[Simultaneous detection of 7 important Rickettsiales pathogens by TaqMan-probe quantitative real-time PCR].

Objective: To establish and optimize a TaqMan-probe quantitative real-time PCR (qPCR) assay for the detection of 7 important Rickettsiales pathogens and simultaneous identification of the infection types. Methods: Based on the ompB gene of Rickettsia prowazekii, Rickettsia mooseri and spotted fever group rickettsiae, the groEL gene of Orientia tsutsugamushi, the 16S rRNA of Ehrlichia chaffeensis, the gltA gene of Anaplasma phagocytophilum and the com1 gene of Coxiella burnetii, we synthesized primers and TaqMan-probes and optimized the reaction system and reaction process to same solution. The sensitivity, specificity and reproducibility of this assay were evaluated and the assay was used for the detection of simulated and actual samples. Results: The Ct value of the standard curves of the 7 pathogens showed a good linear relationship with the number of DNA copies (all R2 >0.990 0), the minimum detection limit was 10 copies/µl, showing good specificity. In the 96 tick nucleic acid extracts, Coxiella burnetii was detected in 1 sampleand spotted fever group Rickettsiae was detected in 3 samples. In the 80 blood samples from patients with undefined febrile illness, Orientia tsutsugamushi was detected in 1 sample and spotted fever group rickettsiae was detected in 2 samples. Conclusions: In this study, based on the established TaqMan-probe qPCR assay, the reaction system and reaction condition of the 7 important pathogens of Rickettsiales were optimized to the same solution. This method overcomes the shortcomings of using different reaction systems and reaction conditions for different pathogens, which can precisely identify the species of 7 important pathogens of Rickettsiales in clinical sample detections and is important for the infection type identification and laboratory detection time reduction to facilitate precise treatment of the patients.

Surface reconstructions and related local properties of a BiFeO3 thin film.

Coupling between lattice and order parameters, such as polarization in ferroelectrics and/or polarity in polar structures, has a strong impact on surface relaxation and reconstruction. However, up to now, surface structures that involve the termination of both matrix polarization and polar atomic planes have received little attention, particularly on the atomic scale. Here, we study surface structures on a BiFeO3 thin film using atomic-resolution scanning transmission electron microscopy and spectroscopy. Two types of surface structure are found, depending on the polarization of the underlying ferroelectric domain. On domains that have an upward polarization component, a layer with an Aurivillius-Bi2O2-like structural unit is observed. Dramatic changes in local properties are measured directly below the surface layer. On domains that have a downward polarization component, no reconstructions are visible. Calculations based on ab initio density functional theory reproduce the results and are used to interpret the formation of the surface structures.

Alpha-amino acid behaves differently from beta- or gamma-amino acids as treated by trimetaphosphate.

The condensation reactions of sodium trimetaphosphate with single amino acids, namely glycine, L-alanine, beta-alanine and gamma-aminobutyric acid or pairs of these amino acids were reinvestigated by electrospray ion-trap mass spectrometry and high performance liquid chromatography. It was found when mixtures were treated by sodium trimetaphosphate only in the presence of alpha-amino acid dipeptides were formed. Without addition of alpha-amino acids, the beta-amino acid or gamma-aminobutyric acid could not form peptide either by themselves or with their mixtures under the same conditions. From the data it is concluded that phosphate might select alpha-amino acids to produce the peptides being important precursors for the origin of life.

Variation in proton affinity of the guanidino group between free and blocked arginine.

In this paper, the analog of arginine residues in peptides was synthesized and characterized by ESI-MS/MS (electrospray ionization with tandem mass spectrometry), (31)P NMR, (1)H NMR, IR and high-resolution mass spectrometry. When the Todd reaction activity of the guanidino group in free arginine and the arginine peptide analog were compared, it was found that the proton affinity of the guanidino group was decreased when both the N- and the C-terminal were blocked. As a result, the guanidino group of arginine residues in peptides could be phosphorylated under the Todd reaction condition, but not the free arginine. This result was further proved by the theoretical calculation of their proton affinity.

Orientation-dependent transparency of metallic interfaces.

As devices are reduced in size, interfaces start to dominate electrical transport, making it essential to be able to describe reliably how they transmit and reflect electrons. For a number of nearly perfectly lattice-matched materials, we calculate from first principles the dependence of the interface transparency on the crystal orientation. Quite remarkably, the largest anisotropy is predicted for interfaces between the prototype free-electron materials silver and aluminum, for which a massive factor of 2 difference between (111) and (001) interfaces is found.

Identification of a distinct family of genes encoding atypical odorant-binding proteins in the malaria vector mosquito, Anopheles gambiae.

We performed a genome-wide analysis for candidate odorant-binding protein (OBP) genes in the malaria vector Anopheles gambiae (Ag). We identified fifty-seven putative genes including sixteen genes predicted to encode distinct, higher molecular weight proteins that lack orthologues in Drosophila. Expression analysis indicates that several of these atypical AgOBPs are transcribed in chemosensory organs in adult and immature stages. Phylogenetic analysis of the Anopheles and Drosophila OBP families reveals these proteins fall into several clusters based on sequence similarity and suggests the atypical AgOBP genes arose in the mosquito lineage after the divergence of mosquitoes and flies. The identification of these AgOBP genes is the first step towards determining their biological roles in this economically and medically important insect.

Molecular effects of Eya1 domain mutations causing organ defects in BOR syndrome.

Eya1 is a critical gene for mammalian organogenesis. Mutations in human EYA1 cause branchio-oto-renal (BOR) syndrome, an autosomal dominant disorder characterized by varying combinations of branchial, otic and renal anomalies, whereas deletion of mouse Eya1 results in the absence of multiple organ formation. Eya1 and other Eya gene products share a highly conserved 271 amino acid Eya domain that is required for protein-protein interaction. Recently, several point mutations that result in single amino acid substitutions in the conserved Eya domain region of EYA1 have been identified in BOR patients; however, the molecular and developmental basis of organ defects that occurred in BOR syndrome is unclear. To understand how these point mutations cause disease, we have analyzed the functional importance of these Eya domain missense mutations with respect to protein complex formation and cellular localization. We have demonstrated that these point mutations do not alter protein localization. However, four mutations are crucial for protein-protein interactions in both yeast and mammalian cells. Our results provide insights into the molecular mechanisms of organ defects detected in human syndromes.

A modified cryosection method for mouse testis tissue.

We have previously described a modified cryosection technique that improved the quality of histological sections as well as increasing the sensitivity to detect specific mRNA expression during early insect embryogenesis. Here, we report the use of this technique to produce high-quality sections of mouse testis tissue. The possibility of applying this technique to section the loose rat testis tissue is also discussed.

Pax-6 expression and activity are induced in the reepithelializing cornea and control activity of the transcriptional promoter for matrix metalloproteinase gelatinase B.

Recent evidence supports the idea that matrix metalloproteinases (MMPs) act as morphogenetic regulators in embryonic and adult events of tissue remodeling. MMP activity is controlled primarily at the level of gene expression. In a recent study we characterized the transcriptional promoter of the MMP gene, gelatinase B (gelB), in transgenic mice, demonstrating the requirement for DNA sequences between -522 and +19 for appropriate activity. In this study we investigated factors required for gelB promoter activity in the developing eye and reepithelializing adult cornea. Pax-6 is a homeobox and paired domain transcription factor that acts at the top of the hierarchy of genes controlling eye development. Pax-6 is also expressed in the adult eye. We show here that the tissue expression pattern of Pax-6 overlaps extensively with gelB promoter activity in the developing and adult eye. In addition Pax-6 is observed to be upregulated in repairing corneal epithelium, as is gelB promoter activity. In cell culture transfection experiments, we identified two promoter regions which mediate positive response to Pax-6. By electrophoretic mobility shift assay, we further pinpoint two Pax-6 binding sites within these response regions and demonstrate direct interaction of the Pax-6 paired domain with one of these sites. These data suggest a mechanism by which Pax-6 may direct gelB expression in an eye-specific manner.

Eya1-deficient mice lack ears and kidneys and show abnormal apoptosis of organ primordia.

Haploinsufficiency for human EYA1, a homologue of the Drosophila melanogaster gene eyes absent (eya), results in the dominantly inherited disorders branchio-oto-renal (BOR) syndrome and branchio-oto (BO) syndrome, which are characterized by craniofacial abnormalities and hearing loss with (BOR) or without (BO) kidney defects. To understand the developmental pathogenesis of organs affected in these syndromes, we inactivated the gene Eya1 in mice. Eya1 heterozygotes show renal abnormalities and a conductive hearing loss similar to BOR syndrome, whereas Eya1 homozygotes lack ears and kidneys due to defective inductive tissue interactions and apoptotic regression of the organ primordia. Inner ear development in Eya1 homozygotes arrests at the otic vesicle stage and all components of the inner ear and specific cranial sensory ganglia fail to form. In the kidney, Eya1 homozygosity results in an absence of ureteric bud outgrowth and a subsequent failure of metanephric induction. Gdnf expression, which is required to direct ureteric bud outgrowth via activation of the c-ret Rtk (refs 5, 6, 7, 8), is not detected in Eya1-/- metanephric mesenchyme. In Eya1-/- ear and kidney development, Six but not Pax expression is Eya1 dependent, similar to a genetic pathway elucidated in the Drosophila eye imaginal disc. Our results indicate that Eya1 controls critical early inductive signalling events involved in ear and kidney formation and integrate Eya1 into the genetic regulatory cascade controlling kidney formation upstream of Gdnf. In addition, our results suggest that an evolutionarily conserved Pax-Eya-Six regulatory hierarchy is used in mammalian ear and kidney development.

Regulation of Pax6 expression is conserved between mice and flies.

Pax6 plays a key role in visual system development throughout the metazoa and the function of Pax6 is evolutionarily conserved. However, the regulation of Pax6 expression during eye development is largely unknown. We have identified two physically distinct promoters in mouse Pax6, P0 and P1, that direct differential Pax6 expression in the developing eye. P0-initiated transcripts predominate in lens placode and corneal and conjunctival epithelia, whereas P1-initiated transcripts are expressed in lens placode, optic vesicle and CNS, and only weakly in corneal and conjunctival epithelia. To further investigate their tissue-specific expression, a series of constructs for each promoter were examined in transgenic mice. We identified three different regulatory regions which direct distinct domains of Pax6 expression in the eye. A regulatory element upstream of the Pax6 P0 promoter is required for expression in a subpopulation of retinal progenitors and in the developing pancreas, while a second regulatory element upstream of the Pax6 P1 promoter is sufficient to direct expression in a subset of post-mitotic, non-terminally differentiated photoreceptors. A third element in Pax6 intron 4, when combined with either the P0 or P1 promoter, accurately directs expression in amacrine cells, ciliary body and iris. These results indicate that the complex expression pattern of Pax6 is differentially regulated by two promoters acting in combination with multiple cis-acting elements. We have also tested whether the regulatory mechanisms that direct Pax6 ocular expression are conserved between mice and flies. Remarkably, when inserted upstream of either the mouse Pax6 P1 or P0 promoter, an eye-enhancer region of the Drosophila eyeless gene, a Pax6 homolog, directs eye- and CNS-specific expression in transgenic mice that accurately reproduces features of endogenous Pax6 expression. These results suggest that in addition to conservation of Pax6 function, the upstream regulation of Pax6 has also been conserved during evolution.

Mouse Eya genes are expressed during limb tendon development and encode a transcriptional activation function.

Vertebrate limb tendons are derived from connective cells of the lateral plate mesoderm. Some of the developmental steps leading to the formation of vertebrate limb tendons have been previously identified; however, the molecular mechanisms responsible for tendinous patterning and maintenance during embryogenesis are largely unknown. The eyes absent (eya) gene of Drosophila encodes a novel nuclear protein of unknown molecular function. Here we show that Eya1 and Eya2, two mouse homologues of Drosophila eya, are expressed initially during limb development in connective tissue precursor cells. Later in limb development, Eya1 and Eya2 expression is associated with cell condensations that form different sets of limb tendons. Eya1 expression is largely restricted to flexor tendons, while Eya2 is expressed in the extensor tendons and ligaments of the phalangeal elements of the limb. These data suggest that Eya genes participate in the patterning of the distal tendons of the limb. To investigate the molecular functions of the Eya gene products, we have analyzed whether the highly divergent PST (proline-serine-threonine)-rich N-terminal regions of Eya1-3 function as transactivation domains. Our results demonstrate that Eya gene products can act as transcriptional activators, and they support a role for this molecular function in connective tissue patterning.

Double-segment defining role of even-skipped homologs along the evolution of insect pattern formation.

Recent studies on insect patterning suggest that the genetic hierarchy may be roughly conserved in phylogenetically divergent species, but pair-rule genes may not function identically in all insects. In order to understand potential evolutionary changes in the role of the pair-rule genes, a Bombyx even-skipped homolog was cloned and its expression pattern during early embryogenesis studied. Eight stripes of Bombyx even-skipped were progressively expressed in an antero-posterior order. Later, these stripes disappeared anteriorly. Under this detection system, Bombyx even-skipped stripes clearly do not resolve into the corresponding secondary stripes, an obvious difference from Drosophila and Tribolium. These results suggest that Bombyx even-skipped may serve a double-segment defining role and may determine the odd-numbered engrailed stripes.

Mouse Eya homologues of the Drosophila eyes absent gene require Pax6 for expression in lens and nasal placode.

We have identified and mapped three members of a new family of vertebrate genes, designated Eya1, Eya2 and Eya3, which share high sequence similarity with the Drosophila eyes absent (eya) gene. Comparison of all three murine Eya gene products and that encoded by the Drosophila eya gene defines a 271 amino acid carboxyl terminal Eya domain, which has been highly conserved during evolution. Eya1 and Eya2, which are closely related, are extensively expressed in cranial placodes, in the branchial arches and CNS and in complementary or overlapping patterns during organogenesis. Eya3 is also expressed in the branchial arches and CNS, but lacks cranial placode expression. All three Eya genes are expressed in the developing eye. Eyal is expressed in developing anterior chamber structures, including the lens placode, the iris and ciliary region and the prospective corneal ectoderm. Eyal is also expressed in retinal pigment epithelium and optic nerve. Eya2 is expressed in neural retina, sclera and optic nerve sheath. Moreover, Eya1 and Eya2 expressions in the lens and nasal placode overlap with and depend upon expression of Pax6. The high sequence similarity with Drosophila eya, the conserved developmental expression of Eya genes in the eye and the Pax6 dependence of Eya expression in the lens and nasal placode indicates that these genes likely represent functional homologues of the Drosophila eya gene. These results suggest that members of the Eya gene family play critical roles downstream of Pax genes in specifying placodal identity and support the idea that despite enormous morphological differences, the early development of insect and mammalian eyes is controlled by a conserved regulatory hierarchy.

Spatial and temporal expression pattern of POU-M1/SGF-3 in Bombyx mori embryogenesis.

The expression pattern of the POU-M1/SGF-3 gene during Bombyx embryogenesis has been analysed using in situ hybridization and immunohistochemistry. At the early embryo retraction stage, both the transcripts and protein were first detected in precursor cells of the prothoracic glands in the labial segment, in the oenocytes in the A1-A8 segments and in invaginated regions in the mandibular and maxillary segments. The invaginated regions in the mandibular segment develop into the abductor plates in the lateral anterior region and the adductor plates in the posterior region. From the latter plates, the salivary glands elongate. The invaginated regions in the maxillary segment develop into the corpora allata in the anterior region and the subbuccal glands in the posterior region, which unite with tissues of the anterior region of the mandibular segment at later stages. After the embryo retraction stage, the transcripts and protein products also become detectable in the silk gland invagination points and, after the blastokinesis stage, the products are restricted to the entire anterior silk glands and to the anterior and middle parts of the middle silk glands. Expression can also be detected in a part of the hindgut, in the tracheal system and in some cells of the central nervous system. These results indicate that POU-M1/SGF-3 might play roles in the development of the silk glands, nervous system, tracheal system and other organs like the prothoracic glands and oenocytes.

A maternal homeobox gene, Bombyx caudal, forms both mRNA and protein concentration gradients spanning anteroposterior axis during gastrulation.

We have isolated a caudal (cad) homologue from a cDNA library of Bombyx mori embryos. The Bombyx cad cDNA encodes a protein of 244 amino acids. The homology between Drosophila and Bombyx homeodomains is 80%. Similar to Drosophila cad, there is no YPWM peptide sequence along the upstream of homeodomain. Northern blot hybridization with a Bombyx cad probe revealed the presence of single maternal transcript of 2.3 kb. A stronger signal of the transcripts was detected in unfertilized eggs and in eggs up to 36 hours after deposition. The transcripts decreased rapidly by 2 days and a weak signal was maintained until hatching. To analyse its spatial expression pattern, we have established a novel frozen sectioning method for in situ hybridization and immunohistochemistry experiments. The results showed that Bombyx cad transcripts accumulated first in the nurse cells and transferred into the oocyte at a defined time during oogenesis. The maternal transcripts of Bombyx cad formed a concentration gradient spanning the anteroposterior axis during the gastrulation stage and were restricted to the anal pad, the most posterior domain, after 2 days of embryogenesis; the Drosophila cad mRNA revealed the corresponding expression profile during the syncytial blastoderm stage. The Bombyx cad protein was not detected in the ovary and the first 9 hours of eggs, but was first detected evenly during cellular blastoderm stage. During gastrulation, Bombyx cad protein concentration gradients shifted along the anteroposterior axis coinciding with the shifting of the mRNA concentration gradients.(ABSTRACT TRUNCATED AT 250 WORDS)

Promoter of the POU-M1/SGF-3 gene involved in the expression of Bombyx silk genes.

To characterize the transcription regulation of the POU-M1/SGF-3 gene, we have cloned a genomic DNA fragment encompassing the whole coding region and its flanking sequences. This gene does not contain any intron. The 5'-flanking region of the gene contains several interesting motifs, such as homeodomain-binding motifs, sequences resembling the transcriptional factor Sp1-binding site, and TGTTT motifs, but lacks some of the typical transcriptional regulatory sequences, such as TATA and CCAAT boxes. Transcriptional analysis of a series of deletion mutants of the gene in the nuclear extracts prepared from the middle silk gland of 2-day-old fifth instar larvae revealed the presence of multiple cis-regulatory elements located both upstream and downstream of the initiation site. One of these elements, the homeodomain-binding element, was identified to mediate negative regulation. By mobility shift assay using the POU-M1 specific antibodies, we found that this negative element interacts with the POU-M1/SGF-3. Transcription analysis in vitro using templates mutagenized in the PB region and one of the POU-M1 antibodies indicated that the PB region is an autoregulatory element responsible for SGF-3-dependent transcriptional repression.

Molecular cloning of a POU domain-containing factor involved in the regulation of the Bombyx sericin-1 gene.

The POU domain is a highly conserved region found in a number of transcription factors and products of developmental control genes. We report here the isolation and characterization of a POU domain-containing cDNA (POU-M1) from the middle silk gland of Bombyx mori. It encodes a protein with a POU domain identical to that of the Drosophila Cf1-a protein. By mobility shift and nuclease protection assays, the POU-M1 protein and the putative silk gland factor 3 (SGF-3) were found to interact in an indistinguishable manner with the SC region (positions -204 to -183) of the sericin-1 gene, a key cis-acting element involved in the regulation of the gene through the interaction with SGF-3. Antibodies raised against the synthetic oligopeptide corresponding to the COOH-terminal region of the putative POU-M1 sequence reacted specifically to both the POU-M1 protein and SGF-3. Northern blot hybridization and Western blotting revealed that POU-M1 expression is regulated both temporally and spatially during silk gland development. We conclude that the POU-M1 protein is SGF-3 and propose that the differential expression of the POU-M1 gene is probably involved in the transcriptional regulation of the silk protein genes.