Increased Vδ1γδT cells predominantly contributed to IL-17 production in the development of adult human post-infectious irritable bowel syndrome.

BACKGROUND: γδT cells play an important role in the mucosa inflammation and immunity-associated disorders. Our previous study reported that γδ T cells producing IL-17 were involved in the pathogenesis of post-infectious irritable bowel syndrome (PI-IBS). However, their subset characteristic profile in this kind of disease remains unclear. Thus the current study's aim is to investigate the functionally predominant subset and its role in PI-IBS. METHODS: The total T cells were collected from the peripheral blood of patients with PI-IBS. The peripheral proportion of Vδ1 and Vδ2 subset was detected by FACS after stained with anti δ1-PE and anti δ2-APC. The local colonic proportion of this two subsets were measured under laser confocal fluorescence microscope. Vδ1 γδ T cells were enriched from the total peripheral T cells by minoantibody-immuno-microbeads (MACS) method and cultured, functionally evaluated by CCK-8 assay (proliferation), CD69/CD62L molecules expression assay (activation) and ELISA (IL-17 production) respectively. RESULTS: 1. Vδ1 γδ T cells significantly increased while Vδ2 γδ T cells remained unchanged in both the peripheral blood and local colonic tissue from PI-IBS patients (p < 0.05). 2. When cultured in vitro, the Vδ1 γδ T cells remarkably proliferated, activated and produced IL-17 (p < 0.05). CONCLUSIONS: Our results suggest that Vδ1 γδ T cells was the predominant γδ T cells subset in both peripheral and intestinal tissue, and was the major IL-17 producing γδ T cells in PI-IBS.

Cloning and characterization of the dehydration-responsive element-binding protein 2A gene in Eruca vesicaria subsp sativa.

Eruca vesicaria subsp sativa is one of the most tolerant Cruciferae species to drought, and dehydration-responsive element-binding protein 2A (DREB2A) is involved in responses to salinity, heat, and particularly drought. In this study, a gene encoding EvDREB2A was cloned and characterized in E. vesicaria subsp sativa. The full-length EvDREB2A cDNA sequence contained a 388-bp 5'-untranslated region (UTR), a 348-bp 3'-UTR, and a 1002-bp open reading frame that encoded 334 amino acid residues. The theoretical isoelectric point of the EvDREB2A protein was 4.80 and the molecular weight was 37.64 kDa. The genomic sequence of EvDREB2A contained no introns. Analysis using SMART indicated that EvDREB2A contains a conserved AP2 domain, similar to other plant DREBs. Phylogenetic analysis revealed that EvDREB2A and DREB2As from Brassica rapa, Eutrema salsugineum, Arabidopsis thaliana, Arabidopsis lyrata, and Arachis hypogaea formed a small subgroup, which clustered with DREB2Bs from A. lyrata, A. thaliana, Camelina sativa, and B. rapa to form a larger subgroup. EvDREB2A is most closely related to B. rapa DREB2A, followed by DREB2As from E. salsugineum, A. thaliana, A. hypogaea, and A. lyrata. A quantitative real-time polymerase chain reaction indicated that EvDREB2A expression was highest in the leaves, followed by the roots and hypocotyls, and was lowest in the flower buds. EvDREB2A could be used to improve drought tolerance in crops.

Cloning and characterization of the nicotianamine synthase gene in Eruca vesicaria subsp sativa.

Nicotianamine (NA) is a ubiquitous metabolite in plants that bind heavy metals, is crucial for metal homeostasis, and is also an important metal chelator that facilitates long-distance metal transport and sequestration. NA synthesis is catalyzed by the enzyme nicotianamine synthase (NAS). Eruca vesicaria subsp sativa is highly tolerant to Ni, Pb, and Zn. In this study, a gene encoding EvNAS was cloned and characterized in E. vesicaria subsp sativa. The full-length EvNAS cDNA sequence contained a 111-bp 5'-untranslated region (UTR), a 155-bp 3'-UTR, and a 966-bp open reading frame encoding 322-amino acid residues. The EvNAS genomic sequence contained no introns, which is similar to previously reported NAS genes. The deduced translation of EvNAS contained a well-conserved NAS domain (1-279 amino acids) and an LIKI-CGEAEG box identical to some Brassica NAS and to the LIRL-box in most plant NAS, which is essential for DNA binding. Phylogenetic analysis indicated that EvNAS was most closely related to Brassica rapa NAS3 within the Cruciferae, followed by Thlaspi NAS1, Camelina NAS3, and Arabidopsis NAS3. A reverse transcription-polymerase chain reaction indicated that EvNAS expression was greatest in the leaves, followed by the flower buds and hypocotyls. EvNAS was moderately expressed in the roots.

Production and genetic characterization of interspecific hybrids among Crambe abyssinica, Crambe hispanica and Crambe kralikii.

In this paper, interspecific crosses among Crambe abyssinica, Crambe hispanica, and Crambe kralikii were reported. In the C. hispanica x C. abyssinica (H x A) cross, 118 F1 hybrids were produced without embryo rescue, while 5 F1 hybrids were obtained with embryo rescue, when C. hispanica was used as the female parent. In the reciprocal cross (A x H), 232 hybrids were obtained without embryo rescue. From more than 1000 C. kralikii flowers pollinated with pollen grains of C. abyssinica (K x A), only 2 F1 hybrids were obtained with embryo rescue, whereas the reciprocal cross produced no hybrids, even with embryo rescue. The hybrids were confirmed at the morphological, cytological, and molecular levels. In the combinations of A x H and H x A, many BC1 hybrids were obtained without embryo rescue. In contrast, in the K x A cross, only 7 BC1 plants were obtained with embryo rescue, while no seed set was achieved under self-pollination or in backcrosses without embryo rescue. In the H x A F1 hybrids, the pollen stainability was 65.4-86.0%, with an average of 76.9%. In comparison, the pollen viability of hybrids in the reciprocal cross (A x H) ranged from 66.2 to 81.1%, with an average of 75.4%. Fertile pollen grains were not found in the K x A F1 hybrids. All F1 hybrids of the 3 crosses (H x A, A x H, and K x A) had the expected 2n = 75 chromosomes. AFLP analyses indicated that all F1 hybrids and their progenies had typical bands of the parents. These hybrids and progenies are anticipated to be valuable for future C. abyssinica improvement in breeding programs.

Small RNAs of Sequoia sempervirens during rejuvenation and phase change.

In this work, the population of small RNAs (sRNAs) was studied in the gymnosperm Sequoia sempervirens during phase changes, specifically in the juvenile, adult and rejuvenated plants obtained in vitro. The potential target genes of Sequoia sRNAs were predicted through bioinformatics. Rejuvenation is a pivotal process in woody plants that enables them to regain their growth potential, which results in the recovery of physiologic and molecular characteristics that were lost when the juveniles mature into adult plants. The results from the five repeated graftings of juvenile, adult and rejuvenated plants in vitro showed that sRNAs could be classified into structural RNAs (Group I), small interfering RNAs (Group II), annotated microRNAs (Group III, and unannotated sRNAs (Group IV). The results indicate that only 573 among 15,485,415 sRNAs (Groups III and IV) had significantly different expression patterns associated with rejuvenation and phase change. A total of 215 sRNAs exhibited up-regulated expression patterns in adult shoots, and 358 sRNAs were down-regulated. Expression profiling and prediction of possible target genes of these unique small RNAs indicate possible functions in the control of photosynthetic efficiency and rooting competence abundance during plant rejuvenation. Moreover, the increase in SsmiR156 and decrease in SsmiR172 during plant rejuvenation suggested that these two microRNAs extensively affect phase transition.

Understanding bamboo flowering based on large-scale analysis of expressed sequence tags.

Unlike other plants, bamboo (Bambusoideae) flowering is an elusive physiological phenomena, because it is unpredictable, long-periodic, gregarious, and uncontrollable; also, bamboo plants usually die after flowering. The flowering mechanism in Arabidopsis thaliana, a eudicot model species, is well established, but it remains unknown in bamboo species. We found 4470 and 3878 expressed sequence tags in the flower bud and vegetative shoot cDNA libraries, respectively, of the bamboo species, Bambusa oldhamii. Different genes were found expressed in bamboo flower buds compared to vegetative shoots, based on the Munich Information Center for Protein Sequences functional categorization; flowering-related genes were also identified in this species. We also identified Arabidopsis flowering-specific homologs that are involved in its photoperiod in this bamboo species, along with autonomous, vernalization and gibberellin-dependent pathways, indicating that bamboos may have a similar mechanism to control floral transition. Some bamboo expressed sequence tags shared high similarity with those of rice, but others did not match any known sequences. Our data lead us to conclude that bamboo may have its own unique flowering genes. This information can help us understand bamboo flowering and provides useful experimental methods to study the mechanisms involved.

First Report of Rhizoctonia Blight of a Coastal Redwood Tissue-Culture-Derived Saplings Caused by Rhizoctonia solani AG-IV in Taiwan.

Coastal redwood (Sequoia sempervirens (D. Don) Endl.) is native to North America. This tall tree species is used for forestation and lumber; its wood is also used for furniture, its burls for art ware, and its bark for fuel, insulation, and mulch. In August 2005, an instance of wilt was observed among 2-year-old tissue-culture-cloned plants (2) in the Sitou Forest of central Taiwan. Essentially, all plants were infected. The leaves or stems near the ground were affected first, but the wilt soon spread over the entire plant with the leaves becoming grayish brown and water soaked, and then wilting, drying, and finally defoliation occurred. Aerial hyphae were present over the affected areas, aerial mycelium was cob-web-like, hyaline, later becoming slightly brown. Hyphae were 6.5 to 10.4 µm wide with right-angle branching and septal constriction at their bases. Sclerotia were hemispherical, subglobose, to irregular in shape, 1 to 2 mm, and brown. The perfect stage of the fungus was not found. The fungus was identified as Rhizoctonia solani Kühn (3). Vegetative cells were stained with alkaline safranin solution and identified as multinucleate (1). Portions of the stem that displayed symptoms, together with adjacent healthy tissue, were disinfested for 1 min in 0.5% NaOCl and plated on to potato-dextrose agar (PDA) (Merck, Darmstadt, Germany) supplemented with 100 mg/L of ampicillin (Sigma, St. Louis, MO). Single hyphal tips were transferred to PDA and two isolates were established as pure cultures. On the basis of hyphal anastomosis with AG-IV tester isolates (exfop234, exfop241, and exfop250) (1), the fungus was identifed as R. solani AG-IV. Pathogenicity of the fungal isolates was confirmed by inoculating 2-month-old tissue-culture-derived S. sempervirens plants that were grown in pots and incubated in a growth chamber maintained at 28°C with a relative humidity above 95%. Inoculum consisted of a single mycelial 5-day-old 0.5-cm disc grown on PDA of the pathogen placed on the soil surface touching the base of each plant. Four plants were inoculated with mycelium and the four control plants were noninoculated. Inoculated plants wilted gradually over 4 days and all plants developed severe stem rot and were dead in 6 days, whereas control plants remained symptomless. The Rhizoctonia solani AG-IV was reisolated from all inoculated plants. This fungus has been observed to cause disease in many species of plants (4), but to our knowledge, this is the first report of Rhizoctonia blight of coastal redwood tissue-culture-derived saplings caused by Rhizoctonia solani AG-IV in Taiwan. References: (1) T. T. Chang. Taiwan J. For. Sci. 12:47, 1997. (2) L. C. Huang et al. Plant Physiol. 98:166, 1992. (3) B. Sneh et al. Identification of Rhizoctonia species. The American Phytopathological Society, St. Paul, MN, 1991. (4) S. T. Su et al. List of Plant Diseases in Taiwan. The Phytopathological Society of the Republic of China, 2002.

Mouth cell collection device for newborn mice.

For efficient and accurate genotyping of transgenic and knockout mice, the ability to reduce pain and suffering and to obtain DNA early in life are critical. We have developed a novel method to sample buccal cells from neonatal mice to obtain DNA. Our mouse mouth cell collection process includes an oral speculum and collection device which enables rapid extraction of enough DNA for up to 50 PCRs from each buccal sampling. This cell collection device fills a clear need for buccal sampling from neonatal mice, greatly facilitating research in mouse models of human disease. Eliminating the pain, distress, and death caused by invasive and mutilating procedures lessens the potential for confounding variables between control and experimental animals. In conclusion, our mouse mouth cell collection process can be applied to very small animals for which there exists no current device.

Human ARX gene: genomic characterization and expression.

Arx is a homeobox-containing gene with a high degree of sequence similarity between mouse and zebrafish. Arx is expressed in the forebrain and floor plate of the developing central nervous systems of these vertebrates and in the presumptive cortex of fetal mice. Our goal was to identify genes in Xp22.1-p21.3 involved in human neuronal development. Our in silico search for candidate genes noted that annotation of a human Xp22 PAC (RPCI1-258N20) sequence (GenBank Accession No. AC002504) identified putative exons consistent with an Arx homologue in Xp22. Northern blot analysis showed that a 3.3kb human ARX transcript was expressed at high levels in fetal brain. A 5.9kb transcript was expressed in adult heart, skeletal muscle, and liver with very faint expression in other adult tissues, including brain. In situ hybridization of ARX in human fetal brain sections at various developmental stages showed the highest expression in neuronal precursors in the germinal matrix of the ganglionic eminence and in the ventricular zone of the telencephalon. Expression was also observed in the hippocampus, cingulate, subventricular zone, cortical plate, caudate nucleus, and putamen. The expression pattern suggests that ARX is involved in the differentiation and maintenance of specific neuronal cell types in the human central nervous system. We also mapped the murine Arx gene to the mouse genome using a mouse/hamster radiation hybrid panel and showed that Arx and ARX are orthologues. Therefore, investigations in model vertebrates may provide insight into the role of ARX in development. The recent identification of ARX mutations in patients with various forms of mental retardation make such studies in model organisms even more compelling.

Genetic network identification by high density, multiplexed reversed transcriptional (HD-MRT) analysis in steroidogenic axis model cell lines.

Transcriptional network analysis in steroidogenic axis cell lines requires an understanding of cellular network composition and complexity. Previous studies have shown that absence of transcriptional network components in a cell line compromises that cell line's functional capacity for transcriptional regulation. Our goal was to analyze qualitatively steroidogenic axis-derived cell lines' expression of a putative transcriptional network involved in human and mouse development. To pursue this analysis we used Northern blots and a high density-multiplexed reverse transcription-polymerase chain reaction (HD-MRT-PCR) approach. Our results revealed that, while some members of this putative network were universally expressed, only a minority of the non-constitutive targeted transcripts were present in any single line. Based on our data and previously published results for contextual expression of these transcription factors, a model was constructed possessing the topology suggestive of a scale-free network: certain network members were highly connected nodes and would represent critical sites of vulnerability. The importance of these highly connected nodes for network function is supported by the severe phenotypes exhibited by human patients and animal models when these genes are mutated. We conclude that knowledge of network composition in specific cell lines is essential for their use as models to investigate functional interactions within selected subnetworks.

Nine novel mutations in NR0B1 (DAX1) causing adrenal hypoplasia congenita.

X-linked adrenal hypoplasia congenita (AHC) is caused by mutations in the NR0B1 gene. This gene encodes an orphan member of the nuclear receptor superfamily, DAX1. Ongoing efforts in our laboratory have identified nine novel NR0B1 mutations in X-linked AHC patients (Y81X, 343delG, 457delT, 629delG, L295P, 926-927delTG, 1130delA, 1141-1155del15, and E428X). Two additional families segregate previously identified NR0B1 mutations (501delA and R425T). Sequence analysis of the mitochondrial D-loop indicates that the 501delA family is unrelated through matrilineal descent to our previously analyzed 501delA family.

Glycerol kinase deficiency: evidence for complexity in a single gene disorder.

Glycerol kinase deficiency (GKD) occurs as part of an Xp21 contiguous gene syndrome or as isolated GKD. The isolated form can be either symptomatic with episodic metabolic and central nervous system (CNS) decompensation or asymptomatic with hyperglycerolemia and glyceroluria only. To better understand the pathogenesis of isolated GKD, we sought individuals with point mutations in the GK coding region and measured their GK enzyme activities. We identified six individuals with missense mutations: four (N288D, A305V, M428T, and Q438R) among males who were asymptomatic and two (D198G, R405Q) in individuals who were symptomatic. GK activity measured in lymphoblastoid cell lines or fibroblasts was similar for the symptomatic and the asymptomatic individuals. Mapping of the individuals' missense mutations to the three-dimensional structure of Escherichia coli GK revealed that the symptomatic individuals' mutations are in the same region as a subset of the mutations among the asymptomatic individuals, adjacent to the active-site cleft. We conclude that, like many other disorders, GK genotype does not predict GKD phenotype. We hypothesize that the phenotype of an individual with GKD is a complex trait influenced by additional, independently inherited genes.

Primate DAX1, SRY, and SOX9: evolutionary stratification of sex-determination pathway.

The molecular evolution of DAX1, SRY, and SOX9, genes involved in mammalian sex determination, was examined in six primate species. DAX1 and SRY have been added to the X and Y chromosomes, respectively, during mammalian evolution, whereas SOX9 remains autosomal. We determined the genomic sequences of DAX1, SRY, and SOX9 in all six species, and calculated K(a), the number of nonsynonymous substitutions per nonsynonymous site, and compared this with the K(s), the number of synonymous substitutions per synonymous site. Phylogenetic trees were constructed by means of the DAX1, SRY, and SOX9 coding sequences, and phylogenetic analysis was performed using maximum likelihood. Overall measures of gene and protein similarity were closer for DAX1 and SOX9, but DAX1 exhibited nonsynonymous amino acid substitutions at an accelerated frequency relative to synonymous changes, similar to SRY and significantly higher than SOX9. We conclude that, at the protein level, DAX1 and SRY are under less selective pressure to remain conserved than SOX9, and, therefore, diverge more across species than does SOX9. These results are consistent with evolutionary stratification of the mammalian sex determination pathway, analogous to that for sex chromosomes.

[High current microsecond pulsed hollow cathode lamp excited inductively coupled plasma ionic fluorescence spectrometry of Eu, Yb, Ca, Sr and Ba with an extended-sleeve torch].

Inductively coupled plasma (ICP) is a powerful atomizer for atomic fluorescence spectrometry (AFS), but high degree of ionization in high temperature of plasma at lower observation height and easy combination with oxygen at high observation height significantly decrease fluorescence intensity of refractory elements. Inoic fluorescence spectrometry (IFS) of Eu, Yb, Ca, Sr and Ba was investigated with high current microsecond pulsed (HCMP) hollow cathode lamps (HCLs) and an extended-sleeve torch adopted in commercial Baird Plasma AFS 2000 fluorescence spectrometer. Without introduction of any organic gas or solvent into the plasma, the detection limits (DLs) with HCMP-HCL-IFS was improved by about 1.5 order of magnitude (37x) for Sr, by over 2 orders of magnitude for Ba, and by nearly 1 order of magnitude for Eu and Yb as compared to those of conventionally pulsed (ICP-HCL-AFS). The HCMP IFS DLs of Eu and Yb were even superior to those of dye laser excited IFS reported in literatures.

AluY insertion (IVS4-52ins316alu) in the glycerol kinase gene from an individual with benign glycerol kinase deficiency.

Glycerol kinase deficiency has three distinct forms: an isolated form which may be benign or symptomatic, and a complex form which is symptomatic and part of an Xp21 contiguous gene syndrome. Here we report the case of a male with benign isolated glycerol kinase deficiency who was incidentally identified after observation of pseudohypertriglyceridemia. DNA sequencing of this subject's glycerol kinase gene showed the insertion of an AluY sequence in intron 4 of the glycerol kinase gene. Although Alu insertions have been implicated in other diseases, and a closely related AluY element is found as an insert in the C1 inhibitor gene in patients with hereditary angioedema, this is the first case of glycerol kinase deficiency caused by an Alu insertion.

Midkine is expressed early in rat fetal adrenal development.

Adrenal gland development is complex and poorly understood at the molecular level. Only a subset of patients with adrenal hypoplasia congenita (AHC) carry mutations in DAX1, a member of the nuclear hormone receptor superfamily. Therefore we set out to identify other candidate genes responsible for AHC by characterizing genes involved in fetal adrenal development. To identify these genes, we studied the differential expression of genes in fetal rat adrenals comparing tissues at 14 and 15 days postcoitum (dpc) since this period encompasses major morphological change in rat adrenal development. Fetal rat adrenals were dissected, cDNAs were prepared, and suppressive subtractive hybridization was performed. We isolated 126 clones of putatively differentially expressed clones and approximately 250 bp of each of the clones was sequenced. The most interesting putative developmental genes were examined. One member of the extracellular PTN/MDK (pleiotrophin/midkine) heparin-binding protein family involved in regulation of growth and differentiation was selected for initial study. We obtained full-length transcript by 3' rapid amplification of cDNA ends and performed Northern analysis on rat adrenal RNA from fetuses at 13, 14, 15, 17, and 19 dpc and newborns. Results from those analyses demonstrated the highest Mdk expression at days 13 and 14 followed by a moderate decrease of expression during the fetal stages thereafter. In the newborn, Mdk expression is nearly undetectable. Our results indicate that Mdk has a very specific pattern of fetal expression in the adrenals. We conclude that Mdk is involved early in fetal development of the rat adrenal. Therefore, MDK is a candidate gene for AHC not due to DAX1 mutations.

[Primary surgical operation in the treatment of varicosis of lower limb accompanied by chronic leg ulcer].

OBJECTIVE: Retrospective clinical analysis of primary surgical operation in the treatment of lower limb accompanied by chronic leg ulcer were adopted in this study. METHODS: From September 1990 to June 1998, there were 31 males and 20 females, aged 68 years in average, the area of ulcer varied from 5 cm x 3 cm to 22 cm x 11 cm. The ligation and strip of saphenous vein, debridement and free skin flap grafting were finished in primary operation. RESULTS: The skin flaps were survived completely in 50 cases, only 1 case was necrosis partially and healed after changing dressing. Forty-two cases were followed up for 16 months to 9 years (66 months in average), the varicosis and ulcer were healed in 39 cases and only 3 relapsed in ulceration. CONCLUSION: Primary surgical operation in the treatment of varicosis of lower limb accompanied by chronic ulcer is practicable in clinic. The curative efficacy is satisfactory and the operative manipulation is simple.

Bacterial species identification after DNA amplification with a universal primer pair.

The diagnosis of bacterial infections can be difficult and time consuming. Rapid and reliable molecular triage of potentially infected patients, particularly the young and the elderly, would prevent unnecessary hospitalizations, reduce associated medical costs, and improve the quality of care. Polymerase chain reaction (PCR) amplification utilizing a universal bacterial primer pair, followed by hybridization with species-specific probes, would allow rapid identification of the presence or absence of bacterial DNA, along with an identification of the bacterial species present. Molecular microbiological analyses will require access to bacterial strain standards that can be catalogued and distributed to clinical laboratories. We amplified template DNA in filter paper spots containing boiled bacteria from 14 clinical isolates using a universal primer pair for the 16S ribosomal RNA (rDNA) coding sequence. Species-specific probes were hybridized to the amplification products for bacterial species identification. We conclude that template DNA can be identified with species-specific probes after universal bacterial amplification with a single primer pair. We also demonstrate a rapid and efficient method for the long-term storage and cataloguing of bacterial DNA for use in quality control at clinical laboratories adopting molecular diagnostic methodologies. We speculate that PCR amplification combined with species-specific probe hybridization not only will represent an improvement over culture-based methods in terms of speed, sensitivity, and cost, but will also allow for the identification of unculturable bacteria and emerging or reemerging pathogenic organisms.

Derepression of human embryonic zeta-globin promoter by a locus-control region sequence.

A multiple protein-DNA complex formed at a human alpha-globin locus-specific regulatory element, HS-40, confers appropriate developmental expression pattern on human embryonic zeta-globin promoter activity in humans and transgenic mice. We show here that introduction of a 1-bp mutation in an NF-E2/AP1 sequence motif converts HS-40 into an erythroid-specific locus-control region. Cis-linkage with this locus-control region, in contrast to the wild-type HS-40, allows erythroid lineage-specific derepression of the silenced human zeta-globin promoter in fetal and adult transgenic mice. Furthermore, zeta-globin promoter activities in adult mice increase in proportion to the number of integrated DNA fragments even at 19 copies/genome. The mutant HS-40 in conjunction with human zeta-globin promoter thus can be used to direct position-independent and copy number-dependent expression of transgenes in adult erythroid cells. The data also supports a model in which competitive DNA binding of different members of the NF-E2/AP1 transcription factor family modulates the developmental stage specificity of an erythroid enhancer. Feasibility to reswitch on embryonic/fetal globin genes through the manipulation of nuclear factor binding at a single regulatory DNA motif is discussed.