ß-catenin downregulates Dicer to promote ovarian cancer metastasis.

Ovarian cancer is a nearly uniform lethal disease and its highly aggressive metastatic phenotype portends a poor prognosis. Lack of a well-controlled, relevant experimental model has been a major obstacle to identifying key molecules causing metastasis. Here we describe the creation of a new isogenic model of spontaneous human ovarian cancer metastasis exhibiting opposite phenotypes-highly metastatic (HM) and non-metastatic (NM)-both in vitro and in vivo. HM was unique in its ability to metastasize consistently to the peritoneum, mimicking the major dissemination route of human ovarian cancer. In contrast, NM failed to form detectable metastases, although it was equally tumorigenic. Using comparative label-free quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS), we identified ß-catenin, which we demonstrated for the first time as having a direct role in the pathogenesis of ovarian cancer metastasis. Our studies also revealed a previously unrecognized role of ß-catenin in the downregulation of multiple microRNAs (miRNAs) through attenuating miRNA biogenesis by targeting Dicer, a key component of the miRNA-processing machinery. One such downregulated miRNAs was miR-29s involved in epithelial-to-mesenchymal transition and subsequent stem cell traits. Silencing ß-catenin or overexpressing Dicer or miR-29 mimics in HM significantly reduced the ability of these cells to migrate. ß-catenin-knockdown cells also failed to metastasize in an orthotopic model of ovarian cancer. Meta-analysis revealed an increase in CTNNB1 and a decrease in DICER1 expression levels in the high-risk group. These results uncover ß-catenin as a critical factor in promoting ovarian cancer aggressiveness and a new mechanism linking between ß-catenin and miRNA downregulation underlying this process.

Electron ionization dissociation of singly and multiply charged peptides.

A new tandem mass spectrometry technique, electron ionization dissociation (EID), employs irradiation of trapped cations [M + nH](n+) (n > or = 1) by fast electrons with energy at least 10 eV higher than the ionization threshold of the cations. Such irradiation causes simultaneous ionization and electronic excitation of the irradiated species, which is equivalent of double-ionization to [M + nH]((n+2)+) followed by electron capture to form electronically excited [M + nH]((n+1)+*)* ions. Subsequent fragmentation of the latter species gives both side-chain losses and backbone fragmentation. Theoretical efficiency of such fragmentation calculated as a ratio of the fragment ion current and the precursor ion depletion can reach (n+1)/n, that is, can exceed 100%. EID often leads to backbone N-C(alpha) bond cleavage, giving c-/z-type fragments. C-C bond cleavage, giving preferentially a-/x-type fragments, is also observed. EID could be used in bottom-up proteomics for both electrospray and matrix-assisted laser desorption ionization (MALDI) -produced ions. The energy incorporated into the precursor ion in a single interaction with electrons can exceed 10 eV, which makes EID suitable for top-down analysis of folded gas-phase protein conformations.

Histidine-containing radicals in the gas phase.

Radicals containing the histidine residue have been generated in the gas phase by femtosecond electron transfer to protonated histidine-N-methylamide (1H+), Nalpha-acetylhistidine-N-methylamide (2H+), Nalpha-glycylhistidine (3H+), and Nalpha-histidylglycine (4H+). Radicals generated by collisional electron transfer from dimethyldisulfide to ions 1H+ and 2H+ at 7 keV collision energies were found to dissociate completely on the microsecond time scale, as probed by reionization to cations. The main dissociations produced fragments from the imidazole side chain and the cleavage of the C(alpha)CO bond, whereas products of NCalpha bond cleavage were not observed. Electron transfer from gaseous potassium atoms to ions 3H+ and 4H+ at 2.97 keV collision energies not only caused backbone NCalpha bond dissociations but also furnished fractions of stable radicals that were detected after conversion to anions. Ion structures, ion-electron recombination energies, radical structures, electron affinities, and dissociation and transition-state energies were obtained by combined density functional theory and Møller-Plesset perturbational calculations (B3-PMP2) and basis sets ranging from 6-311+G(2d,p) to aug-cc-pVTZ. The Rice-Ramsperger-Kassel-Marcus theory was used to calculate rate constants on the B3-PMP2 potential energy surfaces to aid interpretation of the mass spectrometric data. The stability of Nalpha-histidylglycine-derived radicals is attributed to an exothermic isomerization in the imidazole ring, which is internally catalyzed by reversible proton transfer from the carboxyl group. The isomerization depends on the steric accessibility of the histidine side chain and the carboxyl group and involves a novel cation radical-COO salt-bridge intermediate.

Bifurcating fragmentation behavior of gas-phase tryptic peptide dications in collisional activation.

Collision-activated dissociation (CAD) of tryptic peptides is a cornerstone of mass spectrometry-based proteomics research. Principal component analysis of a database containing 15,000 high-resolution CAD mass spectra of gas-phase tryptic peptide dications revealed that they fall into two classes with a good separation between the classes. The main factor determining the class identity is the relative abundance of the peptide bond cleavage after the first two N-terminal residues. A possible scenario explaining this bifurcation involves trans- to cis-isomerization of the N-terminal peptide bond, which facilitates solvation of the N-terminal charge on the second backbone amide and formation of stable b(2) ions in the form of protonated diketopiperazines. Evidence supporting this scenario is derived from statistical analysis of the high-resolution CAD MS/MS database. It includes the observation of the strong deficit of a(3) ions and anomalous amino acid preferences for b(2) ion formation.

Electron capture dissociation of peptides metalated with alkaline-earth metal ions.

The possible use of divalent alkaline-earth metal ions, including Mg2+, Ca2+, Sr2+, and Ba2+, as charge carrier for electron capture dissociation of peptides was investigated. Model peptides of RGGGVGGGR and NGGGWGGGN were used to simplify the interpretation of spectral information. It was demonstrated that useful electron capture dissociation (ECD) tandem mass spectra of these metalated peptides could be generated. Interestingly, peptides metalated with different alkaline-earth metal ions generated very similar ECD tandem mass spectra. Metalated c-ions and z-ions were the predominant fragment ions. Only Mg2+-metalated peptides gave somewhat different results. Some nonmetalated c-ions were observed from ECD of [RGGGVGGGR + Mg]2+ but not from [NGGGWGGGN + Mg]2+. Together with some ab initio calculations, it was established that the bound metal ions might activate the acidity of the amide hydrogen. With the presence of high proton affinity moiety, such as N-terminal amino group and/or side chain of the arginine residues, the metalated peptide ions could exist predominantly in their zwitterion forms, in which one or two backbone amide group(s) was deprotonated and the high proton affinity functional group(s) was protonated. It was believed that electron capture leads primarily to the reduction of the mobile proton rather than the metal ions. With this zwitterion model, the formation of nonmetalated c-fragments and the generation of similar ECD spectra for peptides metalated with various alkaline-earth metal ions could readily to be explained. Another interesting observation in the ECD mass spectra of metalated peptides is related to the enhanced formation of the minor ECD products, i.e., (c - 1)(+*) and (z + 1)+ ions. Together with ab initio calculations using a truncated peptide model, various possible reaction mechanisms for the formation of these minor ECD products were evaluated. It was concluded that hydrogen transfer between the initiated formed c and z(.) species plays an important role in the formation (c - 1)(+*) and (z + 1)+ ions. Although peptides metalated with these metal ions do not have better ECD efficiency compared to the multiply-protonated peptides, it provides practical accessibility of ECD methods to analyze small peptides with no basic amino acid residues.

Experimental and theoretical investigations of the loss of amino acid side chains in electron capture dissociation of model peptides.

Loss of side chains from different amino acid residues in a model peptide framework of RGGGXGGGR under electron capture dissociation conditions were systematically investigated, where X represents one of the twenty common amino acid residues. The alpha-carbon radical cations initially formed by N-Calpha cleavage of peptide ions were shown to undergo secondary dissociation through losses of even-electron and/or odd-electron side-chain moieties. Among the twenty common amino acid residues studied, thirteen of them were found to lose their characteristic side chains in terms of odd-electron neutral fragments, and nine of them were found to lose even-electron neutral side chains. Several generalized dissociation pathways were proposed and were evaluated theoretically with truncated leucine-containing models using ab initio calculations at B3-PMP2/6-311++G(3df,2p)//B3LYP/6-31++G(d,p) level. Elimination of odd-electron side chain was associated with the initial abstraction of the hydrogen from the alpha-carbon bearing the side chain by the N-terminal alpha-carbon radical. Subsequent formation of alpha-beta carbon-carbon double bond leads to the elimination of the odd-electron side chain. The energy barrier for this reaction pathway was 89 kJmol-1. This reaction pathway was 111 kJmol-1 more favorable than the previously proposed pathway involving the formation of cyclic lactam. Elimination of even-electron side chain was associated with the initial abstraction of the gamma-hydrogen from the side chain by the N-terminal alpha-carbon radical. Subsequent formation of beta-gamma carbon-carbon double bond leads to the elimination of the even-electron side chain and the migration of the radical center to the alpha-carbon. The energy barrier for this fragmentation reaction was found to be 50 kJmol-1.

A comparative study of the collision induced dissociation and the electron capture dissociation of model peptides using ESI-FTMS.

Tandem mass spectra of several model peptides, including KG3WG3K, NG3WG3N, RG7R, RG3WG3R, RG3DG3R, RG3EG3R, RG3FG3R and RG5WG5R were studied using both SORI-CID and ECD methods. By cross comparing the fragmentation pattern of these peptides using the same dissociation method and the same peptide using different dissociation methods, interesting spectral features that are related to the mechanisms of dissociation under SORI-CID and ECD conditions were extracted. Both dissociation methods were believed to be charge-directed. Due presumably to the stepwise ion activation, peptide ion dissociation under SORI-CID conditions was influenced mainly by "localized" hydrogen bonds. Consistent with previous literature findings, mobility proton model could be used to account for the spectral features observed. Substantial changes in the fragmentation patterns of these peptides were observed by using ECD methods. By postulating that the initial tertiary structures of the peptide ions were retained prior to electron capture process, the changes in fragmentation pattern could be attributed to the directing effect of the "global" hydrogen-bonding network. From the present results, no special preference was observed for cleavage at the backbone linkages adjacent to tryptophan residue over other inter-residual linkages. The previous reported nine-times cleavage preference at the C-terminal side of the tryptophan residue should therefore be attributed to some sequence specific phenomena.

A study of fast and metastable dissociations of adenine-thymine binary-base oligonucleotides by using positive-ion MALDI-TOF mass spectrometry.

In the present study, fast and metastable dissociations of a number of adenine-thymine binary-base oligonucleotides under the conditions of UV matrix-assisted laser desorption/ionization mass spectrometry were investigated. 2-Aminobenzoic acid/ammonium fluoride (ABA/NH4F) matrix system was used. The spectra obtained under metastable and fast dissociation conditions exhibit distinctive dissociation products. From the post-source-decay analysis, all oligonucleotides underwent predominantly metastable dissociations at the 3' C-O linkages to form [a(n)-B]+ and w(n)+ complimentary ion series. Based on the present results, the so-called "[wn+80]+" ions were postulated to be the complimentary [Z(8-n)AH]+ ions rather than the expected phosphate rearrangement products. In addition, these oligonucleotides were found to generate fast dissociation products of b(n)+, d(N)+, w(N)+ and y(N)+ ions through backbone cleavages at 5' C-O, 5' O-P, 3' C-O and 3' P-O linkages, respectively. Product ion series formed under PSD conditions were not observed. The implications of this mutually exclusive occurrence of the two sets of fragment ions under fast and metastable conditions using ABA/NH4F matrix would be discussed. A model of ion activation under UV-MALDI conditions was also proposed.

Genital prolapse: a legacy of the West?

A retrospective study was undertaken to determine if there has been a change in the incidence of genital prolapse and urinary stress incontinence in Chinese women in Hong Kong. The medical records of 2,312 consecutive patients admitted for major gynaecological surgery for benign conditions in 5 out of 7 major public teaching hospitals in Hong Kong for the calendar year of 1993 were reviewed by way of an audit of all gynaecological operations performed in these hospitals. A total of 578 cases (25%) were identified as having genital prolapse and or urinary problems.

Polyhydramnios and hypercalcemia associated with congenital mesoblastic nephroma: case report and a new appraisal.

BACKGROUND: Polyhydramnios and hypercalcemia are known complications of congenital mesoblastic nephroma. Hypercalcemia has been shown to cause polyuria. Polyuria is believed to be the probable cause for the polyhydramnios, but the exact mechanism remains unknown. To our knowledge, this is the first reported case of a relationship between hypercalcemia and polyhydramnios. CASE: A 26-year-old primigravida was diagnosed with congenital mesoblastic nephroma and polyhydramnios at 36 weeks' gestation. Neonatal hypercalcemia was detected immediately after delivery at 40 weeks' gestation. After removal of the nephroma, the calcium level normalized. CONCLUSION: Hypercalcemia may be the mechanism underlying polyhydramnios in cases of congenital mesoblastic nephroma. Calcium levels should be monitored after delivery, and prompt surgical removal of the tumor should be performed.

A prospective study of the treatment of cervical intraepithelial neoplasia by loop electrosurgical excision procedure (LEEP) in Hong Kong population.

From May to December 1990, 72 patients suffering from cervical intraepithelial neoplasia confirmed with guided biopsy were treated with loop electrosurgical excision procedure (LEEP) as outpatients under local anaesthesia. Overall success rate of a single treatment at 6 weeks follow-up was 88.9%. All the specimens obtained from the procedure were of good quality for histological examination. The overall significant complication rate of the procedure was 4.2% during treatment and 8.4% after treatment. The results suggested that LEEP is an effective and safe outpatient treatment for CIN lesions. It is easy to use and readily acceptable to patients. It also has the advantage of providing histological confirmation and more economical than the use of laser.

Malignant lymphocyst after Wertheim's operation.

Lymphocyst is a well-known complication after Wertheim's operation with an incidence varying from 2 to 20%. The majority are asymptomatic. However, when complications occur, the symptoms depend on the location and the pressure effects created. Most of the lymphocysts occur within 1 year after surgery and need to be differentiated from a hematoma or urinoma. At present, there is no standard management of early lymphocysts. Conservative management, ultrasound-guided needle aspiration, or percutaneous insertion of an indwelling catheter have been successfully employed. Intraperitoneal marsupilization with or without omental falp is also highly effective. When pelvic lymphocysts occur later than normal, the diagnostic dilemma is to differentiate benign collections from those involving recurrent tumor. Fine-needle biopsy of the cyst wall under ultrasound guidance is more effective in identifying recurrence than cytological evaluation of the fluid. Nonetheless, if such facility is not readily available, surgical drainage and excision of the cyst wall should be considered to ensure early diagnosis of recurrence.

Atopic disease in the Hong Kong Chinese.

One hundred and sixty Chinese men and 96 women resident in Hong Kong completed a questionnaire about atopic disease and their responses were compared with replies from 500 Britons. Asthma and hayfever were less common in the Chinese and this could not be attributed to medical awareness as the results were similar in Chinese surgical patients and Chinese medical students. The role of heredity and environment may be assessed by studying Chinese people who have moved to Britain.

Filter-mediated color vision with one visual pigment.

The compound eye of the grasshopper Phlaeoba has alternating bands that appear clear or brown. Electroretinograms recorded from the individual bands have different action spectra: The spectrum of the clear band peaks at 525 nanometers and that of the brown band at 545 nanometers. Spectrally selective whole-eye adaptation with light of eight long of short wavelength yields identical action spectra. This evidence suggests that this eye has only one visual pigment, whose spectrum is altered in the brown bands by a screening pigment. In behavioral tests of spontaneous choices between stimuli that appear green to the normal human and those that appear red, the green stimuli are preferred even when the relative intensity is varied by 0.9 log units around the equal-brightness level (determined by the electroretinogram). When some red light is mixed with the green light, the preference for the mixture is less than for the green light alone, even though the mixture is more intense. True color vision therefore seems to exist. Painting the bands shows that behavioral color vision requires the presence of both types. These data suggest that Phlaeoba has true color vision mediated by one visual pigment and suitable optical filters.