Highly efficient multipotent differentiation of human periodontal ligament fibroblasts induced by combined BMP4 and hTERT gene transfer.

Because periodontal ligament (PDL) cells are reported to contain progenitor or stem cell populations, they are considered a beneficial cell source for clinical periodontal regeneration. Both bone morphogenetic protein 4 (BMP4) and human telomerase reverse transcriptase (hTERT) have essential roles in the modulation of stem cell properties. In this study we report for the first time that the combined ectopic expression of BMP4 and hTERT significantly enhanced the multipotent differentiation efficiency and capacity of human PDL fibroblasts (PFs), as shown by osteogenic, adipogenic and neurogenic differentiation in vitro, and cementum/PDL-like tissue regeneration in vivo. These findings may be attributed, at least in part, to the original upregulation of important stem cell markers, such as scleraxis, Stro-1 and CD146, and the extremely lowered threshold for BMP concentration to activate BMP signaling by enhanced basal phosphorylation levels of Smad 1/5/8. In addition, the significantly reduced expression levels of CD146 and CD90 with the presence of Noggin confirms the direct effect of BMP4 on the stem cell-like phenotype of genetically modified PF cells (BT-PFs). Furthermore, BT-PFs exhibited a high neural differentiation capacity (>75%). After transplantation into NOD/SCID mice, genetically modified-PFs generated cementum/PDL-like structures on the surface of the carrier. The multipotency of these modified cells potentially provides an attractive source of stem cells for therapeutic purposes and regenerative medicine.

Proteomic identification of membrane proteins regulating antimicrobial peptide resistance in Vibrio parahaemolyticus.

AIMS: To identify proteins regulating antimicrobial peptide (AMP) resistance in Vibrio parahaemolyticus using membrane subproteome analysis. METHODS AND RESULTS: Three synthetic AMPs (Q4, Q6 and H1) and a natural one from fish (pleurocidin) were used for selection of AMP-resistant strains. Differential expression patterns of the outer and inner membrane proteins (OMPs and IMPs) among wild-type and the resistant strains were obtained using two-dimensional gel electrophoresis. Two OMPs (TolC and flagellin) and five IMPs [transcription termination factor (NusA), long-chain fatty acid transport protein (FadL), elongation factor Tu (EF-Tu), ATP synthase F1, alpha subunit (F1-ATPa) and dihydrolipoamide dehydrogenase (DLD)] were identified using LC-ESI-Q-TOF MS/MS and Mascot program. Real-time quantitative polymerase chain reaction was also performed to determine the mRNA expression level of the target genes. All seven membrane proteins except FadL were upregulated in the AMP-resistant clones, both in the translational and transcriptional levels. CONCLUSIONS: Our results suggested that V. parahaemolyticus may obtain their resistance against AMPs through upregulation of the multidrug efflux transporter, effective repair of damaged membranes and prevention of cellular penetration of AMPs. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report describing bacterial AMP resistance mechanism using proteomic methodologies. Elucidating the mechanism could help in the development of more sustainable antimicrobial agents.

Analysis of protein profiles in human epithelial ovarian cancer tissues by proteomic technology.

BACKGROUND: Screening in ovarian cancer is progressively finding out candidate genes and proteins which may work as screening biomarkers and play a role in tumor progression. We examined the protein expression patterns of ovarian cancer tissues using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of fight mass spectrometry (MALDI-TOF MS). METHODS: Tissues from 36 ovarian cancers and 20 normal ovaries were examined by 2-DE. The images of silver stained gels were analyzed by ImageMaster 2D Elite. The peptide mixtures, after in-gel digestion, were determined by MALDI-TOF MS for fingerprinting. The de-isotope tryptic peptide profiles were matched by using the Mascot search engine based on the entire NCBI and Swiss-Prot protein databases. Western/dot blots were then applied to verify the findings. RESULTS: In ovarian cancer, 12 proteins that showed differential expressions were identified unequivocally. Among these proteins, five proteins (galectin-1, cathepsin B, ubiquitin carboxy-terminal hydrolase L1, HLA class II antigen DRB1-11 and heat shock protein 27) were up-regulated and seven proteins (cellular retinol-binding protein, transthyretin, SH3 binding glutamic-rich-like protein, tubulin-specific chaperone A, DJ-1, gamma-actin and tropomyosin 4) were down-regulated. CONCLUSION: The present study is the first to report the up-regulation of ubiquitin carboxy-terminal hydrolase L1 and the down-regulation of SH3 binding glutamic-rich-like protein, tubulin-specific chaperone A, and tropomyosin 4 in human ovarian cancer tissues. Further cloning and functional analysis of these salient proteins will provide more information on their pathophysiologic roles in ovarian cancer.

Patients' Reports of Traumatic Experience and Posttraumatic Stress in Psychiatric Settings.

OBJECTIVE: To determine the prevalence of traumatic experience (TE) among patients in psychiatric settings in Hong Kong and the associations between TE and levels of distress and anxiety and depressive symptoms. METHODS: 129 patients who have received inpatient psychiatric services were recruited. Their lifetime TE was assessed using the Life Event Checklist (LEC), and TE in psychiatric settings using the Psychiatric Experiences Questionnaire (PEQ). Their level of distress symptoms was assessed using the Impact of Event Scale-Revised (IES-R), and the level of anxiety and depressive symptoms using the Hospital Anxiety and Depression Scale (HADS). RESULTS: The prevalence of direct and indirect TE was 84.5%, as was the prevalence of TE in psychiatric settings. Common TE in psychiatric settings included witnessing another patient being taken down (61.2%), being put in restraints of any kind (41.1%), and witnessing another patient being physically assaulted by another patient (36.4%). TE in psychiatric settings associated with high prevalence of severe or extreme distress 1 week after the event included being forced to take medication against their will (52.2%), being threatened with physical violence (52.2%), and experiencing a physical assault (50.0%). Lifetime TE (the total number of LEC items reported) was associated with severity of distress and anxiety and depressive symptoms, whereas TE in psychiatric settings (the total number of PEQ items reported) was associated with severity of distress only. The total number of LEC items reported is the only predictor of levels of distress and anxiety and depressive symptoms. CONCLUSIONS: Lifetime TE and TE in psychiatric settings are common among patients with SMI. Trauma-informed care is suggested for mental health services.

Salt-responsive outer membrane proteins of Vibrio anguillarum serotype O1 as revealed by comparative proteome analysis.

AIMS: Vibrio anguillarum is a universal marine pathogen causing vibriosis. Vibrio anguillarum encounters different osmolarity conditions between seawater and hosts, and its outer membrane proteins (OMPs) play a crucial role in the adaptation to changes of the surroundings. In the present study, proteomic approaches were applied to investigate the salt-responsive OMPs of V. anguillarum. METHODS AND RESULTS: Lower salinity (0.85% NaCl) is more suitable for growth, survival and swimming motility of the bacterium. Comparative two-dimensional electrophoresis (2-DE) analysis reveals six differentially expressed protein spots among three different salinities, which were successfully identified as OmpU, maltoporin, flagellin B, Omp26La, Omp26La and OmpW respectively. CONCLUSIONS: OmpW and OmpU were highly expressed at 3.5% salinity, suggesting their role in the efficient efflux of NaCl. Maltoporin was downregulated in higher salinity, indicating that higher osmolarity inhibits carbohydrate transport and bacterial growth. Omp26La, the homologue of OmpV, functions as a salt-responsive protein in lower salinity. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report describing salt stress-responsive proteins of V. anguillarum using proteomic approaches. Our results provide a useful strategy for delineating the osmoregulatory mechanism of the marine pathogens.

Kinetics and functional assay of liver repopulation after human cord blood transplantation.

BACKGROUND AND AIMS: To evaluate donor cell engraftment and the kinetics of cell repopulation in the injured mouse liver following human umbilical cord blood cell transplantation. METHODS: Nonobese diabetic/severe immunodeficient mice were treated with allyl alcohol to induce liver injury. Twenty-four hours later, umbilical cord blood derived mononuclear cells were transplanted by intra-splenic injection. Mice were sacrificed from 1 to 180 days after transplantation. Temporal changes in the ratio of human cells and fluorescence counts of human sex-determining region Y alleles in mouse liver were determined to evaluate the kinetics of cell repopulation. Mouse liver and sera were examined for the presence of human albumin. RESULTS: Human cell repopulation was extremely rapid in the first week following transplantation, with a doubling time of 1.16-1.39 days apparent. Thereafter cell doubling rate slowed significantly. Cells displaying characteristics of human hepatocytes were still evident at 180 days. Human albumin was detected in mouse liver and sera. CONCLUSION: These findings confirm those from previous studies demonstrating that cells derived from human umbilical cord blood have the capacity to differentiate into cells with human hepatocyte characteristics in mouse liver following injury. Moreover, the detailed information collected regarding the kinetics of human cell repopulation in mouse liver will be of relevance to future studies examining the use of umbilical cord blood cells in liver transplantation therapy.

Validation of the Hong Kong Cantonese Version of World Health Organization Five Well-Being Index for People with Severe Mental Illness.

OBJECTIVE: The World Health Organization Five Well-Being Index (WHO-5) has been developed to measure psychological wellbeing. Translation and linguistic validation of the WHO-5 into a Cantonese version has been accomplished for local use but it is not yet validated in people with severe mental illness in Hong Kong. This study aimed to examine the applicability of WHO-5 in measuring the psychological wellbeing dimension of people with severe mental illness. A brief and easily administrated tool to measure psychological wellbeing of people with severe mental illness can be used to provide an outcome measure in research studies and clinical trials. METHODS: Subjects were randomly recruited from the Extended-Care Patient Intensive Treatment, Early Diversion and Rehabilitation Stepping-Stone Project (EXITERS) and the Rehabilitation Activity Centre (RAC) of Kwai Chung Hospital in Hong Kong. They were invited to complete the abbreviated version of Hong Kong Chinese World Health Organization Quality of Life (WHOQOL-BREF [HK]) and WHO-5 (Cantonese version) separately and concurrent validity was examined. RESULTS: A total of 84 subjects were recruited, 42 each from EXITERS and RAC. In all, 49 (58%) were male and 35 (42%) were female. The mean ± standard deviation age was 43.2 ± 9.7 years. Their mean duration of mental illness was 16.4 ± 10.5 years and the mean years of education was 10.17 ± 2.5 years, i.e. about junior secondary school level in Hong Kong. The internal consistency of the WHO-5 was satisfactory (0.86) and was comparable with previous reports. Regarding validity, 1-factor structure with an eigenvalue of 3.24 explained 64.8% of total variance of WHO-5 for people with severe mental illness. Concurrent validity was established with moderate correlation (0.41-0.51) between WHO-5 and 4 domains of the WHOQOL-BREF (HK). CONCLUSION: The WHO-5 (Cantonese version) is a reliable and valid tool to assess the psychological wellbeing of people with severe mental illness in Hong Kong. It can be used to monitor the effectiveness of psychological intervention aimed at improving the wellbeing of such patients.

A sheep hydatid cyst glycoprotein as receptors for three toxic lectins, as well as Abrus precatorius and Ricinus communis agglutinins.

The binding properties of a glycoprotein with blood group P1 specificity isolated from sheep hydatid cyst fluid with Gal and GalNAc specific lectins was investigated by quantitative precipitin and precipitin inhibition assays. The glycoprotein completely precipitated Ricinus communis agglutinin (RCA1), Abrus precatorius agglutinin (APA) and Mistletoe toxic lectin-I (ML-I). Only 1.0 microgram of P1 glycoprotein was required to precipitate 50% of 5.1 micrograms ML-I nitrogen. It also reacted well with abrin-a and ricin, precipitating over 73% of the lectin nitrogen added, but poorly or weakly with Dolichos biflorus (DBL), Vicia villosa (VVL, a mixture of A4, A2B2 and B4), VVL-B4, Arachis hypogaea (PNA), Maclura pomifera (MPL), Bauchinia purpurea alba (BPL) and Wistaria floribunda (WFL) lectins. When an inhibition assay in the range of 5.1 micrograms N to 5.9 micrograms N of lectins (ML-I, abrin-a; ricin, RCA1, and APA, and 10 micrograms P1 active glycoprotein interaction was performed; from 76 to 100% of the precipitations were inhibited by 0.44 and 0.52 mumol of Gal alpha 1-->4Gal and Gal beta 1-->4GlcNAc, respectively, but not or insignificantly with 1.72 mumol of GlcNAc. The Gal alpha 1-->4Gal disaccharide found in this P1 active glycoprotein is a frequently occurring sequence of many glycosphingolipids located at the surface of mammalian cell membranes, especially human erythrocytes and intestinal cells for ligand binding and microbial toxin attachment. The present finding suggests that the Gal alpha 1-->4Gal beta 1-->4GlcNAc sequence in this P1 active glycoprotein is one of the best glycoprotein receptors for three toxic lectins (ricin, abrin-a, and ML-I) as well as for APA, and RCA1, and the result of inhibition assay implies that these lectins are recognizing part or all of the Gal alpha 1-->4Gal beta 1-->4GlcNAc sequence in the P1 active glycoprotein.

Orbitofrontal cholesterol granuloma.

Cholesterol granuloma of the orbital bones is a rare but readily recognisable condition. It is an osteolytic lesion with a granulomatous reaction surrounding cholesterol crystals, old haemorrhage and a fibrous capsule. There is a male preponderance and it usually occurs in young or middle-aged men. It is treatable with drainage and curettage via an orbitotomy, and craniotomy or wide bone removal is almost never required. Six cases of this condition were reviewed to highlight the typical clinical presentation, computed tomography and magnetic resonance results, and surgical management.

The complete primary structure of abrin-a B chain.

The complete 267 amino acid sequence of abrin-a B chain was determined by analysis of peptides obtained by digestion with trypsin, chymotrypsin, lysyl endopeptidase, Staphylococcus aureus V8 protease and thermolysin. The sequence is not identical with that predicted previously by nucleotide sequencing, indicating the presence of isoforms of abrin. Comparison of the amino acid sequence of abrin-a B chain with that of ricin-D B chain reveals a high degree of sequence identity (59%). Abrin-a B chain appears to consist of two domains, each domain with subdomains (alpha, beta, gamma) of about 40 amino acid residues.

Native and/or asialo-Tamm-Horsfall glycoproteins Sd(a+) are important receptors for Triticum vulgaris (wheat germ) agglutinin and for three toxic lectins (abrin-a, ricin and mistletoe toxic lectin-I).

The binding properties of human Tamm-Horsfall Sd(a+) urinary glycoprotein (THGP) and asialo-THGP with Triticum vulgaris agglutinin(WGA) and three toxic lectins (abrin-a, ricin, and Mistletoe toxic lectin-I) were investigated by quantitative precipitin and precipitin inhibition assays. Both glycoproteins reacted strongly with abrin-a, precipitating over 80% of the lectin nitrogen tested. THGP also bound well to mistletoe toxic lectin-I and precipitated 86% of this lectin added, while the precipitability of its asialo product decreased by 28%. The native glycoprotein completely precipitated the WGA added, but its reactivity was reduced dramatically after desialylation. On the contrary, the poor reactivity of THGP with ricin increased substantially after removal of sialic acid and completely precipitated the lectin added. The glycoprotein-lectin interactions were inhibited by one or several of the following haptens, p-NO2-phenyl alpha GalNAc, p-NO2-phenyl beta GalNAc, Gal beta 1-->4GlcNAc, Gal beta 1-->4Glc, GlcNac beta 1-->4GlcNAc and/or GlcNAc. From the above results, it is concluded that native and/or Tamm-Horsfall glycoproteins serve as important receptors for these three toxic lectins and for WGA.

Bond-specific chemical cleavages of peptides and proteins with perfluoric acid vapors: novel peptide bond cleavages of glycyl-threonine, the amino side of serine residues and the carboxyl side of aspartic acid residues.

Peptide bond cleavages by vapors composed of various from aqueous solutions of perfluoric acid were studied using synthetic peptides and proteins, and specific conditions were established for peptide bond cleavages including a novel cleavage of the glycyl-threonine bond. The peptide bonds on the aminosides of serine residues were cleaved by exposure to a vapor of 75% aqueous heptafluorobutyric acid at 30 or 50 degrees C for 24 h. Glycyl-threonine peptide bonds were cleaved with vapors of various concentrations (5, 75, and 90%) of heptafluorobutyric acid at 30-40 degrees C for 24 h. The peptide bonds on the carboxylsides of aspartic acid residues were cleaved by exposure to a vapor of 0.2% heptafluorobutyric acid at 90 degrees C for 4 to 24 h. The same vapor cleaved aspartyl-proline bonds under milder conditions such as at 60 degrees C for 16 h, under which the other aspartyl bonds were uncleaved. These specific chemical cleavages were applied to several proteins including newly characterized proteins.

Partial Amino Acid Sequences of Peptidyl-Prolyl Isomerases ofFusarium sporotrichioides.

Peptidyl-proprylyl cis-trans isomerase (PPIase) activity was observed from crude extract of Fusarium sporotrichioides. Proteins from this fungi were separated by two-dimensional polyacrylamide gel electrophoresis and more than one thousand protein spots were separated. Two cytosolic PPIases were found by the N-terminal sequencing from the two separated spots. The N-terminal 41 residues of the major protein spot showed high sequence identity (63.4%) with PPIase from Neurospora crassa. This protein was designated as PPIase a, having an apparent molecular mass of 20 kD and pI 7.0. The minor other protein spot, having a similar molecular mass but distinguishable pI 6.4, was also sequenced and the N-terminal twenty residues were almost identical to PPIase a and was designated as PPIase b. Copyright 1995 S. Karger AG, Basel

Amino Acid Sequence of Trichoanguina, a Ribosomal-Inactivating Protein from Trichosanthes anguinea Seeds.

In this study, we sequenced a new type I ribosome-inactivating protein, trichoanguina, from the seeds of Trichosanthes anguina (snake gourd). Trichoanguina is a basic glycoprotein having an apparent molecular mass of 35.0 kD and possessing strong ribosome-inactivating activity. Trichoanguina was cleaved with cyanogen bromide and partially digested with thermolysin, chymotrypsin, trypsin and Staphylococcus aureus V8 protease. The subsequent peptide fragments were separated by SDS-polyacrylamide gel electrophoresis, followed by electroblotting to polyvinylidene difluoride membranes and then sequencing. The sequencing of trichoanguina was completed, consisting of 245 amino acid residues. The sequencing of trichoanguina revealed a considerable homology to trichosanthin and alpha-trichosanthin, which are known as abortifacient, ribosome-inactivating and antihuman immunodeficiency virus proteins, with 46.7% and 55.6% amino acid identities, respectively. The sequence conserves two active sites: Glu-158 and Arg-161. Copyright 1996 S. Karger AG, Basel

Resolution of Fusarium sporotrichioides Proteins by Two-Dimensional Polyacrylamide Gel Electrophoresis and Identification by Sequence Homology Comparison in Protein Data Base.

Proteins from Fusarium sporotrichioides M-1-1, a T2-toxin-producing strain, were separated by two-dimensional polyacrylamide gel electrophoresis. One thousand two hundred and forty-four protein spots were resolved and 103 protein spots were subjected to N-terminal sequencing. Fifty-eight protein spots were sequenced and 48 proteins were observed to have blocked N termini. Forty out of 58 sequenced proteins were identified by homology search against the PIR protein sequence data base and protein superfamily data base, while the residual 18 sequences were not identified. Twenty-seven of the N-terminal-blocked proteins were subjected to mild anhydrous hydrazine vapor deblocking. Twenty-four spots were not deblocked indicating the presence of acyl groups at the N termini, while 3 proteins were deblocked showing the blocked group to be pyrroglutamyl carboxylic acid residues. The results can provide a more global view of cellular genetic expression than any other technique. The created data may offer a unique opportunity to link information with DNA sequence data. Copyright 1995 S. Karger AG, Basel

Carbohydrate specificity of a toxic lectin, abrin A, from the seeds of Abrus precatorius (jequirity bean).

To elucidate of the mechanism of intoxication, the affinity of a toxic lectin, abrin A, from the seeds of Abrus precatorius for mammalian carbohydrate ligands, was studied by enzyme linked lectinosorbent assay and by inhibition of abrin A-glycan interaction. From the results, it is concluded that: (1) abrin A reacted well with Gal beta1-->4GlcNAc (II), Gal alpha1-->4Gal (E), and Gal beta1-->3GalNAc (T) containing glycoproteins. But it reacted weakly with sialylated gps and human blood group A,B,H active glycoproteins (gps); (2) the combining site of abrin A lectin should be of a shallow groove type as this lectin is able to recognize from monosaccharides with specific configuration at C-3, C-4, and deoxy C-6 of the (D)Fuc pyranose ring to penta-saccharides and probably internal Gal alpha,beta-->; and (3) its binding affinity toward mammalian structural features can be ranked in decreasing order as follows: cluster forms of II, T, B/E (Gal alpha1-->3/4Gal) > monomeric T > monomeric II > monomeric B/E, Gal > GalNAc > monomeric I >> Man and Glc (inactive). These active glycotopes can be used to explain the possible structural requirements for abrin A toxin attachment.

Oral contraceptive use and diseases of the circulatory system in Taiwan: an analysis of mortality statistics.

Whether or not increased prevalence of oral contraceptive use in developing countries is associated with increased morbidity and mortality from circulatory system diseases is a serious concern for health and family planning administrators. An analysis of Taiwan's mortality statistics to determine whether such an association existed was undertaken, taking advantage of the excellent vital statistics and medical care systems existing there. The analysis failed to demonstrate that death rates from circulatory system diseases among Taiwanese women of childbearing age have increased since the promotion of oral contraceptive use began in 1967. In fact, mortality from all forms of circulatory system diseases has generally declined since 1967 and the slopes of declines for females of childbearing age who were exposed to oral contraceptives were somewhat greater than those of males of the corresponding age groups.

PIP: Whether or not increased prevalence of oral contraceptive use in developing countries is associated with increased morbidity and mortality from circulatory system diseases is a serious concern for health and family planning administrators. Analysis of Taiwan's mortality statistics to determine whether such an association existed was undertaken, taking advantage of the excellent vital statistics and medical care systems there. The analysis failed to demonstrate that death rates from circulatory system disease have generally declined since 1967, and the slopes of declines for females of childbearing age who were exposed to oral contraceptives were somewhat greater than males of the corresponding age groups. Another study conducted by the World Health Organization found that cases of deep-vein thrombosis were less common among Thai women than among English women. Few women in Taiwan smoke cigarettes. The prevalence of oral contraceptive use in Taiwan was low, never exceeding 10% of the married women of childbearing age. The average duration of pill use by Taiwanese women has been short, 10.5 months. The mortality rate from circulatory system disease among childbearing women, which averaged 38.9 during the prepill period (before 1967) has declined significantly to 27.6 in 1976 and 25.8 in 1977.

Oral contraceptives and stroke: a preliminary report on an epidemiologic study in Taiwan, China.

To investigate risk of stroke following use of oral contraceptives (OC) in less developed countries, a case-control study was conducted in Taiwan involving 323 cases hospitalized in 1978-1980, 250 hospital controls, and 646 neighborhood controls matched with cases on several variables. All were alive at interview. For the total sample, no increased risk was found. However, when comparison is with hospital controls, the risk for the recent OC users was increased (OR = 3.93, 95% C.L. 1.21, 5.46). This latter finding is consistent with studies in developed countries.

Oral contraceptives and mortality from circulatory system diseases: an epidemiologic study in Taiwan.

An epidemiological study on the association between oral contraceptive (OC) use and circulatory system disease (CSD) mortality undertaken in Taiwan by proxy interview of closest family members of 306 deceased cases, 305 deceased controls, and 611 healthy neighborhood controls indicated that the relative risk between the cases and health controls was 0.98 (0.62-1.54) and that between cases and health controls was 0.82 (0.56-1.21). Use of OC by Taiwanese women has not produced any overwhelming adverse effects on CSD mortality.