Isolation and characterization of STI1, a stress-inducible gene from Saccharomyces cerevisiae.

We have isolated a gene from the yeast Saccharomyces cerevisiae that encodes a 2.0-kilobase heat-inducible mRNA. This gene, which we have designated STI1, for stress inducible, was also induced by the amino acid analog canavanine and showed a slight increase in expression as cells moved into stationary phase. The STI1 gene encodes a 66-kilodalton protein, as determined from the sequence of the longest open reading frame. The putative STI1 protein, as identified by two-dimensional gel electrophoresis, migrated in the region of 73 to 75 kilodaltons as a series of four isoforms with different isoelectric points. STI1 is not homologous to the other conserved HSP70 family members in yeasts, despite similarities in size and regulation. Cells carrying a disruption mutation of the STI1 gene grew normally at 30 degrees C but showed impaired growth at higher and lower temperatures. Overexpression of the STI1 gene resulted in substantial trans-activation of SSA4 promoter-reporter gene fusions, indicating that STI1 may play a role in mediating the heat shock response of some HSP70 genes.

RAD4 gene of Saccharomyces cerevisiae: molecular cloning and partial characterization of a gene that is inactivated in Escherichia coli.

In contrast to other Saccharomyces cerevisiae RAD genes involved in nucleotide excision repair of DNA, the RAD4 gene could not be isolated by screening a yeast genomic library for recombinant plasmids which complement the UV sensitivity of rad4 mutants (Pure et al., J. Mol. Biol. 183:31-42, 1985). We therefore attempted to walk to RAD4 from the neighboring SPT2 gene and obtained an integrating derivative of a plasmid isolated by Roeder et al. (Mol. Cell. Biol. 5:1543-1553, 1985) which contains a 4-kilobase fragment of yeast DNA including a mutant allele of SPT2. When integrated into several different rad4 mutant strains, this plasmid (pR169) complements UV sensitivity at a frequency of approximately 10%. However, a centromeric plasmid containing rescued sequences which include flanking yeast DNA no longer complements the phenotype of rad4 mutants. Complementing activity was restored by in vivo repair of a defined gap in the centromeric plasmid. The repaired plasmid fully complements the UV sensitivity of all rad4 mutants tested when isolated directly from yeast cells, but when this plasmid is propagated in Escherichia coli complementing activity is lost. We have mapped the physical location of the RAD4 gene by insertional mutagenesis and by transcript mapping. The gene is approximately 2.3 kilobases in size and is located immediately upstream of the SPT2 gene. Both genes are transcribed in the same direction. RAD4 is not an essential gene, and no increased transcription of this gene is observed in cells exposed to the DNA-damaging agent 4-nitroquinoline-1-oxide. The site of inactivation of RAD4 in a particular plasmid propagated in E. coli was localized to a 100-base-pair region by gene disruption and gap repair experiments. In addition, we have identified the approximate locations of the chromosomal rad4-2, rad4-3, and rad4-4 mutations.

SSC1, an essential member of the yeast HSP70 multigene family, encodes a mitochondrial protein.

SSC1 is an essential member of the yeast HSP70 multigene family (E. Craig, J. Kramer, and J. Kosic-Smithers, Proc. Natl. Acad. Sci. USA 84:4156-4160, 1987). Analysis of the SSC1 DNA sequence revealed that it could encode a 70,627-dalton protein that is more similar to DnaK, an Escherichia coli hsp70 protein, than other yeast hsp70s whose sequences have been determined. Ssc1p was found to have an amino-terminal extension of 28 amino acids, in comparison with either Ssa1p, another hsp70 yeast protein, or Dnak. This putative leader is rich in basic and hydroxyl amino acids, characteristic of many mitochondrial leader sequences. Ssc1p that was synthesized in vitro could be imported into mitochondria and was cleaved in the process. The imported protein comigrated with an abundant mitochondrial protein that reacted with hsp70-specific antibodies. We conclude that Ssc1p is a mitochondrial protein and that hsp70 proteins perform functions in many compartments of the cell.

Does training general practitioners to elicit patients' illness representations and action plans influence their communication as a whole?

OBJECTIVE: To examine whether the discussion of illness representations and action plans during medical encounters affects the way patients and general practitioners (GPs) communicate. METHODS: In a quasi-experimental design, 10 GPs first performed care-as-usual conversations with patients. After a 6 h training they performed consultations either emphasizing patients' illness representations or action plans. Data were collected from 70 videotaped consultations with hypertensive patients, which were analyzed using the Roter Interaction Analysis System. RESULTS: Compared with care-as-usual consultations, communication in the action plan condition resulted in an increased discussion of lifestyle issues whereas communication in the illness representation condition resulted in more discussion of patient concerns. In both experimental conditions the proportion of affective GP utterances was higher while patients contributed more to the conversation. When GPs changed their communication style, patients did accordingly. CONCLUSION: The explicit address of illness representations or action plans during consultations results in more attention to patient concerns and lifestyle issues and an overall improvement in patient-GP communication in terms of affective atmosphere and patient involvement. PRACTICE IMPLICATIONS: These findings show that after a brief training GPs are able to change their communication style in a way that allows for a more thorough consideration of patient self-management.

The RAD2 gene of Saccharomyces cerevisiae: nucleotide sequence and transcript mapping.

We have determined the nucleotide (nt) sequence of a segment of the yeast chromosome carrying the RAD2 gene. The coding region consists of 2925 bp and could encode a protein of 975 amino acids with a calculated Mr of 111 100. A major transcriptional start point was mapped to a position of approx. 22 bp upstream from the first ATG codon. A number of minor transcriptional start points were also identified in this region, all of them 5' to the putative translational start codon. We noted a number of consensus nt sequences in the 5' and 3' non-coding regions of the RAD2, RAD1 and RAD3 genes of Saccharomyces cerevisiae. In addition, three regions of amino acid sequence homology in the putative RAD1 and RAD2 polypeptides were observed.

Expression of a tumor-reactive antibody-interleukin 2 fusion protein after in vivo particle-mediated gene delivery.

We have used a particle-mediated gene transfer method to analyze the posttransfection expression pattern of an antibody-cytokine fusion protein (FP) in vivo. The FP, denoted CC49-IL2, consists of a single-chain antibody containing the antigen recognition domain from the murine monoclonal antibody CC49 (recognizing the tumor-associated antigen TAG-72), a human IgG1 constant heavy chain, and human interleukin-2 (IL-2). This FP can bind to TAG-72-expressing tumor cells and exhibits IL-2 activity. To induce systemic levels of this FP in vivo, we have transferred the FP gene into murine epidermal cells by direct delivery of DNA-coated gold particles using a transcutaneous "gene gun." After the pericutaneous delivery of the FP gene via gold particles, production of the exogenous FP was detected at the epidermal target site. The FP produced in vivo at the site of gene delivery has cytokine activity and antigen recognition capabilities similar to those present in CC49-IL2 FP purified from hybridoma culture supernatants in vitro. FP was also detectable in the serum from test animals treated with particle-mediated gene transfer. Time course experiments indicated that serum levels of FP reached a peak level within 8 hours after DNA delivery, whereas the epidermal target tissue levels continued to increase for 24 hours before plateauing. Our results indicate that exogenous protein levels consistent with immunotherapeutic effects of the FP can be readily achieved at the skin tissue site of gene delivery, with the potential for achieving therapeutic levels systemically.

Models and theories in studies on educating and counseling children about physical health: a systematic review.

The aim of this review is to gain a comprehensive view on the theories and models referred to in studies on educating and counseling children about physical health. A computer search was conducted using PubMed Medline, and Silverplatter Webspirs Psycinfo. Original studies, reviews, and theoretical papers published between 1992-2003 were included. The review presents the results of the 35 studies in which the majority of the subjects were between 0 and 12 years of age. A classification system is proposed that helped grouping the models, and the interrelationship between this classification and the characteristics of the reviewed studies is explored. The classification could function as an introductory guide and help to select appropriate theories and models when defining future research agenda's. The results of this review may attribute to the refinement of the theoretical underpinning of child education and counseling in physical health.

Manipulation of patient-provider interaction: discussing illness representations or action plans concerning adherence.

According to Leventhal's Self-Regulatory Model of Illness, patients have ideas and action plans related to the management of their disease. The aim of this study is to examine whether ideas and action plans relating to hypertension change as a result of general practitioner's (GP's) discussing them during consultation, and whether these changed ideas and actions plans affect adherence. The study employed an experimental design, highlighting three conditions: (0) care-as-usual consultation; (1) discussing patient's ideas about their disorder; and (2) discussing patient's action plans. Ten GP-trainees performed care-as-usual consultations, were subsequently assigned to a training in either Condition 1 or 2, and performed the trained conversations. Hundred and eight patients with hypertension were consecutively assigned to the conditions, and completed questionnaires a week before, immediately after the consultation, and 1 month later. The training resulted in two new, feasible and different types of conversations that managed to affect some of the patient's ideas and action plans. It is concluded that the study provided GPs with a tool to discuss illness representations and actions plan of patients with hypertension. Implications for the management of hypertension adherence in primary care are discussed.

No effect of ambient odor on the affective appraisal of a desktop virtual environment with signs of disorder.

BACKGROUND: Desktop virtual environments (VEs) are increasingly deployed to study the effects of environmental qualities and interventions on human behavior and safety related concerns in built environments. For these applications it is essential that users appraise the affective qualities of the VE similar to those of its real world counterpart. Previous studies have shown that factors like simulated lighting, sound and dynamic elements all contribute to the affective appraisal of a desktop VE. Since ambient odor is known to affect the affective appraisal of real environments, and has been shown to increase the sense of presence in immersive VEs, it may also be an effective tool to tune the affective appraisal of desktop VEs. This study investigated if exposure to ambient odor can modulate the affective appraisal of a desktop VE with signs of public disorder. METHOD: Participants explored a desktop VE representing a suburban neighborhood with signs of public disorder (neglect, vandalism and crime), while being exposed to either room air or subliminal levels of unpleasant (tar) or pleasant (cut grass) ambient odor. Whenever they encountered signs of disorder they reported their safety related concerns and associated affective feelings. RESULTS: Signs of crime in the desktop VE were associated with negative affective feelings and concerns for personal safety and personal property. However, there was no significant difference between reported safety related concerns and affective connotations in the control (no-odor) and in each of the two ambient odor conditions. CONCLUSION: Ambient odor did not affect safety related concerns and affective connotations associated with signs of disorder in the desktop VE. Thus, semantic congruency between ambient odor and a desktop VE may not be sufficient to influence its affective appraisal, and a more realistic simulation in which simulated objects appear to emit scents may be required to achieve this goal.

Quality of life in adolescents born small for gestational age: does growth hormone make a difference?

BACKGROUND/AIMS: To evaluate quality of life (QoL) in adolescents born SGA without spontaneous catch-up growth, treated with and without long-term growth hormone (GH) therapy. Additionally, to assess whether GH treatment has a positive effect on QoL, besides improving adult height and height SDS during childhood. METHODS: Two groups of adolescents born SGA without spontaneous catch-up growth participated in the QoL evaluation; a GH-treated group (n = 44, mean GH duration: 8.8 (1.7) years) and an untreated group (n = 28), both mean age 15.8 (2.1) years. QoL was measured by self-reports of the TACQOL-S, a disorder-specific questionnaire, and the CHQ, a generic questionnaire. RESULTS: The GH group scored significantly better health status and health-related QoL on several scales of the TACQOL-S. On all TACQOL-S scales the GH group scored better QoL than the untreated group, with effect sizes of moderate to large, not all differences reaching statistical significance. The generic CHQ did not reveal significant differences in QoL between the GH group and the untreated group. CONCLUSIONS: Firstly, adolescents born SGA, with a GH-induced improved height, had in many aspects a better QoL than untreated adolescents born SGA, according to the disorder-specific questionnaire. Secondly, we advise to use, in addition to a generic questionnaire, a disorder-specific questionnaire for measuring QoL in children treated for short stature, as the generic CHQ did not reveal such differences.

Promoter analysis of an interferon-inducible gene associated with macrophage activation.

We have investigated the regulation of an activation-associated guanylate-binding protein gene (mGBP-1/mag-1) in murine macrophage cell lines in response to the cytokine IFN-gamma. One of the cell lines utilized (RAW 264.7) acquires the ability to kill tumor cells after IFN-gamma and LPS treatment, whereas the other (WEHI-3) does not. We previously have demonstrated that mGBP-1 is induced by IFN-gamma in RAW 264.7 but not WEHI-3 cells. Here we present information concerning the cloning, sequencing, and initial characterization of the upstream region of the mGBP-1 gene as a first step towards understanding the differential control of this gene in RAW 264.7 versus WEHI-3 cells. Genomic fragments encompassing a portion of the mGBP-1 5' flanking region were inserted into vectors containing a luciferase reporter gene. 928 bp of upstream sequence were found to be sufficient for IFN-gamma-mediated induction of luciferase activity in the RAW 264.7 cell line. Furthermore, sequences within 100 bp of the major transcription initiation site conferred strong IFN-gamma responsiveness to the reporter gene. A perfect match to the interferon-stimulated response element (ISRE) was present within this region, and was shown to be essential for interferon-induced expression. An oligonucleotide corresponding to the mGBP-1 ISRE bestowed interferon-inducible expression on a heterologous minimal promoter. Site-specific mutation of the ISRE within the 106-bp upstream region eliminated interferon inducibility of this construct. Taken together, the results indicate the ISRE is necessary and sufficient for IFN-gamma induction of the mGBP-1 gene. Transient transfection assays carried out with the WEHI-3 cell line indicated that all promoter constructs were transcriptionally inactive in these cells, including the ISRE-minimal promoter construct. The inability of the WEHI-3 cell line to utilize an ISRE after IFN-gamma induction may underlie the functional differences exhibited by the two cell lines after IFN-gamma stimulation.

Functional analysis of a conserved amino-terminal region of HSP70 by site-directed mutagenesis.

Hsp70 proteins have been highly conserved throughout evolution. As a first step in a structure-function analysis of hsp70, we constructed and analysed the consequences of mutations in a portion of the SSA1 gene, a member of the Saccharomyces cerevisiae HSP70 multigene family, that encodes a nearly invariant region near the amino terminus. Analysis of strains expressing SSA1 proteins with alterations at positions 8, 11 and 15 showed that these conserved residues within this region are critical for normal functioning of the protein. SSA1 protein containing either of two changes at position 15 was able to slightly complement the inviability of an ssa1ssa2ssa4 strain, but was inactive in other complementation assays. The other mutant proteins tested were unable to complement any tested phenotype. Effective interallelic complementation of several phenotypes was observed when a mutant protein substituted at position 8 was expressed in the same cell with either of two proteins carrying substitutions at position 15, suggesting that hsp70 acts as a multimer. Evidence from previous studies suggests that hsp70 proteins engage in ATP-driven cycles of binding and release from peptides. The ability of the mutant proteins to bind ATP and a peptide was tested. The Ssa1p carrying a substitution at position 8, which inhibits growth of cells carrying wild-type SSA proteins, showed a defect in release from a peptide relative to wild type. Two mutations, one each at position 8 and 15, resulted in accumulation of phosphorylated isoforms which may be a normal, transient hsp70 intermediate.

Overexpression of the RAD2 gene of S. cerevisiae: identification and preliminary characterization of Rad2 protein.

The cloned RAD2 gene of S. cerevisiae was tailored into regulatable expression vectors for overexpression of Rad2 protein in E. coli and in yeast. In E. coli both Rad2/beta-galactosidase fusion protein and native Rad2 protein are insoluble, but are extractable with 1% Sarkosyl. In yeast some of the overexpressed native Rad2 protein is also insoluble; however, soluble protein is readily detected by immunoblotting with Rad2-specific antibodies. All forms of the protein detected in transformed or untransformed yeast cells and the insoluble species in E. coli migrate in denaturing polyacrylamide gels with an apparent molecular weight considerably larger than the size predicted from the sequence of the RAD2 coding region. This property is not the result of post-translational glycosylation detectable by binding of concanavalin A, or of phosphorylation of the protein. Overexpression of the RAD2 gene is toxic to yeast. Transformed yeast cells grow much more slowly than untransformed controls and when yeast transformants are serially propagated cultures show considerably colony heterogeneity and concomitant selection for rapidly growing variants which express less Rad2 protein. Antisera raised against Rad2/beta-galactosidase fusion protein expressed in E. coli do not cross-react with Rad1, Rad3 or Rad10 protein in crude extracts of yeast, nor with purified E. coli UvrA, UvrB or UvrC proteins.

Incorporation of 32P-phosphate into membrane phospholipids during infection of cultured human fibroblasts by herpes simplex virus type 1.

Infection of cultured human skin fibroblasts by herpes simplex virus leads to increased incorporation of labeled inorganic phosphate into host membrane sphingomyelin. Incorporation into the other major membrane phospholipids, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine, was unaffected. Since sphingomyelin has been shown to serve as precursor to ceramides (via sphingomyelinase) in monkey kidney cells and since enhanced synthesis of ceramide-based glycolipids has been shown to occur after herpes simplex virus infection, we suggest that the observed increase in labeling of sphingomyelin may reflect mobilization for glycolipid synthesis. The distribution of phosphate label among the phospholipids of the viral envelope was identical to that among the phospholipids of the cellular cytoplasmic membrane fraction and differed from that of the nuclear fraction.

Identification and characterization of a new gene family induced during macrophage activation.

In this report, we describe the primary structure and regulation of two novel IFN-gamma-inducible genes expressed during the process of macrophage activation. We used the RAW 264.7 cell line to prepare a cDNA library, from which inducible genes were selected by differential hybridization. Two cDNA clones, mag-1 and mag-2 (for macrophage-activation gene-1 and -2), were induced by IFN-gamma treatment in both RAW 264.7 cells and thioglycolate-elicited peritoneal macrophages, but not in the noncytolytic cell line, WEHI-3. A comparison of the nucleotide and deduced amino acid sequences of clones mag-1 and mag-2 with sequences in available data bases revealed no homologs. However, comparison of mag-1 and mag-2 sequences with each other revealed that these genes are homologous, with conserved residues concentrated at the amino terminus. Kinetic analyses revealed similar temporal patterns of induction of mRNA expression for these genes after IFN-gamma treatment. In addition, the genes showed distinct response patterns to the macrophage-activating stimuli IFN-gamma and LPS used either alone or in combination. Analysis of a panel of cell types of various lineages demonstrated that expression of these genes was associated with cellular activation in multiple cell types. As a result of the sequence similarities between these genes, we propose that they define a new family of IFN-gamma-regulated genes in macrophages.

Differential utilization of IFN-gamma-responsive elements in two maturationally distinct macrophage cell lines.

We have characterized the transcriptional response to IFN-gamma in two maturationally distinct macrophage populations: the mature RAW 264.7 cell line, phenotypically identical to thioglycollate-elicited peritoneal macrophages, and the less mature WEHI-3 cell line. We first investigated the use of two IFN-gamma-responsive regulatory elements, the interferon-stimulated response element (ISRE) and the gamma-activated sequence (GAS), in these cells. Transient transfection assays revealed that synthetic promoter constructs containing either the ISRE or GAS regulatory motif fused to a luciferase reporter gene were transcriptionally inactive in the WEHI-3 cell line. We then analyzed the expression in the two cell lines of a panel of known IFN-gamma-responsive genes that are transcriptionally controlled by different regulatory elements. RT-PCR analysis revealed that both cell lines responded to IFN-gamma treatment by up-regulating genes that are transcriptionally controlled by kappa B or W box DNA binding motifs. However, genes regulated by ISRE or GAS elements were induced by IFN-gamma only in the RAW 264.7 cell line. Kinetic analysis of the transcriptional activity of synthetic promoter constructs in the RAW 264.7 cell line showed rapid IFN-gamma induction through both the ISRE and GAS motifs, indicating that both elements are utilized early after IFN-gamma stimulation in mature macrophages. These results suggest that cis-acting DNA response element utilization, and the subsequent profiles of IFN-gamma-induced gene expression, differ in macrophages at different stages of maturation.