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PubMed2005

Proteome analysis of the medicinal plant Catharanthus roseus.

A proteomic approach is undertaken aiming at the identification of novel proteins involved in the alkaloid biosynthesis of Catharanthus roseus. The C. roseus cell suspension culture A11 accumulates the terpenoid indole alkaloids strictosidine, ajmalicine and vindolinine. Cells were grown for 21 days, and alkaloid accumulation was monitored during this period. After a rapid increase between day 3 and day 6, the alkaloid content reached a maximum on day 16. Systematic analysis of the proteome was performed by two-dimensional polyacrylamide gel electrophoresis. After day 3, the proteome started to change with an increasing number of protein spots. On day 13, the proteome changed back to roughly the same as at the start of the growth cycle. 88 protein spots were selected for identification by mass spectrometry (MALDI-MS/MS). Of these, 58 were identified, including two isoforms of strictosidine synthase (EC 4.3.3.2), which catalyzes the formation of strictosidine in the alkaloid biosynthesis; tryptophan synthase (EC 4.1.1.28), which is needed for the supply of the alkaloid precursor tryptamine; 12-oxophytodienoate reductase, which is indirectly involved in the alkaloid biosynthesis as it catalyzes the last step in the biosynthesis of the regulator jasmonic acid. Unique sequences were found, which may also relate to unidentified biosynthetic proteins.

Alkaloids↗
PubMed2005

Gambol and Tc1 are two distinct families of DD34E transposons: analysis of the Anopheles gambiae genome expands the diversity of the IS630-Tc1-mariner superfamily.

Tc1 is a family of DNA transposons found in diverse organisms including vertebrates, invertebrates and fungi. Tc1 belongs to the IS630-Tc1-mariner superfamily, which is characterized by common 'TA' target site and conserved D(Asp)DE(Glu) or DDD catalytic triad. All functional Tc1-like transposons contain a transposase with a DD34E catalytic triad. We conducted a systematic analysis of DD34E transposons in the African malaria mosquito, Anopheles gambiae, using a reiterative and exhaustive search program. In addition to previously described Tc1-like elements, we uncovered 26 new DD34E transposons including a novel family that we named gambol. Designation of family status to gambol is based on phylogenetic analyses of transposase sequences that showed gambol and Tc1 transposons as distinct clades that were separated by mariner and other families of the IS630-Tc1-mariner superfamily. The distinction between Tc1 and gambol is also consistent with the unique TIRs in gambol elements and the presence of a 'W[I/L/V]DEDC' signature near their N-termini. This signature is predicted as part of the 'RED' domain, a component of the 'PAI' and 'RED' DNA binding domains in Tc1 and possibly mariner. Although gambol appears to be related to a few DD34E transposons from cyanobacteria and fungi, no gambol has been reported in any other insects or animals thus far. Several gambol and Tc1 elements have intact ORFs and different genomic copies with high sequence identity, which suggests that they may have been recently active.

Amino Acid Sequence↗
PubMed1986

AIDS risk-reduction guidelines: a review and analysis.

Until an effective treatment or vaccine for AIDS is developed, the principal strategy for controlling its spread will remain persuading at-risk and diseased populations to modify behaviors implicated in the transmission of the disease. In the case of homosexual and bisexual men the "risk-reduction" or "safe-sex" brochure has emerged as the most widely used public health intervention modality. While there has been a proliferation of such brochures, to date no systematic analysis of the content or potential efficacy of these materials has been undertaken. The authors carry out two kinds of analyses of a sample of risk-reduction brochures. First, these materials are characterized in terms of 13 characteristics related to their content, and style. Second, the extent to which they incorporate the elements of a standard model of health communication is assessed. Limitations of existing brochures are identified and the implications of the findings for the development of future materials are discussed.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Hospital food hygiene: the application of Hazard Analysis Critical Control Points to conventional hospital catering.

The Hazard Analysis Critical Control Point (HACCP) concept is a preventive approach to quality control. It is based on a logical, structured exploration of potential hazard points in a food operation and the introduction of control and monitoring measures. HACCP studies have been extensively applied to manufacturing systems. It has been difficult to extend this detailed systematic analysis to conventional catering, partly due to the wide range of foods being processed. We describe here the application of HACCP methods to a hospital department that uses conventional catering methods. Basic principles, based on the work flow and the range of products are established. The guidelines produced can be applied to any kitchen using similar catering methods. Examples of log charts used for monitoring are provided.

England↗
PubMed1989

Analysis of language-speech samples with salt and pepper.

Microcomputer-aided analysis of spontaneous language-speech samples offers researchers an efficient means of analyzing large amounts of data. It may be necessary, however, to format samples for more than one software program in order to obtain comprehensive morpho-syntactic and phonetic/phonologic analyses. This paper suggests a procedure for the combined use of SALT (Systematic Analysis of Language Transcripts, Miller & Chapman, 1985) and PEPPER (Programs to Examine Phonetic and Phonologic Evaluation Records, Shriberg, 1986) that is designed to minimize the duplication of effort involved in following two different formatting procedures. Results of a study undertaken to explore methodological issues in the combined use of SALT and PEPPER generally support the validity, reliability, and efficiency of the procedure. Results also raise some issues concerning the use of narrow phonetic transcription as opposed to standard orthographic transcription of continuous language-speech samples.

Data Collection↗
PubMed2001

[Organizational and operational work of death analysis committees: review of experiences].

OBJECTIVES: Among deaths occurring in hospital, some are unexpected at the time of admission and unexplained by the course of the illness at the time of death. Health care givers agree that analysis of the circumstances of death is a powerful method for improving the quality of medical care but are nevertheless reluctant to set up mortality conferences. We present institution-wide experience with mortality conferences, detailing methods used and reviewing adverse care events and avoidable organizational and medical errors associated with unexpected deaths. METHODS: We reviewed literature cited in the Medline and Pascal Biomed databases and completed our findings with consultations with key-informants of hospital evaluation units. Institution-wide experiences alone were analyzed. RESULTS: The death analysis committees were composed of health care givers who review the deaths occurring in their units (internal committees). In rare cases, hospitals set up in addition an external committee for methodological assistance. Implicit criteria were almost always used for death case analysis. DISCUSSION: Death analysis by internal committees appears to contribute to physician's knowledge and provokes a global improvement in patient care. This analysis relies however on implicit criteria which leads to minimal reliability. The lack of reliability has two consequences: the impossibility of identifying causes of preventable deaths and related factors, and the impossibility of evaluating the impact of the internal committee. As there is no available epidemiological data, a sentinel system cannot be proposed: a systematic analysis of all deaths is therefore advisable. Other limitations on internal committees are the difficulty of obtaining autopsies and the problem of the confidentiality of death analysis, a serious handicap recognized by all physicians. A national guide on methodology should be developed.

Cause of Death↗
PubMed1991

[Fetal echocardiography. II. Normal and pathological anatomy in real-time ultrasonography].

The paper is a practical approach to fetal echocardiography using real-time ultrasound. The normal sonoanatomy of the fetal heart is presented for the non-cardiologist. The basic cross-sectional views are explained as well as the systematic analysis of the different fetal heart structures. The main heart malformations are reviewed with a description of their appearance in prenatal real-time-ultrasound illustrated by some figures. The examination of the fetal heart could be easy learned, but the assessment and the differentiation of congenital heart defects are only possible by a systematic approach, experience and patience. The examination of the fetal heart should be therefore involved in the screening-ultrasound performed by the prenatal sonographer and suspicious findings has to be referred to a perinatal center.

Echocardiography↗
PubMed2005

Prediction of recurrence in advanced gastric cancer patients after curative resection by gene expression profiling.

The prognosis of patients with advanced gastric cancer remains unfavorable. Even after curative resection, 40% of patients with advanced gastric cancer die of recurrence. Conventional clinicopathlogic findings are sometimes inadequate for predicting recurrence in individuals. Hence, we tried to construct a new diagnostic system, which predicts recurrence in patients with advanced gastric cancer after curative resection based on molecular analysis. Gastric cancer progression is a function of multiple genetic events that may affect the expression of large number of genes. We performed gene expression profiling with 2,304 genes in 60 advanced gastric cancer patients who underwent curative resection using a PCR array technique, a high-throughput quantitative RT-PCR technique. The diagnostic system, which was constructed from the learning set comprised of 40 patients with the most informative 29 genes, classified each case into a good-signature or poor-signature group. Then, we confirmed the predictive performance in an additional test set comprised of 20 patients, and the prediction accuracy for recurrence was 75%. Kaplan-Meier analysis revealed significant difference between the good-signature and the poor-signature group (p = 0.0125). Especially in patients with smaller tumor (< or = 5 cm), less developed LN metastasis (N(0,1)), or earlier stage (stages I and II), the prediction accuracy was high (88.9%, 84.6%, or 81.8%, respectively). Our diagnostic system based on systematic analysis of gene expression profiling can predict the recurrence at clinically meaningful level. By combining our system with conventional clinicopathologic factors, we can improve the prediction of recurrence in patients with advanced gastric cancer who underwent curative surgery.

Aged↗
PubMed2006

Physiological anthropology design: a comparative study between Germany and Japan.

With the recent globalization of industrial products, there is doubt as to whether the methodology of Physiological Anthropology has also been standardized. The purpose of this study is to assess signs of standardization through a comparative analysis of Physiological Anthropology design in Germany and Japan. This survey investigates its characteristics through four factors: comfort, usability, sensation and aesthetics. Both nations regard the first three indicators as important. The difference in assessment is, however, considerable. While German physiological anthropologists use subjective evaluation by means of questionnaires, somatometry and biomechanical analysis, their Japanese counterparts apply physiological measurements of the higher nervous system and the autonomic nervous system. Polymorphism and improving functional potentiality have recently gained increasing respect in Japan. Notions of aesthetics are not consciously analyzed in both countries. If the sense of beauty of product design relates to a physical and mental response, developing a systematic analysis on this factor would be a useful task for Physiological Anthropology.

Anthropology, Physical↗
PubMedDate not supplied

An analysis of allegations of sexual abuse in a multi-victim day-care center case.

Sexual abuse in day-care centers is a phenomenon of increasing concern, both in terms of its prevalence and its serious effects on child victims. Legal intervention in this area is problematic because criminal investigations are usually beset with difficulties: Characteristics of children and of the abuse situation affect disclosure rates and credibility. Research on children as witnesses is amassing; however, so far, little systematic analysis has been conducted on multi-victim child sexual abuse cases. The present study concerns a case of sexual abuse in a day-care center with over 100 alleged victims. Criteria derived from Statement Validity Analysis (SVA) protocols were applied to aggregate record review data to assess the veritability of the abuse allegations. Individual children's abuse disclosures were also analyzed, vis-à-vis characteristics of the children (age, gender, recency of attendance) and of the investigation (agency doing the interview, number of interviews, use of anatomically detailed dolls). The use of SVA criteria supported the veritability of allegations in this day-care abuse case. Even acknowledging the limitations of archival data collected for other purposes, the aggregate data analysis reflected consistency, logical structure, and spontaneity; however, little evidence of accommodation was found. Multivariate comparison of disclosing and nondisclosing children found an unexpected inverse relationship of disclosure with age, as well as positive associations with number of interviews and use of dolls. Alternative interpretations of these results are discussed.

Age Factors↗
PubMed2006

Proteome analysis of human plasma and amniotic fluid by Off-Gel isoelectric focusing followed by nano-LC-MS/MS.

This paper presents a comparative proteomic analysis of human maternal plasma and amniotic fluid (AF) samples from the same patient at term of pregnancy in order to find specific AF proteins as markers of premature rupture of membranes, a complication frequently observed during pregnancy. Maternal plasma and the corresponding AF were immunodepleted in order to remove the six most abundant proteins before the systematic analysis of their protein composition. The protein samples were then fractionated by IEF Off-Gel electrophoresis (OGE), digested and analyzed with nano-LC-MS/MS separation, revealing a total of 73 and 69 proteins identified in maternal plasma and AF samples, respectively. The proteins identified in AF have been compared to those identified in the mother plasma as well as to the reference human plasma protein list reported by Anderson et al. (Mol. Cell. Proteomics 2004, 3, 311-326). This comparison showed that 26 proteins were exclusively present in AF and not in plasma among which 10 have already been described to be placenta or pregnancy specific. As a further validation of the method, plasma proteins fractionated by OGE and analysed by nano-LC-MS/MS have been compared to the Swiss 2-D PAGE reference map by reconstructing a map that matches 2-D gel and OGE experimental data. This representation shows that 36 of 49 reference proteins could be identified in both data sets, and that isoform shifts in pI are well conserved in the OGE data sets.

Amniotic Fluid↗
PubMed1999

Microfabricated modules for sample handling, sample concentration and flow mixing: application to protein analysis by tandem mass spectrometry.

The comprehensive analysis of biological systems requires a combination of genomic and proteomic efforts. The large-scale application of current genomic technologies provides complete genomic DNA sequences, sequence tags for expressed genes (EST's), and quantitative profiles of expressed genes at the mRNA level. In contrast, protein analytical technology lacks the sensitivity and the sample throughput for the systematic analysis of all the proteins expressed by a tissue or cell. The sensitivity of protein analysis technology is primarily limited by the loss of analytes, due to adsorption to surfaces, and sample contamination during handling. Here we summarize our work on the development and use of microfabricated fluidic systems for the manipulation of minute amounts of peptides and delivery to an electrospray ionization tandem mass spectrometer. New data are also presented that further demonstrate the potential of these novel approaches. Specifically, we describe the use of microfabricated devices as modules to deliver femtomole amounts of protein digests to the mass spectrometer for protein identification. We also describe the use of a microfabricated module for the generation of solvent gradients at nl/min flow rates for gradient chromatography-tandem mass spectrometry. The use of microfabricated fluidic systems reduces the risk of sample contamination and sample loss due to adsorption to wetted surfaces. The ability to assemble dedicated modular systems and to operate them automatically makes the use of microfabricated systems attractive for the sensitive and large-scale analysis of proteins.

Equipment Design↗
PubMed2001

Analysis of sequence signature defining functional specificity and structural stability in helix-loop-helix proteins.

Specific functional properties of many proteins directing developmental responses via transcriptional regulation are orchestrated by their characteristic helix-loop-helix (HLH) structural motif. The entire HLH motif in all these proteins assumes a common conformation irrespective of their individual biological effects. The motif controls the affinity of HLH proteins for homo- or heterodimerization, permitting mixing and matching of regulatory factors, and thereby expanding the functional repertoire. Systematic analysis of molecular contacts at the dimer interface using the models built for the functional dimers combined with the pattern of conserved/nonconserved residues within different categories of HLH proteins helped in understanding the differential role played by different residues at the dimer interface for expressing corresponding functions. The residues associated with the self and partner interactions were identified, and the signature residues contributing toward dimeric stability and functional specificity were defined. It is evident that most of the residues involved in self interactions are common among all the HLH proteins. However, while certain residues involved in partner interactions are common among all the HLH proteins, certain others are common within a category, and still others vary widely defining specificity signature at different levels.

Amino Acid Sequence↗
PubMed2006

Interphase FISH analysis of PTEN in histologic sections shows genomic deletions in 68% of primary prostate cancer and 23% of high-grade prostatic intra-epithelial neoplasias.

Prostate cancer (CaP) is characterized by the accumulation of both genetic and epigenetic alterations that transform premalignant lesions to invasive carcinoma. However, the molecular events underlying this critical transition are poorly understood. One of the important genes that might play a role in CaP development is the PTEN gene. At the present time, there has been no systematic analysis of the incidence of genomic PTEN deletion by fluorescence in situ hybridization (FISH) in CaP and associated preneoplastic histologic lesions. This study assesses the frequency of PTEN deletion by interphase FISH analysis in CaP and prostatic intra-epithelial neoplasia (PIN). Dual-color FISH was performed using DNA probes for bands 10q23.3 (PTEN locus) and chromosome 10 centromere using 35 radical prostatectomy specimens. PTEN deletions were not found in 3/3 of stroma, 6/6 samples of benign glandular epithelium, and 12/12 samples of low-grade PIN. However, PTEN deletions were found in 3/13 (23%) of high-grade PIN and 24/35 (68%) of CaP. Concordance was observed between PTEN deletion status and the overall cellular PTEN protein expression levels, as assessed by immunohistochemistry. The high frequency of PTEN deletion observed in CaP versus precursor lesions implicates a pivotal role for PTEN haploinsufficiency in the transition from preneoplastic PIN to CaP. Moreover, this observation is an important consideration for novel therapeutic trials in CaP in which biologic efficacy is influenced by the activity level of PTEN. These findings draw attention to the usefulness of this relatively simple FISH assay for future applications in clinical laboratories.

Gene Deletion↗
PubMed2002

Anatomical analysis of different hemispherotomy procedures based on dissection of cadaveric brains.

OBJECT: Functional hemispherectomy, itself a modification of anatomical hemispherectomy, has been further modified to a less invasive method (hemispherotomy), in which cortical resection is minimized and the rest of the affected hemisphere is functionally isolated by transecting its projection and commissural fibers. Although descriptions of three different types of hemispherotomy procedures have been published, the authors believe that it is important to develop a common and universally acceptable method based on a systematic analysis of topographic anatomy and neuronal connections. To this end, they have analyzed the three aforementioned procedures on the basis of meticulous fiber dissections in previously frozen formalin-fixed human brains. METHODS: The brain anatomy pertinent to surgical hemispherotomy is described in conjunction with dissection studies in 14 previously frozen, formalin-fixed human brains. The anatomical landmarks necessary for performing particular neuronal fiber resections are identified, and their relationships with operative methods are discussed, with an emphasis on commonalities among the three hemispherotomy procedures. CONCLUSIONS: In this analysis the authors confirmed that hemispherotomy typically consists of four common procedures: 1) interruption of the internal capsule and corona radiata; 2) resection of the medial temporal structures; 3) transventricular corpus callosotomy; and 4) disruption of the frontal horizontal fibers. After meticulous dissection of cadavers, the authors have designated a reliable method for performing these four operations that may be applicable as a commonly used procedure.

Adult↗
PubMed2002

Migration behavior of metal complexes in capillary zone electrophoresis. Interpretation in terms of quantitative structure-mobility relationships.

Significant advances in metal ion analysis by capillary zone electrophoresis (CZE) have occurred as a consequence of using metal complexes with various organic and inorganic ligands. Metal-ligand complexes that contribute to metal speciation in solution have also gained the attention of researchers. An understanding of the molecular properties that control the separation and further insight into the migration mechanism call for a systematic analysis of relationships between migration parameters and charge and size characteristics of metal complexes. To perform such an investigation, a number multiparametric migration models derived from a generally valid equation for electrophoretic mobility as a function of charge density were developed. The models operating with tabular or readily calculated structural descriptors (in particular, metal atom electronegativity or effective charge) as well as with the formal charge and ligand number were evaluated using numerous sets of experimental migration data for inorganic and organic ligand complexes. Consistent--in a great many instances--approximation results confirm the separation mechanism for metal complexes in CZE as governed basically by differences in charge-to-size parameters, present a valuable and convincing selection of such parameters, possessing a definite physical meaning, and owing to the general validity of the multivariate regression approach, open new possibilities in its application to more complex (i.e., electrokinetic chromatographic) systems.

Chelating Agents↗
PubMed2002

Mediastinal lymphadenectomy in non-small cell lung cancer: effectiveness in patients with or without nodal micrometastases - results of a preliminary study.

OBJECTIVES: So far it has not clearly been demonstrated that systematic mediastinal lymphadenectomy improves survival in patients with non-small cell lung cancer. One explanation might be that in some patients an early spread of tumor cells has occurred which might not be curable by surgical means. To test this hypothesis lymph nodes of patients which were treated either by lymph node sampling or systematic lymphadenectomy were screened for micrometastatic spread of tumor cells and the influence of nodal micrometastases on the efficacy of lymphadenectomy was analyzed. METHODS: Lymph nodes from patients (n=94) which were included in a randomized trial of lymph node sampling (LS, n=41) versus radical systematic lymphadenectomy (LA, n=53) were screened by immunohistochemistry for disseminated tumor cells using the antibody Ber-Ep4. The median observation time was longer than 5 years and follow-up data were available from all 94 patients. Kaplan-Meier curves were calculated and tested for statistical significance using the log-rank test. RESULTS: Standard histopathological analysis revealed no lymph node involvement (pN0) in 61 patients, pN1 disease in 13 patients and pN2 disease in 20 patients without significant differences between LA and LS with respect to T-stage, N-stage or age and sex of the patients. By immunohistochemistry a minimal nodal spread of tumor cells was detected in 21 out of 94 patients (LS, n=10 (24%); LA, n=11 (21%)). Similar to the entire group of patients also in the subset of patients with nodal micrometastases the type of lymphadenectomy did not significantly influence the long-term survival (P=0.27 and P=0.39, respectively). In contrast, in patients with a negative immunohistochemical analysis systematic lymphadenectomy resulted in an improved overall survival (P=0.044). CONCLUSIONS: Our data provide some evidence that systematic lymphadenectomy improves survival in patients without an early locoregional spread of cancer cells. As long as these patients can not be identified preoperatively all patients should undergo a systematic mediastinal lymphadenectomy.

Carcinoma, Non-Small-Cell Lung↗
PubMed2001

Design, synthesis, and proposed active site binding analysis of monocyclic 2-azetidinone inhibitors of prostate specific antigen.

A homology derived molecular model of prostate specific antigen (PSA) was created and refined. The active site region was investigated for specific interacting functionality and a binding model postulated for the novel 2-azetidinone acyl enzyme inhibitor 1 (IC(50) = 8.98 +/- 0.90 microM) which was used as a lead compound in this study. A single low energy conformation structure II (Figure 2) was adopted as most likely to represent binding after minimization and dynamics calculations. Systematic analysis of the binding importance of all three side chains appended to the 2-azetidinone was conducted by the synthesis of several analogues. A proposed salt bridge to Lys-145 with 4 (IC(50) = 5.84 +/- 0.92 microM) gave improved inhibition, but generally the binding of the N-1 side chain in a specific secondary aromatic binding site did not tolerate much structural alteration. A hydrophobic interaction of the C-4 side chain afforded inhibitor 6 (IC(50) = 1.43 +/- 0.19 microM), and polar functionality could also be added in a proposed interaction with Gln-166 in 5 (IC(50) = 1.34 +/- 0.05 microM). Reversal of the C-4 ester connectivity furnished inhibitors 7 (IC(50) = 1.59 +/- 0.15 microM), 11 (IC(50) = 3.08 +/- 0.41 microM), and 13 (IC(50) = 2.19 +/- 0.36 microM) which were perceived to bind to PSA by a rotation of 180 degrees relative to the C-4 ester of normal connectivity. Incorporation of hydroxyl functionality into the C-3 side chain provided 16 (IC(50) = 348 +/- 50 nM) with the greatest increase in PSA inhibition by a single modification. Multiple copy simultaneous search (MCSS) analysis of the PSA active site further supported our model and suggested that 18 would bind strongly. Asymmetric synthesis yielded 18 (IC(50) = 226 +/- 10 nM) as the most potent inhibitor of PSA reported to date. It is concluded that our design approach has been successful in developing PSA inhibitors and could also be applied to the inhibition of other enzymes, especially in the absence of crystallographic information.

Azetidines↗