Deregulation of MADS-box transcription factor genes in a mutant defective in the WUSCHEL-LIKE HOMEOBOX gene EVERGREEN of Petunia hybrida.

Angiosperm inflorescences develop in two fundamentally different ways. In monopodial plants, for example in Arabidopsis thaliana, the flowers are initiated as lateral appendages of a central indeterminate inflorescence meristem. In sympodial plants, flowers arise by terminal differentiation of the inflorescence meristem, while further inflorescence development proceeds from new sympodial meristems that are generated at the flank of the terminal flower. We have used the sympodial model species Petunia hybrida to investigate inflorescence development. Here, we describe a mutant, bonsai (bns), which is defective in flower formation, inflorescence branching, and control of meristem size. Detailed microscopic analysis revealed that bns meristems retain vegetative charateristics including spiral phyllotaxis. Consistent with a block in flower formation, bns mutants exhibit a deregulated expression of various MADS-box genes. Molecular analysis revealed that the bns mutant carries a transposon insertion in the previously described EVERGREEN (EVG) gene, which belongs to the WUSCHEL-LIKE HOMEOBOX (WOX) transcription factor gene family. EVG falls in the WOX9 subfamily, which has diverse developmental functions in angiosperms. The comparison of WOX9 orthologues in five model species for flowering shows that these genes play functionally divergent roles in monopodial and sympodial plants, indicating that the WOX9 regulatory node may have played an important role in the evolution of shoot architecture.

Five years of experience with the FiLaC™ laser for fistula-in-ano management: long-term follow-up from a single institution.

BACKGROUND: There are limited data available concerning endofistular therapies for fistula-in-ano, with our group reporting the first preliminary outcomes of the use of the radial fibre Fistula laser Closing (FiLaC ™) device. METHODS: The aim of this study was to assess a cohort of anal fistulae managed with laser ablation plus definitive flap closure of the internal fistula opening over a long-term follow-up. Factors governing primary healing success and secondary healing success (i.e. success after one or two operations) were determined. RESULTS: The study analysed 117 patients over a median follow-up period of 25.4 months (range 6-60 months) with 13 patients (11.1%) having Crohn's-related fistulae. No incontinence to solid and liquid stool was reported. Minor incontinence to mucus and gas was observed in two cases (1.7%), and a late abscess treated in one case (0.8%). The primary healing rate was 75/117 (64.1%) overall, and 63.5% for cryptoglandular fistulae versus 69.2% for Crohn's fistulae, respectively. Of the 42 patients who failed FiLaC™ 31 underwent a second operation ("Re-FiLaC™", fistulectomy with sphincter reconstruction or fistulotomy). The secondary healing rate, defined as healing of the fistula at the end of the study period, was 103/117 (88.0%) overall and 85.5% for cryptoglandular fistulae versus 92.3% for Crohn's fistulae. A significantly higher primary success rate was observed for intersphincteric-type fistulae with primary and secondary outcome unaffected by age, gender, presence of Crohn's disease, number of prior surgeries and the type of flap designed to close the internal fistula opening. CONCLUSIONS: There is a moderate primary success rate using first-up FiLaC™ treatment. If FiLaC™ fails, secondary success with repeat FiLaC™ or other approaches was high. The minimally invasive FiLaC™ approach may therefore represent a sensible first-line treatment option for anal fistula repair.

NOD1 gene polymorphisms in relation to aggressive periodontitis.

BACKGROUND: NOD proteins are part of innate immunity mechanisms. They play a role in epithelial barrier functions and inflammatory responses to bacteria. Single nucleotide polymorphisms (SNPs) in the NOD1 gene have proven to be associated with inflammatory bowel disease (IBD) and asthma. OBJECTIVE: To investigate SNPs in the NOD1 gene in relation to aggressive periodontitis (AgP), a multifactorial, inflammatory disease of the supporting tissues of the teeth. MATERIALS AND METHODS: Five SNPs in the NOD1 gene (4 intronic and 1 exonic) were tested for association in a total of 415 AgP patients and 874 controls both of Northern European ancestry. RESULTS: The frequencies of the rare SNP alleles ranged between 21% and 26% among cases, and 20-27% among controls, and were not statistically different between cases and controls. Two SNPs were in strong linkage disequilibrium (r(2) = 0.97 in cases and 0.94 in controls). The overall haplotype distributions did not differ between cases and controls. We observed 8 haplotypes with a frequency of >or=1% among cases and/or controls, but none of these haplotype frequencies differed significantly among cases and controls. Logistic regression analyses with adjustment for gender and smoking status did not reveal significant associations with AgP for any of the 5 SNPs. This study had a power of >or=95% to detect associations with variants carrying relative risks of >or=1.5 for heterozygote carriers and >or=2.25 for homozygote carriers. CONCLUSIONS: Although SNPs in the NOD1 gene have been strongly associated with cases of IBD, the current study failed to show an association of NOD1 SNPs with AgP.

Regulation of autophagy by the inositol trisphosphate receptor.

The reduction of intracellular 1,4,5-inositol trisphosphate (IP(3)) levels stimulates autophagy, whereas the enhancement of IP(3) levels inhibits autophagy induced by nutrient depletion. Here, we show that knockdown of the IP(3) receptor (IP(3)R) with small interfering RNAs and pharmacological IP(3)R blockade is a strong stimulus for the induction of autophagy. The IP(3)R is known to reside in the membranes of the endoplasmic reticulum (ER) as well as within ER-mitochondrial contact sites, and IP(3)R blockade triggered the autophagy of both ER and mitochondria, as exactly observed in starvation-induced autophagy. ER stressors such as tunicamycin and thapsigargin also induced autophagy of ER and, to less extent, of mitochondria. Autophagy triggered by starvation or IP(3)R blockade was inhibited by Bcl-2 and Bcl-X(L) specifically targeted to ER but not Bcl-2 or Bcl-X(L) proteins targeted to mitochondria. In contrast, ER stress-induced autophagy was not inhibited by Bcl-2 and Bcl-X(L). Autophagy promoted by IP(3)R inhibition could not be attributed to a modulation of steady-state Ca(2+) levels in the ER or in the cytosol, yet involved the obligate contribution of Beclin-1, autophagy-related gene (Atg)5, Atg10, Atg12 and hVps34. Altogether, these results strongly suggest that IP(3)R exerts a major role in the physiological control of autophagy.

Gene families from the Arabidopsis thaliana pollen coat proteome.

The pollen extracellular matrix contains proteins mediating species specificity and components needed for efficient pollination. We identified all proteins >10 kilodaltons in the Arabidopsis pollen coating and showed that most of the corresponding genes reside in two genomic clusters. One cluster encodes six lipases, whereas the other contains six lipid-binding oleosin genes, including GRP17, a gene that promotes efficient pollination. Individual oleosins exhibit extensive divergence between ecotypes, but the entire cluster remains intact. Analysis of the syntenic region in Brassica oleracea revealed even greater divergence, but a similar clustering of the genes. Such allelic flexibility may promote speciation in plants.

Alterations in CER6, a gene identical to CUT1, differentially affect long-chain lipid content on the surface of pollen and stems.

Very long chain lipids contribute to the hydrophobic cuticle on the surface of all land plants and are an essential component of the extracellular pollen coat in the Brassicaceae. Mutations in Arabidopsis CER genes eliminate very long chain lipids from the cuticle surface and, in some cases, from the pollen coat. In Arabidopsis, the loss of pollen coat lipids can disrupt interactions with the stigma, inhibiting pollen hydration and causing sterility. We have positionally cloned CER6 and demonstrate that a wild-type copy complements the cer6-2 defect. In addition, we have identified a fertile, intragenic suppressor, cer6-2R, that partially restores pollen coat lipids but does not rescue the stem wax defect, suggesting an intriguing difference in the requirements for CER6 activity on stems and the pollen coat. Importantly, analysis of this suppressor demonstrates that low amounts of very long chain lipids are sufficient for pollen hydration and germination. The predicted CER6 amino acid sequence resembles that of fatty acid-condensing enzymes, consistent with its role in the production of epicuticular and pollen coat lipids >28 carbons long. DNA sequence analysis revealed the nature of the cer6-1, cer6-2, and cer6-2R mutations, and segregation analysis showed that CER6 is identical to CUT1, a cDNA previously mapped to a different chromosome arm. Instead, we have determined that a new gene, CER60, with a high degree of nucleotide and amino acid similarity to CER6, resides at the original CUT1 locus.

Adaptive variation in lactate dehydrogenase-B gene expression: role of a stress-responsive regulatory element.

Although changes in gene regulation may play an important role in adaptive evolution, there have been few attempts to investigate the molecular mechanisms responsible for adaptively significant variation in gene expression. Here we describe the mechanism underlying an adaptive difference in the expression of the lactate dehydrogenase-B gene (Ldh-B) between northern and southern populations of the fish Fundulus heteroclitus. Ldh-B regulatory sequences from northern and southern individuals, coupled to a luciferase reporter gene, were introduced into the livers of live fish. Deletion studies indicated that sequence changes between 400 and 500 bp upstream of the transcription start site resulted in a 2-fold difference in reporter gene transcription. These sequence changes can account for the previously observed 2-fold difference in Ldh-B transcription between populations. Variation in transcription factors did not play an important role. Sequences within the functionally important region resemble a mammary tumor virus glucocorticoid responsive element (MTV-GRE) in southern alleles, whereas northern alleles differ from the consensus by 1 bp. To test the hypothesis that this element is involved in the variation between populations of F. heteroclitus, we exposed transiently transgenic fish containing Ldh-B regulatory sequence/reporter gene constructs to handling stress or injected cortisol. Both treatments increased reporter gene transcription driven by southern alleles but not northern alleles, as expected if an MTV-GRE sequence were involved. This finding suggests that sequence variation in a GRE is the cause of the adaptive differences in Ldh-B gene expression between populations and demonstrates that small changes in gene regulatory sequences can have important evolutionary consequences.

Pancreatic duct stenosis mimicking a tumor due to an aberrant vessel.

A case of a 74-year-old man is reported who had liver cirrhosis and a mass lesion in the left liver lobe. The search for a potential primary tumor showed a filiform duct stenosis in the pancreatic head on ERCP. The presumptive diagnosis of a pancreatic tumor with a liver metastasis was made. Due to the poor general condition of the patient no further diagnostic steps were undertaken and he died four weeks later from progressive liver failure. On autopsy an aberrant vessel originating from the superior mesenteric artery (arteria pancreatico-duodenalis dextra superior) was found to cause the ductal stenosis; an hepatocellular carcinoma in the left liver lobe, but no pancreatic tumor was detected.