrosine kinase (RTKs) family of proteins and is involved in the developmental morphogenesis of the skeletal, cardiovascular and genital systems. Mutations in ROR2 have been shown to cause two distinct human disorders, autosomal recessive Robinow syndrome and dominantly inherited Brachydactyly type B. The recessive form of Robinow syndrome is a disorder caused by loss-of-function mutations whereas Brachydactyly type B is a dominant disease and is presumably caused by gain-of-function mutations in the same gene. We have previously established that all the missense mutations causing Robinow syndrome in ROR2 are retained in the endoplasmic reticulum and therefore concluded that their loss of function is due to a defect in their intracellular trafficking. These mutations were in the distal portion of the frizzled-like cysteine rich domain and kringle domain. Here we report the identification of two novel mutations in the frizzled-like cysteine-rich domain of ROR2 causing Robinow syndrome. We establish the retention of the mutated proteins in the endoplasmic reticulum of HeLa cells and therefore failure to reach the plasma membrane. The clustering of Robinow-causing mutations in the extracellular frizzled-like cysteine-rich domain of ROR2 suggests a stringent requirement for the correct folding of this domain prior to export of ROR2 from the endoplasmic reticulum to the plasma membrane. GenBank accession number ROR2, M97639.