Background
==========

Traditionally, the animal body cavity (coelom) has played a major role in interpretations of metazoan evolution, from groups (e.g., flatworms) lacking a coelom to those (e.g., nematodes) with a false coelom and finally to the bulk of animal phyla having a true coelom (Coelomata) \[[@B1],[@B2]\]. There has never been complete agreement on animal phylogeny and classification, but most researchers have divided living coelomate animals into deuterostomes (echinoderms, hemichordates, urochordates, cephalochordates, and vertebrates) and protostomes (arthropods, annelids, mollusks, and other phyla) based on differences in early embryonic development. An analysis of small subunit ribosomal RNA (18S rRNA) sequences challenged this arrangement by placing acoelomate and pseudocoelomate phyla in more derived positions among the protostomes, and in further defining a clade (Ecdysozoa) of molting animals that includes arthropods and nematodes \[[@B3]\]. This \"Ecdysozoa\" hypothesis has influenced diverse fields \[[@B4]\] and interpretations of developmental evolution in animals \[[@B5]-[@B7]\]. Since its publication, evidence has appeared both for and against this hypothesis \[[@B8]-[@B15]\]. Knowing the branching order of the major animal lineages, especially those three with fully sequenced genomes, is of importance to diverse fields such as medical genetics, physiology, neurobiology, paleontology, and astrobiology. With a genealogy of animals, it will be easier to determine the origins and inheritance of mutations, genes, gene functions, and structures.

The three possible relationships of these animal phyla are: (I) arthropods + vertebrates, (II) arthropods + nematodes, and (III) nematodes + vertebrates. The first hypothesis corresponds to the traditional grouping Coelomata and the second corresponds to Ecdysozoa \[[@B3]\]. For convenience, we will use these names in reference to the two hypotheses while recognizing that this study, by necessity, involves only a subset of all animal phyla. The third hypothesis will be referred to as \"hypothesis III\" (Fig. [1](#F1){ref-type="fig"}). To test each hypothesis, sequence alignments of more than 100 nuclear proteins were assembled and subjected to a series of analyses designed to reveal biases that could result in an incorrect phylogeny.

![The three possible relationships of vertebrates, arthropods, and nematodes.](1471-2148-2-7-1){#F1}

Results and discussion
======================

Analyses of the individual protein datasets using neighbor-joining \[[@B16]\] show that most (62%) support Coelomata while 25% support Ecdysozoa and 13% support hypothesis III ([Supplemental Table 1](#S1){ref-type="supplementary-material"}, four-taxon analysis). Of the 25 proteins in which support for one of the three hypotheses is significant (≥ 95%), the results are 84% (21 proteins), 16% (4), and 0%, respectively; for those ten proteins with a highly significant (≥ 99%) topology, the results are 90% (9 proteins), 10% (1), and 0%, respectively. The four proteins showing significant support (\>95%) for a hypothesis other than Coelomata were reanalyzed using maximum parsimony and maximum likelihood; bootstrap values were not significant using other methods. Such divided results are typical of single-gene analyses because of limited information (\~400 amino acids), necessitating combined analysis. Coelomata was supported significantly (100% bootstrap confidence, posterior probability = 1.0) when the 100 four-taxon protein alignments were concatenated and analyzed using neighbor-joining, maximum parsimony, maximum likelihood, and Bayesian inference (Fig. [2A](#F2){ref-type="fig"}). Using the Shimodaira-Hasegawa (SH) test \[[@B17]\], this maximum likelihood topology was significantly different from the alternative hypotheses of Ecdysozoa (*P* \< 0.001) and Hypothesis III (*P* \< 0.001). These results agree with earlier studies involving 10--36 nuclear proteins \[[@B8],[@B9],[@B11]\].

![Phylogenetic analyses of individual and combined (concatenated) sequence alignments bearing on the position of nematodes. V = vertebrate, A = arthropod, N = nematode, P = platyhelminth. Bootstrap values (\>95%) are shown for neighbor-joining, maximum parsimony, and maximum likelihood, respectively; all are indicated for the node joining *Homo* and *Drosophila* (=Coelomata). Posterior probabilities are not shown (all highlighted nodes = 1.0). (*A*) Four-taxon analysis of 100 combined protein alignments (44,214 amino acids), using nematode *Caenorhabditis elegans* (Chromadorea, Rhabditida, Rhabditoidea, Rhabditidae); the nematode branch is approximately 16% longer than the vertebrate and arthropod branches. (*B*) Five-taxon analysis of 100 combined proteins includes planarian EST sequences (14,041 amino acids); the nematode branch is approximately 23% longer. Other trees show different representative nematodes. (C) *Brugia* (Chromadorea, Spirurida, Filarioidea, Onchocercidae), based on 18 combined proteins (4598 amino acids); nematode branch= 15% longer. (D) *Trichinella* (Enoplea, Trichocephalida, Trichinellidae), based on 6 combined proteins (2261 amino acids); nematode branch = 24% longer than the vertebrate branch and 5% shorter than the arthropod branch. (E) Proportion of individual protein analyses supporting each of the three possible topologies with differing numbers of phyla included (4 taxa = 124 proteins, 5 taxa= 107 proteins, 6 taxa= 66 proteins, \>6 taxa = 12 proteins).](1471-2148-2-7-2){#F2}

To test the stability of Coelomata to taxon sampling, we included new sequences of the planarian *Dugesia japonica* (Phylum Platyhelminthes) in 100 five-taxon protein alignments ([Supplemental Table 1](#S1){ref-type="supplementary-material"}, five-taxon analysis). The results, upon concatenation with this additional taxon, were unchanged (Fig. [2B](#F2){ref-type="fig"}): Coelomata continued to be significantly supported (≥ 98% bootstrap support, posterior probability = 1.0) and the alternative hypotheses were both rejected using the SH test (*P* \< 0.001), although the relationships among the basal phyla (Nematoda and Platyhelminthes) could not be resolved.

Another potential bias is the specific taxa included in an analysis. For example, the original support for Ecdysozoa was obtained only with a particular genus of nematode, *Trichinella,* that had a short branch in the 18S rRNA tree \[[@B3]\]. To test this, phylogenies were constructed using different species of nematodes. The Coelomata hypothesis was significantly supported (≥ 97% bootstrap support, posterior probabilities = 1.0; Ecdysozoa rejected by SH test, *P* \< 0.006; Hypothesis III rejected by SH test, *P* \< 0.027) using either a genus in a different order, *Brugia* (18 proteins), or *Trichinella* (six proteins) (Fig. [2C, 2D](#F2){ref-type="fig"}). To further address the possibility that these results could be biased by taxon sampling, we included representatives from all available phyla for each protein. The results indicate that an increase in the number of taxa does not decrease single-protein support for Coelomata; in fact, the trend is the reverse (Fig. [2E](#F2){ref-type="fig"}). Simulation studies have shown that incomplete taxon sampling does not increase topological errors, and that most error is caused by limited sequence data \[[@B18]\].

In the initial study defining Ecdysozoa \[[@B3]\], rate variation was considered to be the major bias affecting the phylogenetic position of nematodes. In the 18S rRNA gene, nematodes typically have long branches indicating an increased rate of sequence change. Other nuclear genes also show this pattern, but to a lesser degree \[[@B8],[@B9]\]. Phylogenetic methods can accommodate moderate amounts of rate variation among lineages without producing an incorrect phylogeny \[[@B19]\]. However, if the rate of change is sufficiently large, longer branches in a phylogeny will sometimes attract one another \[[@B20]\]. If that happens, an ingroup species with a long branch may move to a more basal position in the tree. In analyses of the 18S rRNA gene, nematodes typically appear basal to arthropods + vertebrates. Because the use of a short branch nematode (*Trichinella*) resulted in a tree whereby nematodes clustered with arthropods, the basal position of nematodes in typical 18S analyses has been interpreted as long-branch attraction \[[@B3]\].

If nematodes cluster basally because of long-branch attraction, then the strongest support for Ecdysozoa should be obtained with the slowest evolving proteins. This was tested in an analysis of 36 nuclear proteins \[[@B8]\], but the results were equivocal. Therefore, we tested this suggestion with our four-taxon data set of 100 proteins, ordered by rate of evolution. Rate orders were determined in two ways: (i) nematode branch length and (ii) vertebrate-arthropod pairwise distance. The 100 proteins were grouped into concatenations of 10 proteins and 20 proteins to increase statistical resolution. The results show support for Coelomata at all rate orders, but the support is significant with the slowest evolving proteins, regardless of rate measure or number of proteins combined (Fig. [3](#F3){ref-type="fig"}). Concatenations of slow evolving proteins also show compositional homogeneity (pairwise disparity index test, *P* \< 0.05) \[[@B21]\], suggesting the basal position of the nematode results from true phylogenetic signal and not compositional bias. Support for Coelomata was weakest with the fastest evolving proteins (which also showed compositional heterogeneity), indicating that Ecdysozoa, not Coelomata, may be the result of a rate bias, compositional bias, or other artifact.

![Effect of genetic distance on bootstrap support for the three hypotheses from analysis of 100 nuclear proteins with four taxa. (*A, B*) show bootstrap support for Coelomata; (*C, D*) for Ecdysozoa; (*E, F*) for Hypothesis III. Proteins were ordered from slowest evolving to fastest evolving based on two criteria: vertebrate-arthropod pairwise distance (diamonds) and nematode branch length (squares). Proteins were concatenated into ten groups often (*A, C, E*) and five groups of twenty (*B, D, F*). Graphs show rate from slowest to fastest evolving (left to right). Trend lines are indicated (solid for vertebrate-arthropod distance, dashed for nematode branch length).](1471-2148-2-7-3){#F3}

To ensure that this result was not affected by mutational saturation in our data set, the mean number of variants per variable site was determined for each protein and averaged for ten groups of proteins ordered by evolutionary rate (Fig [4A](#F4){ref-type="fig"}). As predicted, variable sites in the faster evolving proteins showed a higher number of variants than those in slow-evolving proteins. We also examined the minimum number of nucleotide changes required for sites where only the nematode sequence varied (Fig [4B](#F4){ref-type="fig"}). Slow-evolving proteins showed a smaller number of nucleotide changes required to alter amino acid identity, while faster evolving proteins required more changes. Thus, the nematode sequences in the slow-evolving proteins do not appear to be mutationally saturated.

![Test of mutational saturation in the four-taxon data set. (*A*) The mean number of variants per variable site was averaged for ten groups often according to evolutionary rate (vertebrate-arthropod distance = diamonds, nematode branch length = squares). (*B*) The minimum number of nucleotide changes required for unique nematode variants were also averaged according to evolutionary rate. Trend lines are indicated (solid for vertebrate-arthropod distance, dashed for nematode branch length).](1471-2148-2-7-4){#F4}

Finally, the affect of lineage-specific rate variation on support for Coelomata was tested with the use of relative rate tests. Presumably, the selective elimination of genes with long branches will increase statistical support for the correct topology. Individual proteins from the four-taxon data set were each subjected to two different relative rate tests \[[@B22],[@B23]\]. Proteins determined to be rate-constant at the typically applied stringency level (5% significance), and at two greater stringency levels (10% and 40% significance) were concatenated and bootstrap support was determined using neighbor joining. The results of the two tests were similar. As stringency increased, 40--83% of proteins were rejected, and the relative nematode branch length (to the arthropod and vertebrate branches) dropped from 16% to 0%. However, in all cases, Coelomata remained highly significant (Fig. [5](#F5){ref-type="fig"}). Thus, the suggestion that a basal position of nematodes is the result of long-branch attraction \[[@B3]\] can be rejected.

![Effect of rate constancy on bootstrap support for Coelomata in four-taxon analysis. Graphs show results before application of tests (left, 0-level) followed by increasing stringency (5, 10, 40% significance) of the chi-square test \[[@B22]\] (circles) and Z-test \[[@B23]\] (triangles); the 5% level is normally used. (*A*) number of proteins passing rate constancy at each cutoff level. (*B*) relative nematode branch length upon concatenation of all rate constant proteins at each level. (*C*) bootstrap support for Coelomata for each rate-constant concatenation.](1471-2148-2-7-5){#F5}

The importance of knowing the branching order of these species is illustrated by the immediate and wide acceptance of the Ecdysozoa hypothesis and its use in tracing patterns of developmental evolution \[[@B5]-[@B7],[@B10]\]. However, in the initial analysis of 18S rRNA sequences \[[@B3]\], Ecdysozoa was statistically significant only when a paralinear distance method was used; three other methods did not yield significant bootstrap support. In that study, Ecdysozoa also was not significant, using any method, when the flatworm sequence was included \[[@B3]\]. Subsequent analyses of the 18S rRNA gene have been interpreted differently \[[@B24],[@B25]\], but none has yielded statistically significant results supporting Ecdysozoa. Moreover, the molting cuticles of arthropods (chitin) and nematodes (collagen) are not homologous \[[@B4]\]. The significance of other morphological characteristics bearing on the position of nematodes continues to be debated \[[@B26]\].

Besides the 18S rRNA evidence, other genetic evidence for the grouping of nematodes and arthropods has come from qualitative interpretations of *Hox* gene \[[@B10]\] and β-thymosin \[[@B12]\] evolution. In the case of *Hox* genes, support comes from a single posterior gene sequence (Y75B8A.1) of the nematode *Caenorhabditis elegans* argued to have greater amino acid similarity with a posterior *Hox* genes of *Drosophila* and *Priapulus*\[[@B10]\]. Unfortunately, the *Hox* homeodomain is a short (60 amino acid) region with many sequence differences between these taxa. Definition of \"sequence signatures\" is qualitative and has not been tested statistically. In a subsequent study of nematode posterior *Hox* genes, other researchers were unable to determine if the simple nematode *Hox* cluster of six genes is an ancestral or a derived condition \[[@B13]\].

In the case of β-thymosin, a sequence signature also has been argued to support a grouping of *Drosophila* and *Caenorhabditis*\[[@B12]\]. However, it is a gene family known to have paralogs within animals, the position of introns differs between sequences from the two species, and only four other metazoan taxa were surveyed. In addition, knowing the presence or absence of a gene can be problematic without the complete genome sequence of an organism (in this case, genomes were known only in *Drosophila* and *Caenorhabditis*). Thus, although suggestive, it is too soon to judge the significance of this sequence signature. One difficulty with interpreting such qualitative evidence, including *Hox* gene orthology, is that almost any pattern can be found in nature if one looks. In other words, sequence signatures have not yet been surveyed systematically and objectively. In contrast, sequence evidence from randomly selected genes, analyzed phylogenetically, provides a more unbiased database amenable to statistical analysis.

Conclusions
===========

Although it is possible that a basal position of nematodes is the result of some unknown and widespread bias not yet identified, a simpler explanation is that the grouping of nematodes with arthropods is an artifact that arose from the analysis of a single gene, 18S rRNA. The results presented here suggest caution in revising animal phylogeny from analyses of one or a few genes or sequence signatures. Although many other aspects of animal phylogeny remain unresolved, our results indicate that insects (arthropods) are genetically and evolutionarily closer to humans (vertebrates) than to nematodes.

Materials and methods
=====================

DNA sequences from *Dugesia japonica*\[[@B27]\] were used to search the public protein database (Entrez) for orthologous counterparts in *Drosophila melanogaster, Caenorhabditis elegans, Homo sapiens, Arabidopsis thaliana, Saccharomyces cerevisiae,* and *Schizosaccharomyces pombe.* When available, sequences from other major animal phyla (e.g., Mollusca, Echinodermata, Annelida) also were obtained. In addition, the database was searched for all proteins from two other nematodes, *Brugia* and *Trichinella,* with orthologs in *Drosophila, Homo,* and *Arabidopsis, Saccharomyces,* or *Schizosaccharomyces. Arabidopsis* was used as the primary outgroup (95 out of 100 proteins in the four-taxon analysis, 94 out of 100 proteins for the five-taxon analysis, all proteins in *Brugia* and *Trichinella* analyses); yeast was used as the outgroup when *Arabidopsis* sequences were unavailable or paralogous. This was because many more genes were available for rooting with the plant than with the fungus. All three kingdoms are about equidistant from each other in terms of branch lengths \[[@B9]\] and therefore a plant serves about equally well for rooting an animal phylogeny as does a fungus. Orthology was assessed using reciprocal BLAST searches of the public protein database; those sequences receiving high scores in each search were also analyzed phylogenetically to ensure orthology. Short (\<100 amino acids) sequences were omitted.

Sequences were aligned using Clustal X \[[@B28]\] and each alignment was visually inspected. Primary analyses of aligned protein data sets were conducted in MEGA2 \[[@B29]\]. Phylogenies were reconstructed using neighbor-joining \[[@B16]\] under a Poisson correction and a gamma distance (α = 2, or estimated from the data for combined analyses), with bootstrapping (2000 replications) for all analyses. Gamma parameters were estimated from the combined data using maximum likelihood under a Poisson correction \[[@B30]\] (4-taxon, α = 1.62; 5-taxon, α = 0.94, *Brugia,* α = 0.87; *Trichinella,* α = 0.66). In addition, phylogenetic analyses were conducted with maximum likelihood (JTT-F option) \[[@B31]\] and maximum parsimony (Max-Mini Branch & Bound option) \[[@B29]\] on combined data sets; in all cases they resulted in similar results (topology and significance) to the neighbor-joining analyses. Posterior probabilities of concatenated files were computed using Bayesian inference \[[@B32]\] (Jones model with gamma estimated from data; 10,000 generations; 4 chains with temp = 0.2). Shimodaira-Hasegawa tests \[[@B17]\] were performed in PAML \[[@B30]\] (JTT-F option, fixed gamma); p-values for each topology were recorded.

Rate constancy was assessed using a chi-square test \[[@B22]\] under increasing stringency (5, 10, 40% significance levels); p-values were recorded for each protein. A Z-test \[[@B23]\] was also used under increasing stringency; z-values were recorded for each protein. Proteins determined to be rate constant at different significance levels were concatenated and analyzed in MEGA2 \[[@B29]\]. Nematode position and evolutionary distance were determined for each concatenation. New sequences, accession numbers of sequences, and sequence alignments may be found at the Evogenomics website <>.

Authors\' contributions
=======================

JEB carried out the bioinformatics research and drafted the manuscript. KI and TG supplied the unpublished EST sequences of the platyhelminth *Dugesia japonica.* SBH directed the research and assisted with drafting of the manuscript.

Supplementary Material
======================

::: {.caption}
###### Supplemental Table 1

Proteins analyzed and bootstrap support for each alternative hypothesis.
:::

::: {.caption}
######

Table-SuppInfo.doc
:::

Acknowledgements
================

This work was supported by grants from the NASA Astrobiology Institute and National Science Foundation (to S.B.H.).


Background
==========

There appears to be no one commonly accepted definition for quality of work life. In healthcare organizations, quality of work life (QWL) has been described as referring to the strengths and weaknesses in the total work environment \[[@B1]\]. Characteristics that describe the overall organization are viewed as part of the behaviour and reward system of the staff working in that setting. Organizational features such as policies and procedures, leadership style, operations, and general contextual factors of the setting, all have a profound effect on how staff view the quality of their work life. QWL is an umbrella term which includes many concepts. Therefore, concentrating on only one job characteristic, whether it is wages or management style, is an inadequate approach to assessing QWL. Because the perceptions held by employees play an important role in their decisions to enter, stay with or leave an organization, it is important that staff perceptions be included when assessing QWL. And although job satisfaction is not QWL, perception of QWL is often assessed using job satisfaction surveys.

Previous studies have shown that low job satisfaction is a major cause of turnover among health care providers \[[@B2]-[@B4]\]. In addition, job satisfaction may affect the quality of service and organizational commitment \[[@B5]-[@B9]\] and may be a contributing factor associated with shortages of health care providers \[[@B10]\]. Such findings have recently increased interest in studying job satisfaction among health care providers \[[@B11]\]. The results of a 1993 meta-analysis of 48 studies looking at work satisfaction in over 15,000 nurses revealed that job satisfaction was associated strongly with reduced work stress, organizational commitment, communication with supervisors, autonomy, employee recognition, fairness, locus of control, years of experience, education, and professionalism. This study also found a strong relationship between job satisfaction and QWL for nurses \[[@B12]\].

After reviewing the literature on QWL and job satisfaction, and considering the wide variety of health care settings, situational contexts, and organizational structures (including management styles, reporting structures, staffing complements, and levels of training and experience) in which employees work, we hypothesized that the predictors of job satisfaction would vary depending on the organization. The purpose of this study was to identify organization specific predictors of job satisfaction within a health care system that consisted of six independent and distinct organizations located in five communities in Central West Ontario, Canada.

Methods
=======

Setting
-------

The settings for this study included six independent and distinct health care organizations providing varying levels and types of care. All six organizations were affiliated with the St. Joseph\'s Health System (SJHS) located in five Central West Ontario communities. Collectively, the SJHS is one of the largest corporations in Canada devoted to health care. At the time of the study (2000), the SJHS employed 5,486 full, part and casual time (non physician) staff. Additional information about of each of the six organizations and their respective communities is provided in Table [1](#T1){ref-type="table"}.

::: {#T1 .table-wrap}
::: {.caption}
######

Characteristics of the Organizations within the St. Joseph\'s Health System.
:::

  Site     Type of Organization                               No. of Staff   No. of Beds or Visits/Yr   Community Population^1^
  -------- -------------------------------------------------- -------------- -------------------------- -------------------------
  Site 1   Community Hospital                                 321            101                        84,764
  Site 2   Community Hospital / Long-Term Care Facility       649            186/124                    95,821
  Site 3   Visiting Nurse Organization                        205            140,152                    322,352
  Site 4   Long-Term Care Facility                            481            389                        23,125
  Site 5   Community Hospital                                 889            148                        178,420
  Site 6   Tertiary Care Hospital / Community Health Centre   2,941          459/88,837                 322,352

^1^1996 Census
:::

Questionnaire development
-------------------------

Items included in the \"Quality of Work Life Survey 2000\" were selected after a review of the literature and extensive consultation between research team members and the QWL Task Force (a management group consisting of representatives from each of the six SJHS organizations). The initial selection of items was influenced by a recently published Canadian study \[[@B13]\] and reports from two meta-analyses \[[@B1],[@B12]\]. The QWL Task Force then refined these items to consider, among other things, issues of accuracy, relevance, readability, grammar, potential for offensiveness, and appearance of cultural or gender bias. After several months of development, the instrument was pretested on a small group of staff at two of the participating organizations (Site 2 and Site 4 -- see Table [1](#T1){ref-type="table"}). This pretesting was done to ensure that individuals could follow the instructions associated with the format, to obtain estimates of the time required to complete the survey instrument, to identify items that were poorly written or ambiguous, and to identify an appropriate implementation strategy. The questionnaire and implementation strategies were revised accordingly.

The final 65-item survey contained nine sections representing topic areas considered relevant to assessing QWL in the SJHS. Eight scale scores were developed from the individual items (see below and [Additional File](#S1){ref-type="supplementary-material"}: Statistically Significant Organization Specific (Univariate) Predictors of Job Satisfaction).

The ***Co-worker and supervisor support*** section included 10 closed-ended and 1 open-ended questions. A 3-item supervisor social support scale included questions about supervisor helpfulness, concern about the welfare of employees, and ability to facilitate effective interaction among employees. Co-worker support was measured by a 7-item scale reflecting the extent to which co-workers were seen as competent, understanding, and supportive of employees. Both scales where adapted from Woodward et al. (1999) \[[@B13]\].

The ***Teamwork and Communication*** section included 9 closed-ended and 1 open-ended questions. For determining teamwork, a 7-item scale was adapted from Taylor and Bowers (1972) to measure the extent to which one\'s work unit coordinates efforts, solves problems and works together effectively \[[@B14]\]. A 2-item scale developed for this project measured how communication was practiced within the organization.

The ***Job Demands and Decision Authority*** section included 15 closed-ended and 1 open-ended questions. It included a 4-item scale adapted from Brosnan and Johnson (1980) to measure clarity regarding responsibilities, workloads and conflicting demands \[[@B15]\]. There was also a 9-item scale adapted from Karasek et al. (1998) to measure the extent to which respondents\' jobs gave them autonomy or decision-making latitude \[[@B16]\], and 2 questions which reflected the demands of one\'s work \[[@B17]\].

The ***Characteristics of Your Organization*** section included 6 closed-ended and 1 open-ended questions. This section was adapted from Woodward et al. (1999) and included a 4-item scale that inquired about the extent to which the organization encouraged the best efforts from staff, and how employees were treated \[[@B14]\]. Two additional questions examined the extent to which staff were kept informed, and organizational recognition of employee contributions.

The ***Patient/Resident Care*** section included 5 closed-ended and 1 open-ended questions. The questions (developed for this project) were used to measure employees\' perceptions of the quality and timeliness of care provided for patients and residents at their respective organizations.

The ***Compensation and Benefits*** section included 10 closed-ended and 1 open-ended questions. These questions were developed for this project to determine employee satisfaction concerning a number of employee benefits and level of pay.

The ***Staff Training and Development*** section included 6 closed-ended and 1 open-ended questions. These questions (developed for this project) measured the extent to which each organization supports its staff in training, educational development and opportunities for advancement.

The ***Overall Impressions of Your Organization*** section included 4 closed-ended and 4 open-ended questions. All of the questions (developed for this project) assessed staffs\' impressions of and overall satisfaction with their organization. The question \"Overall, how satisfied are you with your job?\" was used as the outcome variable in this study.

The ***Staff Socio-Demographic Information*** section included 10 closed-ended questions (developed for this project) to collect information on gender, age, marital status, education, length of employment, supervisory status, time spent on job activities, job status and job classification.

Within each of the first 8 sections, employees were asked to circle the response that best described their feelings using 5-point Likert scales. Employees were also asked for written comments pertaining to each of the sections and were provided space to comment on other issues they felt were important.

Survey Procedure
----------------

Because of the diversity of organizations and staff within the SJHS, it was decided by the QWL Task Force, organization administrators and researchers that the implementation of the survey would be customized to best fit each of the organizations. It was felt that a varied approach would be more feasible for the organizations and that this would help maximize response rates. Although the procedures were not identical, all of the organizations provided as a minimum: advance notification (written or voice mail) of the survey to all staff (eligibility was based on whether the worker was active on the organization\'s pay roll at the time of the study and was not a physician); access to questionnaires for all staff (the QWL Task Force felt that each staff member in the SJHS should have the opportunity to complete a questionnaire); one or more reminder notices (e.g., letters, newsletters, voice mail, personal communication); and sealed drop off boxes for completed questionnaires. Pilot testing of the questionnaire revealed that employees felt that tracking individual employees for the purpose of follow-up (i.e., to increase response rate), violated the perception of anonymity and confidentiality. Therefore, to help ensure anonymity and confidentiality, follow-up attempts were limited to general reminder notices to all staff.

Analysis
--------

All closed-ended (or quantitative) responses were entered directly from the questionnaires into SPSS (version 10.0.5 for Windows, SPSS, Inc., Chicago, 1999). Prior to data analysis, most of the survey questions were re-coded. Questions which asked participants to select one response within a five point scale (never to always; very dissatisfied to very satisfied; very poor to very good; no, definitely not to yes, definitely) were collapsed into two categories. For example, for the response scale (1=very dissatisfied, 2=dissatisfied, 3=not sure, 4=satisfied, 5=very satisfied) those who indicated they were either satisfied or very satisfied were re-coded as \"satisfied\" while all others were re-coded \"not satisfied\" by default. In several instances, it was appropriate to combine two or more of the questions into a composite scale score. See \"Questionnaire Development\" section and [Additional File](#S1){ref-type="supplementary-material"}: Statistically Significant Organization Specific (Univariate) Predictors of Job Satisfaction for additional details on how the composite scale scores were calculated. In total, there were eight scale scores (supervisor social support; co-worker support; teamwork; communication; role clarity; decision latitude; organization/staff relations; patient/resident care). Scale scores were generated by summing the participant responses (i.e. one to five) for all questions that made up the scale. In the rare situation where a participant failed to answer one or more of the questions that made up a scale score, missing values were replaced with mean values for that organization. Scale scores were categorized into meaningful dichotomous categories prior to analysis (e.g., satisfied or not satisfied).

For the purpose of this study, QWL was operationally defined using the global question \"Overall, how satisfied are you with your job?\". Employees rated job satisfaction from very dissatisfied to very satisfied using a five point scale (very dissatisfied, dissatisfied, not sure, satisfied, very satisfied). For the analysis, however, those indicating they were either satisfied or very satisfied were considered to be \"satisfied\" with their jobs. All others were considered \"not satisfied\" with their jobs.

Prior to analysis, study researchers reached a consensus on which survey questions to include as potential predictors of job satisfaction. In total, there were eight scale scores and 32 questions that were rationalized *a priori* as potential predictors of job satisfaction. Data from each of the organizations, as well as all of the organizations combined (representing the SJHS), were analyzed separately to identify predictors of job satisfaction. T-test, chi-square analyses and, when appropriate, Fisher exact tests were used to determine which of the variables were statistically associated with job satisfaction i.e., were potential predictors of job satisfaction. Descriptive information (numbers and percentages) for each of the variables was calculated by whether or not staff were satisfied with their jobs. In addition, p-values, odds ratios, and 95% confidence intervals for the odds ratios were calculated for each potential predictor of job satisfaction.

Separate logistic regression analyses were used to identify the best predictors of job satisfaction for each organization and for all organizations combined (SJHS). Only variables which had a statistically significant association with job satisfaction were included in these analyses. Adjusted odds ratios and corresponding 95% confidence intervals are reported for each organization and the SJHS. The logistic regression analyses produces odds ratios which have been simultaneously adjusted for all other variables in their respective final models. The goodness of fit of the logistic regression models were assessed using the rho-squared statistic \[[@B18]\]. A rho-square value between 0.20 and 0.40 suggests a very good fit of the model. A probability level of \<0.05 was used to determine statistical significance. SPSS and Epi-Info (version 6.04a, Centers for Disease Control and Prevention, Atlanta, 1995) were used for statistical computations.

Results
=======

Table [1](#T1){ref-type="table"} provides additional information about each of the six health care organizations, including the type of organization, number of staff, number of beds or visits/year, and the size of the community where the organization was located.

Respondent participation rate
-----------------------------

Response rates are often used as an indicator of the representativeness of a sample of respondents. Of the combined 5,486 staff, 1,819 (33.2%) returned a completed questionnaire. Organization specific response rates varied from 25.3% to 55.3% (Table [2](#T2){ref-type="table"}). In an attempt to assess the representativeness of respondents, a comparison was made of available socio-demographic information between respondents and all staff within each of the organizations. Overall, female employees were more likely to respond than male employees (it should be noted, however, that the vast majority of staff (82% to 98%), were females within each of these organizations), as were full-time employees compared to part-time, casual or temporary employees. There were also some differences in respondents, across organizations, based on job classification. All organizations, however, had respondents within each job classification. A statistical estimating procedure was also used to assess how accurately respondents represent staff at each of the organizations \[[@B19]\]. This calculation suggests that the organization specific findings were accurate plus or minus 3.6% to 8.8%, 19 times out of 20 (Table [2](#T2){ref-type="table"}).

::: {#T2 .table-wrap}
::: {.caption}
######

Response rates and accuracy of responses by organization.
:::

  Site       Number of Staff^1^   Number of Respondents   Response Rate   Accuracy (plus or minus 19 times out of 20)^2^
  ---------- -------------------- ----------------------- --------------- ------------------------------------------------
  Site 1     321                  125                     38.9%           8.8%
  Site 2     649                  210                     32.4%           6.8%
  Site 3     205                  103                     50.2%           6.8%
  Site 4     481                  145                     30.1%           8.1%
  Site 5     889                  492                     55.3%           4.4%
  Site 6     2,941                744                     25.3%           3.6%
  **SJHS**   **5,486**            **1,819**               **33.2%**       **2.3%**

^1^Excludes physicians ^2^Standard error at the 95% confidence interval on a dichotomous variable with a 50/50 distribution.
:::

Potential predictors of job satisfaction
----------------------------------------

Organization specific and combined SJHS (univariate) analyses (t-test, chi-square analyses and, when appropriate, Fisher exact tests) were used to determine which of the potential predictor variables were statistically associated with job satisfaction. Included in these analyses were the 40 potential predictor variables (8 scale scores and 32 individual questions). See [Additional File](#S1){ref-type="supplementary-material"}: Statistically Significant Organization Specific (Univariate) Predictors of Job Satisfaction for a list of all variables. The number of statistically significant variables ranged from 15 to 30 depending on the organization and 32 for all organizations (SJHS) combined (see [Additional File](#S1){ref-type="supplementary-material"}: Statistically Significant Organization Specific (Univariate) Predictors of Job Satisfaction).

Best predictors of job satisfaction
-----------------------------------

Separate logistic regression analyses were then used to identify the best predictors of job satisfaction for each organization and for all organizations combined (SJHS). All variables found to be statistically associated with job satisfaction from the univariate analyses were entered into these logistic regressions analyses. The best predictors of job satisfaction are presented in Table [3](#T3){ref-type="table"}. The ranking assigned to these variables relates to the order in which variables were added to the logistic regression models. For example, the rank \"1\" refers to the first variable that was added to the model i.e., the variable which best improved the fit of the model (or the most important variable). A more detailed description of the magnitude (as represented by the size of the odds ratios) and statistical significance (as represented by the 95% confidence intervals of the odds ratios) of the association between each of these predictors and job satisfaction is presented below for each organization and all organizations combined (SJHS). The best predictors of job satisfaction are again ranked according to their importance. All of the odds ratios presented below have been simultaneously adjusted for all other variables in their respective final logistic regression models. All logistic regression models achieved a rho-square between 0.20 and 0.40 suggesting they were very good (fitting) models for predicting job satisfaction.

::: {#T3 .table-wrap}
::: {.caption}
######

Best Predictors of Job Satisfaction^1^ Ranked by Organization^2^.
:::

  Predictors of Job Satisfaction                             Site 1 Rank   Site 2 Rank   Site 3 Rank   Site 4 Rank   Site 5 Rank   Site 6 Rank   **SJHS^3^ Rank**
  ---------------------------------------------------------- ------------- ------------- ------------- ------------- ------------- ------------- ------------------
  Believes organization carries out its Mission Statement    \-            \-            3             1             1             1             **1**
  Good communication^4^                                      \-            1             \-            \-            \-            2             **2**
  Infrequently asked to do an excessive amount of work       \-            \-            1             .             3             \-            **3**
  Good decision latitude^5^                                  \-            \-            \-            3             4             6             **4**
  Overall satisfaction with pay level                        \-            5             \-            \-            5             \-            **5**
  Satisfied organization recognizes employee contributions   1             \-            \-            \-            \-            7             **6**
  Female employees                                           \-            \-            \-            \-            6             \-            **7**
  Good role clarity^6^                                       4             \-            \-            \-            7             \-            **8**
  Satisfied the organization keeps employees informed        \-            3             2             \-            2             \-            **9**
  Good teamwork^7^                                           \-            \-            \-            \-            \-            \-            **10**
  Given enough time to get job done                          \-            \-            \-            4             \-            3             **11**
  Good organization/staff relations^8^                       \-            \-            \-            \-            \-            \-            **12**
  Good decision authority^9^                                 2             4             4             \-            \-            \-            \-
  Satisfied with patient/resident care^10^                   3             \-            \-            \-            \-            \-            \-
  Good supervisor social support^11^                         \-            2             \-            2             \-            \-            \-
  Hours per week spent on job related activities             \-            \-            \-            5             \-            \-            \-
  Job classification                                         \-            \-            \-            \-            \-            4             \-
  Organization supports training and development                                                                                5             \-

^1^Separate logistic regression analyses were done to identify the best predictors of job satisfaction for each of the organizations and for the System as a whole. ^2^The rank reflects the order that variables were added to the logistic regression models. For example, the rank \"1\" refers to the first variable that was added to the model (i.e. the variable which best improved the fit of the model). ^3^In order to account for the organizational setting, site was forced into the final logistic regression analysis for the SJHS. ^4^Composite score from 2 (5-point) questions. A score of 8 to 10 indicates good communication. ^5^Composite score from 9 (5-point) questions. A score of 36 to 45 indicates good decision latitude. ^6^Composite score from 4 (5-point) questions. A score of 16 to 20 indicates good role clarity. ^7^Composite score from 7 (5-point) questions. A score of 28 to 35 indicates good teamwork. ^8^Composite score from 4 (5-point) questions. A score of 16 to 20 indicates good organization/staff relations. ^9^Decision authority is a subscale of decision latitude. It was found to be a better predictor of job satisfaction for this organization than decision latitude. It is a composite score from 3 (5-point) questions. A score of 12 to 15 indicates good decision authority. ^10^Composite score from 4 (5 point) questions. A score of 16 to 20 indicates satisfaction with patient/resident care. ^11^Composite score from 3 (5 point) questions. A score of 12 to 15 indicates good supervisor social support.
:::

### Site 1 (community hospital)

The most important predictors of job satisfaction were: 1) being satisfied with the organization\'s recognition of employee contributions (OR 5.01, 95% CI 1.59 to 15.81), 2) good decision authority (OR 7.91, 95% CI 1.46 to 42.92), 3) being satisfied with patient resident care (OR 4.66, 95% CI 1.36 to 15.97), and 4) good role clarity (OR 4.24, 95% CI 1.16 to 15.49). The final model achieved a rho-square of 0.30.

### Site 2 (community hospital/long-term care facility)

The most important predictors of job satisfaction were: 1) good open communication between staff (OR 2.55, 95% CI 1.03 to 6.35), 2) good supervisor social support (OR 6.27, 95% CI 1.36 to 29.00), 3) organization keeps staff informed (OR 3.73, 95% CI 1.51 to 9.20), 4) good decision authority (OR 3.49, 95% CI 1.25 to 9.73), and 5) being satisfied with pay level (OR 2.47, 95% CI 1.14 to 5.34). The final model achieved a rho-square of 0.24.

### Site 3 (visiting nurse organization)

The most important predictors of job satisfaction were: 1) less frequently (never/seldom/sometimes) asked to do an excessive amount of work (OR 7.22, 95% CI 2.22 to 23.46), 2) being satisfied or very satisfied that the organization keeps employees informed (OR 4.52, 95% CI 1.43 to 14.32), 3) belief the organization carries out its Mission statement (OR 11.17, 95% CI 2.04 to 61.14, and 4) good decision authority (OR 5.29, 95% CI 1.32, to 21.22). The final model achieved a rho-square of 0.34.

### Site 4 (long-term care facility)

The most important predictors of job satisfaction were: 1) belief the organization carries out its Mission statement (OR 4.63, 95% CI 1.77 to 12.51), 2) good supervisor social support (OR 3.32, 95% CI 1.22 to 9.04), 3) good decision latitude (OR 11.61, 95% CI 1.33 to 101.8), 4) often or always given enough time to get the job done (OR 3.05, 95% CI 1.00 to 9.35), and 5) spending 38 hours or more on the job or job related activities (OR 3.55, 95% CI 1.32 to 9.59). The final model achieved a rho-square of 0.34.

### Site 5 (community hospital)

The most important predictors of job satisfaction were: 1) belief the organization carries out its Mission statement (OR 3.42, 95% CI 1.82 to 6.43), 2) satisfied that the organization keeps staff informed (OR 2.62, 95% CI 1.48 to 4.65), 3) not being asked frequently to do an excessive amount of work (OR 2.41, 95% CI 1.36 to 4.27), 4) good decision latitude (OR 5.65, 95% CI 2.09 to 15.25), 5) being satisfied with pay level (OR 2.41, 95% CI 1.37 to 4.23), 6) being female (OR 2.99, 95% CI 1.29 to 6.90), and 7) good role clarity (OR 2.45, 95% CI 1.02 to 5.86). The final model achieved a rho-square of 0.25.

### Site 6 (tertiary care hospital/community health centre)

The most important predictors of job satisfaction were: 1) belief the organization carries out its Mission statement (OR 3.99, 95% CI 2.52 to 6.31), 2) good communication (OR 3.00, 95% CI 1.85 to 4.88), 3) being given enough time to get the job done (OR 2.63, 95% CI 1.58 to 4.40), 4) being a member of the nursing staff (OR 2.73, 95% CI 1.75 to 4.26), 5) good organization support for training and development (OR 3.51, 95% CI 1.59 to 7.76), 6) good decision latitude (OR 2.57, 95% CI 1.30 to 5.09) and 7) being satisfied with the organization\'s recognition of employee contributions (OR 2.05, 95% CI 1.07 to 3.91). The final model achieved a rho-square of 0.25.

### All sites combined (SJHS)

The most important predictors of job satisfaction after adjusting for site were: 1) belief the organization carries out its Mission statement (OR 2.79, 95% CI 2.07 to 3.77), 2) good communication (OR 1.87, 95% CI 1.33 to 2.62), 3) less frequently being asked to do an excessive amount of work (OR, 1.80, 95% CI 1.33 to 2.43), 4) good decision latitude (OR 3.28, 95% CI 2.09 to 5.17), 5) being satisfied with pay level (OR 1.61, 95% CI 1.21 to 2.15), 6) being satisfied with the organization\'s recognition of employee contributions (OR 1.57, 95% CI 1.07 to 2.29), 7) being female (OR 2.83, 95% CI 1.81 to 4.42), 8) good role clarity (OR 1.73, 95% CI 1.17 to 2.56), 9) being satisfied that the organization keeps employees informed (OR 1.35, 95% CI 1.00 to 1.85), 10) good teamwork (OR 1.45, 95% CI 1.01 to 2.09), 11) being given enough time to get the job done (OR 1.57, 95% CI 1.10 to 2.23), and 12) good organization/staff relations (OR 2.02, 95% CI 1.13 to 3.62). The final model achieved a rho-square of 0.26.

Discussion
==========

The results of this survey were intended to assist decision-makers in identifying key workplace issues, as perceived by employees, in order to develop strategies to address and improve the quality of working conditions for staff within each of the individual health care organizations and the SJHS as a whole. This research represents the first step of an ongoing process to ensure better QWL for employees. In addition to the findings presented here, information from the survey\'s open-ended written comments have also been summarized for each of the six organizations (L Lohfeld, K Brazil, P Krueger, G Edward, D Lewis, E Tjam, E., personal communication, 2001) and the SJHS as a whole (St. Joseph\'s Health System Quality of Work Life Technical Reports 2000). This open-ended information provides additional and complementary information to that which is provided in this report. Together, these findings are currently being used by decision-makers at each of the organizations, and the SJHS, in an effort to improve employee QWL.

It should be noted that at the time of this survey, all of the hospitals included in this study (as well all other hospitals within the Province of Ontario) were operating in an environment of restructuring and change. This was a time of anxiety for many health care professionals, hospital staff and the general public. In 1996, the Ontario government created a Health Services Restructuring Commission (HSRC) with a four year mandate to restructure Ontario\'s hospitals and health services system. The HSRC was given authority under the Public Hospitals Act and The Ministry of Health Act to direct public hospitals to change their roles, transfer services and programs, amalgamate or close. The HSRC completed its mandate, announced its decisions and was terminated in March 2000. The timing for this study was after the decisions of the HSRC were announced. All of the organizations included in this study were impacted to varying degrees either directly or indirectly the HSRC decisions. The most notable impacts occurred at Site 1 and Site 2. Site 1 (a community hospital) was ordered closed effective March 2001 with programs and services to be transferred to the other local community hospital while site 2 (a community hospital/long-term care facility) was ordered to transfer its acute care services to the other local hospital in its community thereby becoming a long-term care facility. During the time of the survey, a new building (adjacent to the current facility) for the new long-term care facility was under construction and was scheduled to open in 2002. These contextual issues could have influenced employee responses and therefore the predictors of job satisfaction for all of these organizations, particularly for site 1 and site 2.

There are several positive attributes of this study. First, to our knowledge, it is the largest QWL investigation of health care workers in Canada with 1,819 completed interviews. Second, it is also unique in that we collected information from staff at six distinct and functionally diverse health care organizations. Third, because we could not find an \"off-the-shelf\" QWL instrument that suited our needs and collected all the information desired by key stakeholders, we developed (through a combination of modifying existing instruments and creating our own questions and scales) our own questionnaire. Finally, although the response rates were not as high as we would have hoped, the findings: appear to be consistent with what we expected *a priori* (the study\'s investigators had offices within 5 of the 6 organizations thus having inside knowledge about these organizations); appear consistent with the published literature; and were judged credible by management and staff at each of the sites. The statistical estimating procedure to assess how accurately respondents represent staff at each of the organizations also suggest that our findings were fairly representative of staff within these organizations, particularly the larger organizations.

Conclusions
===========

The results of this research show that job satisfaction is a multidimensional construct and is a product of the global evaluation of one\'s work place and context. This report provides valuable information about how employees in specific health care settings view their work environment. A number of organization specific predictors of job satisfaction were identified as a result of this study. The implications of these findings are currently being deliberated as they relate to improving QWL within each of the six health care organizations that make up the SJHS. These findings, may also be of relevance and value to employees, researchers, evaluators, human resource planners and administrators of similar health care organizations.

The results of this survey can also be used as baseline measures against which the findings of future job satisfaction surveys can be compared. Such comparisons place this type of research within a continuous quality improvement framework.

Competing interests
===================

None declared.

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

<>

Supplementary Material
======================

::: {.caption}
###### Additional File

Statistically Significant1 Organization Specific (Univariate) Predictors of Job Satisfaction.
:::

::: {.caption}
######

Click here for file
:::

Acknowledgments
===============

We would like to express our sincere appreciation to the QWL Task Force, to those involved in pretesting the survey, to Justin Garbedian, Project Assistant, and most importantly, to all staff who returned a completed questionnaire. This research was funded in part by the St. Joseph\'s Health System and the St. Joseph\'s Health System Research Network, Father Sean O\'Sullivan Research Centre, Hamilton, Ontario.


Background
==========

*Helicobacter pylori (H. pylori)* has been etiologically linked with primary gastric lymphoma (PGL) and gastric carcinoma (GC) \[[@B1],[@B2]\]. Evidences in favor of cause and effect relationship between *H. pylori* and PGL include: higher frequency of isolation in PGL than in controls \[[@B3]\]; development of the disease during long-term follow up of infected subjects \[[@B4]\]; regression after eradication of the organism \[[@B5]\]; and rapid recurrence following re-infection \[[@B6]\]. Therefore, all components of Koch\'s postulates for establishing cause and effect relationship between *H. pylori* and PGL are fulfilled \[[@B7]\]. Evidences in favor of etiological relationship between *H. pylori* and GC are somewhat incomplete. In most studies from developed countries *H. pylori* infection has been correlated with GC \[[@B8]\]; however, the data from developing world where *H. pylori* infection is more frequent and is acquired at an early age is somewhat contradictory \[[@B9],[@B10]\]. There is no report of regression of GC after eradication of *H. pylori.* Recently, GC has been shown to develop in Mongolian gerbils after infection with *H. pylori*\[[@B11]\]. However, development of GC during follow up after fresh infection with *H. pylori* in humans has not been documented despite such report in PGL \[[@B4]\]. Accordingly we report a patient with PGL from India in whom surgical resection of the tumor combined with eradication of *H. pylori* was followed by regression of PGL. This patient developed metachronous GC associated with recrudescence / re-infection of *H. pylori.*

Case presentation
=================

A 32-year-old man presented in May 1998 with epigastric pain of six months duration. The pain was constant without any relation to meal or radiation to back. He also complained of anorexia, weight loss, vomiting of copious amount of stale food without any bile for four months. Melena occurred once three months ago. There was no history of fever. He denied history of gastric cancer in his family. Physical examination revealed emaciation and pallor. There was no lymphadenopathy, hepatosplenomegaly and abdominal lump. Examination of throat did not reveal any abnormality.

Hemoglobin=85g/L, TLC=6.7 × 10^9^/L, ESR=78 mm/h. Chest radiograph was normal. Abdominal ultrasonogram did not reveal lymphadenopathy, hepatosplenomegaly or ascites. Upper gastrointestinal (UGI) endoscopy revealed hyperemic lower esophageal mucosa with multiple non-confluent erosions; there was excessive food residue inside the lumen and multiple hypertrophied gastric folds with erosions over these. Pyloric orifice was narrow and endoscope could not be negotiated into the duodenum. Six biopsies taken from the hypertrophied folds revealed only chronic inflammatory infiltrate and no specific pathology. *H. pylori* was not detected on histologic examination of gastric biopsies. In house rapid urease test (RUT) was positive within 15 minutes and scanning electron microscopy (SEM, Hitachi S530, Japan) from biopsies obtained from relatively normal looking areas revealed multiple round cells (might be lymphocytes, figure [1A](#F1){ref-type="fig"}) but no *H. pylori.* Serum titer for IgG antibody against *H. pylori* using ELISA based commercially available kit (Monobind Incorporation, California) was positive (21 IU/ml). Lansoprazole was started. With a diagnosis of gastric outlet obstruction, surgery was done after 2 weeks. After exploring the abdomen by midline incision thickening and nodularity of antrum were detected. There was no regional lymphadenopathy. Partial gastrectomy, removal of peri-gastric lymph nodes, closure of resected end of the duodenum in double layers and end-to-side ante-grade retro-colic gastrojejunostomy were done. Postoperative course was uneventful. Histological examination of resected specimen revealed transmural infiltration by mononuclear cells diffusely extending through muscularis mucosa into the submucosa without formation of lymphoid follicles, cellular atypia, abnormal mitotic figures (figure [2A](#F2){ref-type="fig"}) and lymphoepithelial lesions. On thorough histologic evaluation no evidence of synchronous GC was seen. Peri-gastric lymph nodes were not infiltrated by lymphoma. Bone marrow examination did not reveal any abnormality. On the basis of these morphological features a diagnosis of PGL (low-grade B cell lymphoma of MALT type by revised European-American classification) \[[@B12]\], stage I E: I~1~ (Musshoffs modification of Ann Arbor classification) \[[@B13]\] and *H. pylori* infection were made. Patient was treated with amoxycillin 1.5 g/d, omeprazole 40 mg/d and tinidazole 1 g/d for two weeks to eradicate the infection with *H. pylori.* UGI endoscopy repeated one month after completion of anti-*H. pylori* treatment revealed small sized stomach, no ulcer. RUT and Giemsa staining of biopsies obtained from proximal stomach were negative for *H. pylori.* However, a repeat UGI endoscopy done after six months revealed a benign-looking ulcer (1.5 cm in diameter) at gastrojejunostomy stoma. RUT and histology (Giemsa stain) on biopsies obtained from proximal stomach for *H. pylori* were still negative. Biopsy obtained by well technique from margin of the ulcer revealed chronic inflammatory infiltrate. Flow cytometry by standard technique \[[@B14]\] from these biopsies using anti-CD 19 (B cell marker) and anti-CD3 (T cell marker) monoclonal antibodies revealed polyclonal infiltrate with 6.6% CD 19 and 10.8% CD3 bearing cells (figure [3](#F3){ref-type="fig"}). With a possibility of benign stomal ulcer patient was treated with lansoprazole 30 mg/d for 3 months. After this period of treatment, healing of the ulcer and absence of *H. pylori* infection were documented by UGI endoscopy and negative RUT respectively (2 weeks after stopping lansoprazole). On follow up during the next six months, he remained well with improved appetite and weight gain (35 to 46 kg). He presented with recurrence of epigastric pain, anorexia and vomiting in August 1999. He had lost body weight (34 kg in August 1999). Examination revealed pallor, no peripheral lymphadenopathy and abdominal lump. UGI endoscopy: friable ulcerated growth involving most of stomach with reduced distensibility of the organ and patent gastrojejunostomy stoma. Since a possibility of recurrent PGL was considered, flow cytometry was done by standard technique \[[@B14]\] with biopsy from the tumor using anti-CD 19 (B cell marker) and anti-CD3 (T cell marker) antibodies; it revealed a polyclonal lymphocytic infiltrate consisting of 0.9% CD 19 and 3.9% CD3 bearing cells (figure [3](#F3){ref-type="fig"}). Biopsies obtained from relatively normal looking mucosa of stomach gave positive result to RUT. Histological examination revealed chronic gastritis but no intestinal metaplasia. SEM (Hitachi S530, Japan) with these biopsies revealed multiple bacilli suggestive of *H. pylori* (figure [1B](#F1){ref-type="fig"}). Histopathology of biopsy from margin of ulcers revealed multiple signet ring cells, cellular pleomorphism, mucin secretion, abnormal mitotic figures and gland formation at certain places (figure [2B](#F2){ref-type="fig"}). A diagnosis of GC and recrudescence / re-infection of *H. pylori* were made. Patient denied further treatment at this stage and left the hospital.

![(A) Scanning electron microphotograph of gastric mucosa at presentation showing multiple round cells (possibly lymphocytes) but No *H. pylori* (× 1000). (B) Scanning electron microphotograph of gastric mucosa during re-infection showing bacillus with polar flagella suggestive of *H. pylori* (× 8000).](1471-230X-2-6-1){#F1}

![(A) Resected specimen of distal stomach showing diffuse infiltration by mononuclear cells without formation of lymphoid follicles with obvious cellular atypia and abnormal mitotic figures (H&E × 275). (B) The high power view of recurrent gastric tumor showing pleomorphic cells, abnormal mitotic figures, mucin secretion and formation of gland at places diagnostic of adenocarcinoma (H&E × 275).](1471-230X-2-6-2){#F2}

![Flow cytometry was performed using FACS calibur (Becton Dickinson) and data were analyzed using Cell Quest program. One parameter histogram is shown.](1471-230X-2-6-3){#F3}

Discussion and conclusion
=========================

High index of suspicion is required for early diagnosis of PGL in developing countries. Endoscopic features of PGL may be very non-specific. Endoscopic findings like slight increase in thickness of gastric folds, multiple benign looking gastric ulcers or erosions \[[@B15]\] are too non-specific to suspect the disease unless endoscopist specifically looks for it. Endoscopic mucosal biopsy may miss the diagnosis \[[@B16]\] and may show only benign looking lymphocytic infiltrate as in the present case. Molecular diagnostic methods essential for confirming the diagnosis are not widely available in developing countries including ours. Early diagnosis is important, as eradication of *H. pylori* has been commonly shown to cause regression in an early stage of the disease \[[@B17]\]. *H. pylori* has been found more often in patients with PGL \[[@B3]\], chronic *H. pylori* infection progresses from polyclonal lymphocytic proliferation to PGL \[[@B4]\], and its eradication leads to regression \[[@B5]\]. Recurrence of PGL following re-infection with *H. pylori* following eradication has been documented in the literature \[[@B6]\]. Presence of *H. pylori* infection in our patient at presentation was evidenced by positive RUT (sensitivity 92%, specificity 80%) \[[@B18]\] and serology; absence of organism in SEM might be due to sampling error as similar phenomenon has been reported earlier in presence of gastric atrophy and intestinal metaplasia, both being common in chronic gastritis and malignancy \[[@B19]\]. Though, biopsy was obtained from endoscopically normal-looking mucosa, SEM showed round cell infiltration suggesting involvement by lymphoma. Absence of *H. pylori* in that area is quite expected. Diagnosis of PGL in our patient could be further confirmed by molecular biological methods before eradication of *H. pylori.* However, typical histology picture is quite diagnostic of PGL \[[@B20]\]. We could demonstrate clinical and histologic regression of the disease following surgical resection followed by eradication of *H. pylori* (documented by negative RUT and histology obtained from unresected proximal stomach). Flow cytometry of gastric mucosal biopsy documented polyclonal lymphocytic infiltrate confirming absence of residual lymphoma. Molecular evidences of the neoplasm may persist after treatment despite histological regression \[[@B21]\]. However, eradication of *H. pylori* removes growth stimulus for PGL. Histological and molecular relapse in absence of re-infection with *H. pylori* are rare and transient \[[@B22]\].

Our patient developed GC on follow up after regression of PGL. This was temporally associated with re-infection with *H. pylori.* Positive RUT and demonstration of typical bacilli in SEM documented re-infection. Since we could not demonstrate a different genotype of *H. pylori* at the time of re-infection a possibility of recrudescence of earlier infection can not be entirely excluded though seems less likely. Development of intestinal metaplasia, a precursor of GC \[[@B23]\] and advanced cancer \[[@B11]\] has been reported after infection with *H. pylori* in Mongolian gerbils recently. This is perhaps the first report of development of GC following recrudescence / re-infection with *H. pylori* in human. We can not rule out a possibility of spontaneous development of GC. However, this seems less likely as in most cases of combined tumor reported earlier GC and PGL were synchronous in nature \[[@B24]-[@B29]\]. In a single series of 12 cases two developed metachronous tumor in both of whom GC preceded PGL \[[@B24]\]. In contrast, development of GC is expected to occur later in the course of *H. pylori* infection in patients with PGL \[[@B30]\]. The latter develops in the active phase of inflammation whereas the former in the pre-atrophic stage of gastritis \[[@B30]\]. In our patient resected specimen of PGL on thorough histologic examination did not reveal presence of synchronous GC. Therefore, as expected, GC in our patient developed later and was temporally associated with recrudescence / re-infection with *H. pylori.* Occurrence of metachronous GC after regression of PGL with eradication of *H. pylori* has been reported rarely \[[@B31],[@B32]\]. Four cases of metachronous GC after regression of PGL with *H. pylori* eradication have been reported till date \[[@B31],[@B32]\]. Development of GC in our patient was temporally associated with re-infection with *H. pylori* following regression of PGL. This is perhaps the first report of such an association. Considering several earlier studies \[[@B33],[@B34]\], which suggested reduction in risk of GC following *H. pylori* eradication, temporal association of development of GC with recrudescence / re-infection with *H. pylori* in our patient is not difficult to explain. However, development of GC within a short period of time after fresh infection with *H. pylori* is somewhat odd as GC takes time to develop after infection with *H. pylori*\[[@B35]\]. A possibility of genetic or immunological predisposition can not be excluded. Further, since the patient was already chronically infected with *H. pylori* he might have been predisposed. Some workers have suggested a possibility of immunological containment of dysplastic epithelial cells by lymphocytic infiltrate \[[@B36]\]. Whether immunological imbalance resulting from regression of lymphocytic infiltrate could contribute to rapid development of GC, which got further triggered by re-infection or recrudescence of pre-existing infection with *H. pylori* remains an open question. Development of GC in this patient might be related to changes induced by many years of *H. pylori* related gastritis rather than a few months of re-infection. However, since there was a temporal relation with re-infection, it could also be related to progression of existing changes induced over many years by re-infection. This remains a conjecture.

In conclusion, we have shown regression of PGL after surgical resection combined with eradication of *H. pylori* and development of GC in association with reinfection with *H. pylori* in the present case. This may suggest the possibility of cause and effect relationship of both these malignancies to *H. pylori.* This case also highlights a unique problem in management of PGL in tropical countries where re-infection with *H. pylori* is supposed to be high \[[@B37]\].

List of abbreviations
=====================

H. pylori: Helicobacter pylori;

PGL: primary gastric lymphoma

GC: gastric carcinoma

TLC: total leukocyte count

ESR: erythrocyte sedimentation rate

UGI: upper gastrointestinal

RUT: rapid urease test

SEM: scanning electron microscopy

IgG: immunoglobulin G

ELISA: enzyme linked immunosorbent assay

IU: international unit

MALT: mucosa-associated lymphoid tissue

CD: cluster of differentiation

Competing interests
===================

None declared

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

<>

Acknowledgements
================

We thank relatives of the patient for giving us written consent for publishing the patient\'s details.


Background
==========

Mos belongs to a small family of cytoplasmic protein serine/threonine kinases having oncogenic activity \[[@B1],[@B2]\]. It is highly expressed in germ cells but barely detectable in a variety of somatic tissues \[[@B3]-[@B5]\]. Studies in Xenopus oocytes have established a role for c-mos in a) initiation of the maturation process and the meiosis I / meiosis II transition and b) in metaphase II arrest in mature oocytes \[[@B6]-[@B12]\]. In mouse c-Mos is apparently not required for initiation of maturation, however, like in Xenopus it is absolutely essential for the metaphase II arrest \[[@B13],[@B14]\].

The 124-v-mos oncogene represents one of several transforming gene isolates of the moloney murine sarcoma virus \[[@B15],[@B16]\] and shows unique constitutive protein kinase activity and enhanced transforming activity when compared to other v-Mos proteins or to c-Mos \[[@B2],[@B17]-[@B19]\]. The transforming mechanism of Mos involves signalling through the MAP kinase pathway as phosphorylation of MEK by c-Mos has been demonstrated \[[@B20]-[@B23]\] and mapping analyses have shown that Mos and Raf phosphorylate identical sites on MEK \[[@B16],[@B24]\]. The upstream events of the Mos/MEK/MAPK signalling cascade have not as yet been identified. In earlier studies we have shown that an activating mechanism of c-Mos is likely to involve a conformational change which is mimicked when a single amino acid is exchanged in the α-helix C loop of the kinase domain (Arg145-Gly) resulting in constitutive active c-Mos \[[@B19]\]. Recently Fisher and co-workers proposed an activating mechanism of c-Mos by sequential association with Hsp70 and Hsp90, in addition to phosphorylation \[[@B25],[@B26]\].

Presence of the activating Arg145-Gly amino acid substitution in 124-v-Mos does not change kinase specificity but is sufficient for constitutive kinase activity \[[@B19]\]. Hence the kinase activity of 124-v-Mos is independent of upstream effectors and we have used this oncogenic Mos derivative to identify substrates for the Mos protein kinase in vitro. Using the baculo virus expression system we have expressed active 124-v-Mos protein kinase, as demonstrated by its ability to auto-phosphorylate, predominantly on serine residues, and to phosphorylate vimentin in vitro. We have analysed a panel of acidic and basic substrates in immunocomplex protein kinase assays and identified two novel in vitro substrates for 124-v-Mos, the protein tyrosine phosphatase 1B and α/β-casein.

Results
=======

Three tryptic 124-v-Mos peptides include target sites for auto-phosphorylation
------------------------------------------------------------------------------

We have expressed 124-v-Mos with the baculovirus system in Sf9 insect cells and immunopurified 124-v-Mos using the anti-Mos N13 antiserum \[[@B19]\]. As a control, a Mos-unrelated protein, a synthetic kinase-inactive construct of PKC, PKCγ^K380R^\[[@B27]\], was expressed in Sf9 cells. Mos kinase assays, completed in the presence of \[γ-^32^P\]ATP, were resolved using SDS-PAGE and the Coomassie blue staining of the protein gel showed visible amounts of immunopurified 124-v-Mos (fig. [1B](#F1){ref-type="fig"}, arrowhead). The corresponding autoradiograph in figure [1A](#F1){ref-type="fig"} demonstrates that 124-v-Mos is expressed as a constitutive active protein kinase indicated by its ability to auto-phosphorylate in vitro. Further, a parallel kinase reaction was used for phosphoamino acid analyses which confirmed that 124-v-Mos auto-phosphorylation occurred predominantly on serine residues (fig. [1C](#F1){ref-type="fig"}) and a two-dimensional resolution of a tryptic digest of auto-phosphorylated 124-v-Mos showed that three tryptic peptides include auto-phosphorylation target sites (fig. [1D](#F1){ref-type="fig"}), demonstrating that auto-phosphorylation occurs on multiple sites of the Mos protein \[[@B28]\].

![**Constitutive kinase activity of immunopurified 124-v-Mos from baculovirus expressing Sf9 insect cells.** Auto-phosphorylation of immunopurified 124-v-Mos expressed in Sf9 cells is shown in B (Coomassie stained 10% SDS-PAGE) and A (corresponding autoradiograph). Parallel 124-v-Mos kinase assays were subjected to a two-dimensional phosphoamino acid analysis (C) or a tryptic digestion followed by a two-dimensional resolution (D). Arrowheads indicate the origin of sample application in (C,D) and the position of 124-v-Mos (A,B).](1471-2091-3-6-1){#F1}

124-v-Mos phosphorylates vimentin but not tubulin in vitro
----------------------------------------------------------

Initially, we tested the kinase activity of 124-v-Mos using previously identified Mos substrates. It has been shown that 124-v-Mos, derived from mos-transformed fibroblasts, phosphorylates vimentin in vitro \[[@B29]\] and as presented here in figure [2C](#F2){ref-type="fig"}, in vitro kinase assays using immunopurified 124-v-mos from Sf9 insect cells showed strong vimentin phosphorylation. In contrast, tubulin which has been shown to be phosphorylated in vivo and in vitro by Xenopus c-Mos \[[@B30]\] was not a substrate for 124-v-Mos in vitro (fig. [2A](#F2){ref-type="fig"}). We have tested tubulin purified from various organs (mouse brain, testis and spleen) either polymerised, unpolymerised or pretreated with phosphatases but in none of these states found tubulin to be phosphorylated by 124-v-Mos (data not shown).

![**124-v-Mos phosphorylates vimentin but not tubulin.** In vitro 124-v-Mos kinase assays with either vimentin (C,D) or purified tubulin from brain (A,B) as substrates were electrophoresed using 10% SDS-PAGE and Coomassie stained (B,D), the corresponding autoradiographs are shown in (A,C). Immunoprecipitates of Sf9 cells expressing the kinase-inactive PKCγ^K380R^ were indicated as controls.](1471-2091-3-6-2){#F2}

Demonstration of alpha and beta-casein phosphorylation by 124-v-Mos
-------------------------------------------------------------------

In search of further substrates for the 124-v-Mos protein kinase we tested MBP; histone HI, H2AS, H3; protamine; protaminsulphate; purified PKC-α/-β II/γ and α- and β-casein. With the exception of α- and β-casein (fig. [3A](#F3){ref-type="fig"}) none of these substrates were phosphorylated by 124-v-Mos (data not shown). The possibility that factors other than 124-v-Mos in the immunoprecipitate might be responsible for the observed casein phosphorylation was eliminated by including a synthetic kinase-inactive construct of 124-v-Mos, 124-v-Mos^K121R^\[[@B19]\], as a control in addition to the Mos-unreleated protein, PKC\_^K380R^. A comparison of background phosphorylation on β-casein in the immunoprecipitates of both controls and 124-v-Mos specific phosphorylation showed that 124-v-Mos phosphorylates β-casein 7fold relative to background (fig. [3B](#F3){ref-type="fig"}). Critically, a tryptic digest of phosphorylated β-casein revealed that 124-v-Mos phosphorylates a specific tryptic peptide in β-casein which shows no background phosphorylation in either controls (fig. [3C](#F3){ref-type="fig"}, arrowhead) strongly supporting that 124-v-Mos is able to phosphorylate β-casein. Further, a two-dimensional phosphoamino acid analysis (fig. [3D](#F3){ref-type="fig"}) showed that 124-v-Mos phosphorylates α- and β-casein on serine and threonine residues at a ratio of 1:1.

![**124-v-Mos phosphorylates α- and β-casein in vitro.** Mos kinase assays, in the presence of α- and β-casein, were resolved using 10% SDS-PAGE; the Coomassie stained protein gel shown in 3A, right panel and the corresponding autoradiograph on the left panel. Arrowheads indicate the position of 124-v-Mos, α- and β-casein and the antibody. Using two control immunoprecipitates of Sf9 cells expressing the synthetic kinase-inactive constructs, 124-v-Mos^K121R^ or PKCγ^K380R^, Mos-specific β-casein phosphorylation was demonstrated in 3B and 3C: Mos kinase assays were blotted on nylon-membrane, the phospho-β-casein bands (B, arrowhead) excised and ^32^P-Cerenkov counts recorded (B). Alternatively, the excised phospho-β-casein bands were digested with trypsin and electrophoresed using 16% SDS-PAGE (C). The arrowhead in 3C indicates the tryptic β-casein peptide phosphorylated by wild-type 124-v-Mos only. Further, two-dimensional phosphoamino acid analyses of 124-v-Mos phosphorylated α-casein (D, left panel) and β-casein (D, right panel) were completed, the arrowheads indicating the origins of sample application.](1471-2091-3-6-3){#F3}

The protein tyrosine phosphatase 1B is a novel in vitro substrate for 124-v-Mos
-------------------------------------------------------------------------------

Protein tyrosine phosphatases constitute a diverse family of enzymes that can be divided into several subgroups, including receptor and non-receptor PTPs \[[@B31]\]. The non-transmembrane protein tyrosine phosphatase PTP-1B, a major intracellular PTP is widely expressed. PTP-1B has been demonstrated to be phosphorylated on multiple sites in a cell cycle specific manner whereby mitotic hyper-phosphorylation occurs, reflected by a protein mobility shift in SDS-PAGE analyses \[[@B32]\]. Using purified PTP-1B as a substrate, we show here that 124-v-Mos can phosphorylate PTP-1B in vitro (fig. [4A](#F4){ref-type="fig"}). We controlled this result by using immunoprecipitates from Sf9 cells expressing the synthetic kinase-inactive 124-v-Mos construct or purified PTP-1B alone in parallel kinase assays (fig. [4A](#F4){ref-type="fig"}). Other kinases such as PKC and CKII that phosphorylate PTP-1B in vitro are unable to induce a mobility shift of PTP-1B as observed in mitotic cells \[[@B32]\]. Likewise, as shown in figure [4B](#F4){ref-type="fig"}, a Mos-dependent phosphorylation did not result in a mobility shift of PTP-1B.

![**PTP-1B is a substrate for 124-v-Mos in vitro.** In vitro Mos kinase assays, using purified PTP-1B as a substrate, were resolved using 10% SDS-PAGE and the autoradiograph is shown in 4A. Immunoprecipitates of Sf9 cells expressing the kinase-inactive 124-v-Mos^K121R^ variant or PTP-1B alone were included as controls (A,B). A parallel kinase assay was blotted on nylon-membrane and PTP-1B was detected (B) using the PTP-1B-specific antiserum FG6 \[[@B29]\], arrowheads indicate the position of 124-v-Mos and PTP-1B.](1471-2091-3-6-4){#F4}

Discussion
==========

In this study we have expressed constitutive active 124-v-Mos using the baculovirus expression system and identified novel in vitro substrates for Mos by immunocomplex kinase assays. It has been shown that 124-v-Mos from mos-transformed mouse fibroblasts phosphorylates vimentin in vitro \[[@B29]\] and that v-Mos is physically associated with vimentin in transformed cells \[[@B33]\]. We have used vimentin as a positive control for 124-v-Mos kinase assays in vitro to demonstrate protein kinase activity of baculovirus expressed 124-v-Mos (fig. [2](#F2){ref-type="fig"}). It is known that the kinase activity of c-Mos is regulated by cellular factors and therefore we have chosen the oncogenic variant of c-Mos, 124-v-Mos, in our study since it is independent of activating mechanisms. Recently it has been shown that Hsp70 and Hsp90 physically interact with c-Mos in Xenopus oocytes and are required for c-Mos activation \[[@B25],[@B26]\]. Another factor controlling c-Mos kinase activity in Xenopus oocytes was identified by Chen and colleagues \[[@B34],[@B35]\] to be CKII, a tetrameric holoenzyme composed of two catalytic α-subunits and two regulatory β-subunits \[[@B36]\]. In Xenopus oocytes c-Mos kinase activity is inhibited by binding to the C-terminus of CKII β-subunit and by over-expression of the α-subunit of CKII this effect can be neutralized suggesting a binding competition between c-Mos and the α-subunit of CKII \[[@B34]\]. Another protein that interacts with c-Mos in Xenopus oocytes is tubulin. Tubulin not only co-precipitates with c-Mos but also serves as an in vivo and in vitro substrate \[[@B30]\]. In contrast, tubulin was not a substrate for 124-v-Mos in our immunocomplex kinase studies (fig. [2A](#F2){ref-type="fig"}). Possibly, this indicates that a cellular factor present in Xenopus oocytes and co-precipitating with c-Mos might be necessary for tubulin phosphorylation by the Mos protein kinase. This factor might not interact with the v-Mos protein, be absent in Sf9 insect cells or unable to interact with v-Mos. Interestingly, we have not detected any co-precipitation of the \_-subunit of CKII from Sf9 cells with 124-v-Mos in our immunoprecipitates (data not shown). However, as previously mentioned, Hsp70 is known to interact also with 124-v-Mos \[[@B26]\].

Having established that our recombinant 124-v-Mos protein is active in vitro, we tested a variety of molecules in immunocomplex kinase assays and identified α- and β-casein as very good substrates in vitro (fig. [3](#F3){ref-type="fig"}). This phosphorylation was specific to active 124-v-Mos as the overall phosphorylation on casein was significantly reduced using the synthetic kinase-inactive construct 124-v-Mos^K121R^ and more importantly, a tryptic peptide of casein was identified to be phosphorylated by 124-v-Mos only and not by either of the controls used in this study. As expected, casein phosphorylation occured on serine and threonine residues. The Mos-specific consensus phosphorylation site has not as yet been identified and only the mos-phosphorylation sites on MAP kinase kinase have been mapped revealing them to be identical to raf-phosphorylation sites \[[@B24]\]. Using the mos substrates identified in this study, it may be possible to determine the specific consensus phosphorylation site for the mos protein kinase as a basis for developing Mos-specific inhibitors.

We have also identified protein tyrosine phosphatase 1B (PTP-1B) as a substrate for 124-v-mos in vitro (fig. [4A](#F4){ref-type="fig"}). PTP-1B is phosphorylated on multiple sites in vivo and during mitosis becomes hyper-phosphorylated resulting in a mobility shift in SDS-PAGE \[[@B32]\]. Protein kinase C and CKII phosphorylate PTP-1B in vitro but neither is responsible for the observed mitotic hyper-phosphorylation in vivo \[[@B32]\]. We show here that likewise PTP-1B phosphorylation by 124-v-mos is insufficient to effect a mobility shift (fig. [4B](#F4){ref-type="fig"}). PTP-1B phosphorylation occurs on serine 386, a phosphoacceptor site for Cdc2/cyclin B in vitro and serine 352, phosphorylated by an unknown kinase. The serine 352 phosphorylation site either might not be a target for Mos in vitro or PTP-1B may be sequentially phosphorylated by multiple kinases in vivo. Interestingly, it has been shown that PTP-1B hyper-phosphorylation does not occur uniquely in mitosis but also during osmotic shock and is induced by several other stress stimuli \[[@B37]\]. Given that activation of c-Mos is dependent on its interaction with the heatshock proteins, Hsp70 and Hsp90, it is tempting to speculate that the Mos kinase may phosphorylate PTP-1B also in vivo.

Conclusions
===========

The crucial biological functions of c-mos during meiosis have been analysed by antisense experiments in Xenopus lavis and by generating mos-deficient mice establishing mos as the main player in metaphase II arrest. In contrast, not much is known about activating mechanisms of mos and biochemical properties such as the mos-specific consensus phosphorylation site. In this study we immunopurified an oncogenic and constitutive active variant of mos, 124-v-Mos, and identified novel phosphorylation substrates, PTP1B and α- and β-casein. Our substrates represent a basis to determine the consensus mos-specific phosphorylation site and further, to analyze this phosphorylation ability functionally in vivo.

Materials and Methods
=====================

Protein expression and in vitro immunocomplex protein kinase assays
-------------------------------------------------------------------

The construction and isolation of recombinant baculoviruses expressing active 124-v-Mos and the synthetic kinase-inactive variant of 124-v-Mos, 124-v-Mos^K121R^, is described in detail elsewhere \[[@B19]\]. According to the standard procedure published by Summers & Smith \[[@B38]\], recombinant proteins were expressed at 27°C in Sf9 cells for 48 hrs. and mos was immunopurified using the anti-Mos N13 antiserum as stated in \[[@B19]\]. Mos kinase assays were carried out in 50 \_l kinase reaction buffer (10 mM HEPES pH 7.3, 150 mM NaCl, 0.1% Triton X-100, 2 mM DTT, 15 mM MnCl~2~, 5 mM MgCl~2~, 2.5 mM β-glycerophosphate, 2.5 mM NaF, 20 μM ATP/ 10 μCi \[\_γ^32^P\]ATP), incubated for 20 min. at 25°C and stopped by the addition of Laemmli buffer. For in vitro substrate kinase assays, 2 μg of substrate was added to each kinase reaction. Phosphoproteins were resolved using 10% SDS-PAGE, Coomassie stained, dried and compared with the corresponding autoradiograph. Immunodetection of western blots were performed using the ECL system and protocol (Amersham).

Substrates for in vitro immunocomplex kinase assays
---------------------------------------------------

α- and β-casein (dephosphorylated, bovine origin) were purchased from Sigma and vimentin from Roche. Purified PTP-1B and the PTP-lB-specific antiserum FG6 were provided by N. Tonks, Cold Spring Harbor \[[@B32]\]. Tubulin was purified from either mouse brain, testis or spleen by F. Propst, Vienna.

Two-dimensional phosphoamino acid analyses
------------------------------------------

Two-dimensional phosphoamino acid analyses were completed according to Boyle and colleagues \[[@B39]\]. Briefly, phosphoproteins were separated using SDS-PAGE, blotted on nylon-membrane and the desired protein bands were excised. The membrane strips were washed sequentially with 100% methanol and water and the phosphoproteins hydrolysed for 60 min. at 110°C in 5.7 N HCl. The hydrolysed samples were lyophilised, resuspended in 2.5% formic acid, 7.8% acetic acid and mixed at 15:1 with a non-radioactive amino acid standard (1 mg/ml of each phospho-serine, -threonine, -tyrosine; Sigma). Finally, samples were spotted on thin-layer chromatography plates and separated in two dimensions using the HTLE-7000 apparatus and manufacture\'s procedure (Two-Dimensional Peptide Mapping And Phosphoamino Acid Analysis, Featuring The Hunter Thin Layer Plate Electrophoresis System. B. Boyle & T. Hunter, C.B.S. Scientific Company, Del Mar, USA). First dimension: 20 min. electrophoresis at 0.8 bar, 1 kV in 2.5% formic acid, 7.8% acidic acid. Second dimension: 16 min. at 0.8 bar, 1.3 kV in 5% acidic acid, 0.5 % pyridine. The phosphoamino acids were fixed for 10 min. at 65°C and the standard non-radioactive amino acids visualised by spraying the chromatography plates with 0.25% ninhydrin followed by incubation for 15 min. at 65°C. The phosphoamino acids were located by comparing the autoradiograph with the stained standard amino acids.

Tryptic digests and one- or two-dimensional separation of tryptic phosphopeptides
---------------------------------------------------------------------------------

According to Boyle and colleagues \[[@B39]\] phosphorylated proteins were proteolytically digested with trypsin by incubating twice for 2 hrs. at 37°C, on each occasion with 10 μg trypsin (Promega, modified trypsin, sequencing grade) in 200 μl 50 mM NH~4~HCO~3~ and a two-dimensional separation of tryptic phosphopeptides was completed using the HTLE-7000 apparatus and manufacture\'s protocol: electrophoretic separation was performed on thin layer chromatography plates for 25 min. at 0.8 bar and 1 kV, followed by conventional chromatography in 39.25% n-butanol, 30.25% pyridine, 6.1% acetic acid. One-dimensional separation of tryptic phosphopeptides was achieved using 16% SDS-PAGE according to Schägger and von Jagow \[[@B40]\].

List of Abbreviations used
==========================

Sf9, *Spodoptera frugiperda* cell line; MAPK, mitogen-activated protein kinase; MEK, MAP and erk kinase; Hsp, heat-shock protein; PTP, protein tyrosine phosphatase; MBP, myelin basic protein; PKC, protein kinase C; CKII, casein kinase II.

Acknowledgements
================

We are most grateful to Friedrich Propst for purifying tubulin and generating the anti-Mos N13 antiserum.