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.

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.

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.

Genomic sequence of the DAX1 gene: an orphan nuclear receptor responsible for X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism.

The gene responsible for X-linked adrenal hypoplasia congenita, DAX1, encodes a member of the nuclear hormone receptor superfamily. We sequenced 8851 bp that contained the DAX1 genomic region. The DAX gene was composed of two exons and one 3.4-kilobase intron. Putative TATA and GC boxes and a putative steroidogenic factor 1 response element were present in the 5'-flanking region. Two potentially polymorphic short tandem repeats were identified. The first exon encoded two putative novel zinc finger motifs within a putative DNA binding domain and part of the ligand binding domain, and the second exon encoded the remainder of the ligand binding domain. Although the putative DNA binding domain of DAX1 does not contain substantial sequence similarity to other nuclear hormone receptor superfamily members, the putative ligand binding domain had remarkable similarity to other family members. Single-strand conformational polymorphism analysis permitted identification of three new mutations in DAX1. In conclusion, single-strand conformational polymorphism analysis facilitates identification of mutations in the DAX1 gene, and the short tandem repeats may permit linkage analysis in families in which mutations are not yet identified. We speculate that DAX1 may be the most primitive member of the nuclear hormone receptor superfamily identified in mammals.

Ahch, the mouse homologue of DAX1: cloning, characterization and synteny with GyK, the glycerol kinase locus.

We cloned the murine full-length cDNA encoding Ahch, the mouse homologue of DAX1 (DSS-AHC Region on Human X Chromosome, Gene1) which is the gene responsible for human X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH). Sequence analysis revealed that the murine and human cDNAs have 65% aa identity and 75% aa similarity overall. The cysteine residues in the putative DNA binding domain, which may interact with Zn2+ ions to form zinc fingers, are 100% conserved between the two species, indicating that the novel zinc-finger structures in DAX1 may be functional. In addition, mouse interspecific backcrosses show that the Ahch gene is closely linked to the glycerol kinase locus, GyK, on the mouse X chromosome, indicating that the order of the loci is conserved in this syntenic region between mouse and human.

Diagnosis of obstructive jaundice in infants: Tc-99m DISIDA in duodenal juice.

Technetium-99m di-isopropylphenylcarbamoylmethylimidodiacetic acid cholescintigraphy, together with measurements of radioactivity in duodenal juice, was used to evaluate 23 infants with prolonged obstructive jaundice. Four patients proved to have biliary atresia. The remainder had neonatal hepatitis. There was distinct differentiation of biliary atresia from neonatal hepatitis when the time-activity curves were analyzed. In neonatal hepatitis the radioactivity in duodenal juice is obviously higher, peaking above 1500 cpm/100 microliter per mCi dose. In biliary atresia the pattern is flattened, with maximal activity below 500 cpm/100 microliter per mCi dose.

An improved technique for coronary vascular anastomosis.

A vein holder is described that is used for anastomosis of both ends of aortocoronary bypass grafts. It minimizes handling of the graft, ensures precision in placing sutures with excellent visualization, and provides maximum patency at the anastomotic sites.

[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.

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.

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.

Ultrasonography and 99mTc-DISIDA cholescintigraphy in the diagnosis of choledochal cyst in children.

Eighteen children with choledochal cyst have underwent abdominal ultrasonography and 99mTc-DISIDA (di-isopropylphenylcarbamoyl-methylimidodiacetic acid) cholescintigraphy before surgery. All patients had typical sonographic findings of a large cystic mass in the porta hepatis with direct continuity to the biliary system. The gallbladder could be easily recognized and distinguished from the cystic mass in every except one case. Dilatation of the intrahepatic ducts was noted in 12 patients (67%). Coexisting biliary stones were found in 3 patients (17%). On the cholecintigrams, 14 patients (78%) had characteristic findings of a round or ovoid photon-deficient area in the porta hepatis with delayed filling and stasis of the tracer in the same area. Three infants (17%) showed a persistent round photon-defect over the porta hepatis without visualization of gallbladder or bowel radioactivity, which was thought to suggest a choledochal cyst with obstruction. A combination of ultrasonography and cholescintigraphy can provide a very accurate preoperative diagnosis of choledochal cyst.

[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.

Determination of femtomole/milliliter concentrations of enprostil acid in human plasma using high-performance liquid chromatography-laser-induced fluorescence detection.

This paper describes the use of multiple-column high-performance liquid chromatography (HPLC) combined with laser-induced fluorescence for the determination of femtomole/milliliter concentrations of enprostil acid, a prostaglandin analogue, in human plasma. The drug is isolated from plasma by phenyl solid-phase extraction and fluorescently labeled at its carboxyl functional group with a large excess of 2-bromoacetyl-6-methoxynaphthalene. A multi-column method using both normal- and reversed-phase chromatography is necessary to separate the labeled drug from the unreacted reagent. Post-column dilution of the mobile phase with water after the reversed-phase chromatography allows on-line concentration of the labeled analyte onto a guard column prior to the microbore HPLC. A loop guard column device provides a simple way to inject up to 1.0 ml of sample solution onto a microbore column without significantly reducing the column efficiency. A 325-nm He-Cd laser is used to excite the labeled drug, and fluorescence emission is monitored at 450 nm. Using this system, we are able to derivatize, detect, and quantify 5 pg of the prostaglandin analogue in 1.0 ml of plasma.