An SH2-domain-containing kinase negatively regulates the phosphatidylinositol-3 kinase pathway.

SHK1 is a novel dual-specificity kinase that contains an SH2 domain in its C-terminal region. We demonstrate that SHK1 is required for proper chemotaxis and phagocytosis. Mutant shk1 null cells lack polarity, move very slowly, and exhibit an elevated and temporally extended chemoattractant-mediated activation of the kinase Akt/PKB. GFP fusions of the PH domain of Akt/PKB or the PH-domain-containing protein CRAC, which become transiently associated with the plasma membrane after a global stimulation with a chemoattractant, remain associated with the plasma membrane for an extended period of time in shk1 null cells. These results suggest that SHK1 is a negative regulator of the PI3K (phosphatidylinositol-3 kinase) pathway. Furthermore, when a chemoattractant gradient is applied to a wild-type cell, these PH-domain-containing proteins and the F-actin-binding protein coronin localize to its leading edge, but in an shk1 null cell they become randomly associated with the plasma membrane and cortex, irrespective of the direction of the chemoattractant gradient, suggesting that SHK1 is required for the proper spatiotemporal control of F-actin levels in chemotaxing cells. Consistent with such functions, SHK1 is localized at the plasma membrane/cortex, and we show that its SH2 domain is required for this localization and the proper function of SHK1.

Safe use of radioisotopes.

In all cases where radioisotopes are used, depending on the quantity and nature of the isotope, certain precautions must be taken to ensure the safety of the scientist. This appendix outlines a few such considerations relevant to the isotopes most frequently used in biological research.

Phosphopeptide mapping and identification of phosphorylation sites.

Protein phosphorylation is a common modification for many proteins in the cell. Phosphorylation can affect localization of a protein, its stability, and its ability to dimerize or form stable complexes with other molecules. To understand the underlying mechanisms behind the phosphorylation of a given protein, it is often necessary to precisely identify which amino acid residues are phosphorylated. This unit describes the technique of phosphopeptide mapping. In this procedure, a radiolabeled protein is proteolytically digested, and the resulting phosphopeptides are separated in two dimensions on a TLC plate. The phosphopeptides are also analyzed by HPLC and mass spectrometry or peptide microsequencing. Such mapping gives information about the number of phosphorylation sites present in the protein, and can also be used to find out if sites of phosphorylation on a protein change upon treatment of cells with specific agents.

Safe use of radioisotopes.

The use of radioisotopes to label specific molecules in a defined way has greatly advanced the discovery and dissection of biochemical pathways. The development of methods to inexpensively synthesize such tagged biological compounds on an industrial scale has enabled them to be used routinely in laboratory protocols, including many detailed in this manual. Although most of these protocols involve the use of only microcurie (mCi) amounts of radioactivity, some (particularly those describing the metabolic labeling of proteins or nucleic acids within cells) require amounts on the order of millicuries (mCi). In all cases where radioisotopes are used, depending on the quantity and nature of the isotope, certain precautions must be taken to ensure the safety of the investigator. It is essential to use good safety practices and proper protection to handle radioactive substances. This unit discusses storage, handling, and disposal of 35S, 32P, 33P, and 125I.

Phosphopeptide mapping and identification of phosphorylation sites.

This unit describes a procedure for proteolytic digestion of a (32)P-labeled protein, followed by separation of the digestion products in two dimensions on a TLC plate, which gives rise to a phosphopeptide map. Phosphopeptides present on the TLC plate are visualized by autoradiography. These maps give information about the number of phosphate-containing peptides in the digest, and this is related to the number of phosphorylation sites present in the protein. Phosphopeptide maps can also be used to find out whether the sites of phosphorylation on a protein change upon treatment of cells with certain agents. Procedures are also provided for the identification of the sites of phosphorylation from the phosphopeptide maps. A support protocol details purification of phosphopeptides by HPLC and subsequent analysis by mass spectrometry or peptide microsequencing.

Prayer and health outcomes in church lay leaders.

This study examined the relationship of frequency of prayer on health outcomes in a national sample of 1,014 church lay leaders. The survey included questions on the frequency of prayer and the Medical Outcomes Study, Short Form 36 Health Survey, measuring eight categories of functional health. The results indicated a high level of functioning overall. Age was strongly related to most aspects of health in this sample. Frequent prayer was associated with poor physical functioning and poor ability to carry out role activities, but these relationships were not significant when the effect of age and gender was controlled. Frequent prayer was also significantly associated with high mental health scores, regardless of age or gender. This study supports the growing body of data suggesting a positive relationship between frequency of spiritual practices and mental health, even in a homogeneous sample of active church members.

CDK9 autophosphorylation regulates high-affinity binding of the human immunodeficiency virus type 1 tat-P-TEFb complex to TAR RNA.

Human immunodeficiency virus type 1 (HIV-1) Tat interacts with cyclin T1 (CycT1), a regulatory partner of CDK9 in the positive transcription elongation factor (P-TEFb) complex, and binds cooperatively with CycT1 to TAR RNA to recruit P-TEFb and promote transcription elongation. We show here that Tat also stimulates phosphorylation of affinity-purified core RNA polymerase II and glutathione S-transferase-C-terminal-domain substrates by CycT1-CDK9, but not CycH-CDK7, in vitro. Interestingly, incubation of recombinant Tat-P-TEFb complexes with ATP enhanced binding to TAR RNA dramatically, and the C-terminal half of CycT1 masked binding of Tat to TAR RNA in the absence of ATP. ATP incubation lead to autophosphorylation of CDK9 at multiple C-terminal Ser and Thr residues, and full-length CycT1 (amino acids 728) [CycT1(1-728)], but not truncated CycT1(1-303), was also phosphorylated by CDK9. P-TEFb complexes containing a catalytically inactive CDK9 mutant (D167N) bound TAR RNA weakly and independently of ATP, as did a C-terminal truncated CDK9 mutant that was catalytically active but unable to undergo autophosphorylation. Analysis of different Tat proteins revealed that the 101-amino-acid SF2 HIV-1 Tat was unable to bind TAR with CycT1(1-303) in the absence of phosphorylated CDK9, whereas unphosphorylated CDK9 strongly blocked binding of HIV-2 Tat to TAR RNA in a manner that was reversed upon autophosphorylation. Replacement of CDK9 phosphorylation sites with negatively charged residues restored binding of CycT1(1-303)-D167N-Tat, and rendered D167N a more potent inhibitor of transcription in vitro. Taken together, these results demonstrate that CDK9 phosphorylation is required for high-affinity binding of Tat-P-TEFb to TAR RNA and that the state of P-TEFb phosphorylation may regulate Tat transactivation in vivo.

Prayer and health outcomes in church members.

CONTEXT: This study adds to the existing research on religion and health by focusing on the specific practice of prayer and its relationship to health outcomes. OBJECTIVES: The purpose of this survey is to examine the relationship of frequency of prayer to 8 categories of physical and mental health. DESIGN: The Presbyterian Church, USA, performed data collection as part of an ongoing research program. Members of the Presbyterian Church were randomly selected from the national population and surveyed by mail on their frequency of prayer and their health status, as measured by the Medical Outcomes Study Short-form 36 Health Survey. RESULTS: Self-reports of health indicated a high level of functioning overall for all 8 categories of physical and mental health. People who prayed more often scored lower in their physical functioning and their ability to carry out role activities, and higher in their reports of physical pain. However, people who prayed more often also had significantly higher mental health scores than did those who prayed less frequently, despite their physical health problems. CONCLUSION: This study supports the relationship of a high frequency of prayer with a more positive mental health. Various explanations of the results are explored.

Faith, prayer, and health outcomes in elderly Native Americans.

This survey questioned 71 Native Americans over age 65 living in the general community on their frequency of prayer, importance of faith, and their health status. The researchers hypothesized that people with higher scores in faith and prayer would experience a more positive health status. Self-reports of health indicated a high level of functioning overall. Older people and those living alone had poorer physical and emotional health outcomes than younger elders and those living with one or more persons, although neither age nor living situation was related to mental health. People who prayed more often and those who indicated a high importance of their faith scored higher in the mental health subscale, confirming the hypothesis for this dimension of health.

Characterization and minimization of extremity doses during 32P metabolic cell labeling.

A study was performed to characterize hand extremity doses received by a laboratory researcher performing a metabolic cell labeling procedure using 10-cm-diameter cell culture dishes containing GBq amounts of 32P-orthophosphate. Specifically, the optimal location for placement of thermoluminescent dosimeters on the extremities was determined as was the phase (time frame) during the cell labeling procedure when the highest dose was received. The cell labeling procedure was divided into four phases. Thermoluminescent dosimeters were placed on three fingers of each hand at the start of each phase and after the completion of that phase, collected, and sent to the processor. It was determined that in the case of this right-handed worker, the left-hand index finger should be used for determining the maximum extremity dose. The time frame during which the highest extremity dose was received was the cell lysis phase. Acrylic shielding was fabricated, which significantly reduced worker extremity dose.

Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1.

Although genetic analysis has demonstrated that members of the winged helix, or forkhead, family of transcription factors play pivotal roles in the regulation of cellular differentiation and proliferation, both during development and in the adult, little is known of the mechanisms underlying their regulation. Here we show that the activation of phosphatidylinositol 3 (PI3) kinase by extracellular growth factors induces phosphorylation, nuclear export, and transcriptional inactivation of FKHR1, a member of the FKHR subclass of the forkhead family of transcription factors. Protein kinase B (PKB)/Akt, a key mediator of PI3 kinase signal transduction, phosphorylated recombinant FKHR1 in vitro at threonine-24 and serine-253. Mutants FKHR1(T24A), FKHR1(S253A), and FKHR1(T24A/S253A) were resistant to both PKB/Akt-mediated phosphorylation and PI3 kinase-stimulated nuclear export. These results indicate that phosphorylation by PKB/Akt negatively regulates FKHR1 by promoting export from the nucleus.

Accidental occupational exposure of intravenous nurses to human immunodeficiency virus. Anticipating the consequences.

This descriptive study randomly surveyed all 302 Massachusetts members of the Intravenous Nurses Society in 1991 regarding their perceptions of nine possible consequences of human immunodeficiency virus (HIV) infection caused by accidental occupational exposure. Areas of highest concern were financial: adequacy of worker's compensation, ability of the employer to cover all healthcare costs, and job security. Nurses also were concerned about confidentiality of their HIV status and personal history jeopardizing their benefits. The i.v. nurses felt most secure in areas of their personal lives: housing and support of family and friends. Although some concerns correlated significantly with fear of contagion, others were unrelated, indicating a need for policy and attitude changes to promote comfort in working with HIV.

Anticipated outcomes of accidental occupational exposure to HIV.

This descriptive study surveyed a random sample of 60 registered nurses (R.N.s) regarding their perceptions of nine possible consequences of an HIV infection resulting from occupational exposure. Areas of highest concern were financial: adequacy of workers' compensation and ability of the employer to cover all health care costs. Nurses were also concerned about confidentiality and ability to trace the event of the occupational exposure. These nurses felt the least threatened in the areas of maintaining their housing and the support of family and friends. Overall, the responses indicate a great deal of uncertainty surrounding the anticipated outcomes of an accidental occupationally derived infection. The results suggest clearer communication is needed between nurses and their employers regarding their benefits and existing support services.

Determination of phosphorylation sites in peptides and proteins employing a volatile Edman reagent.

A manual Edman degradation protocol has been developed that allows the identification of phosphorylation sites in 32P-labeled peptides at the subpicomole level. By using both a volatile reagent, trifluoroethyl isothiocyanate, and volatile buffers, extraction steps are rendered unnecessary and cycle times can be reduced to 45 min. The protocol was employed to identify the site of phosphorylation in phosphoserine- and phosphotyrosine-containing peptides.

Complete structure of the murine p36 (annexin II) gene. Identification of mRNAs for both the murine and the human gene with alternatively spliced 5' noncoding exons.

p36 (also termed annexin II) is a 39 kDa Ca2+/phospholipid-binding, membrane-associated protein that is a protein-tyrosine kinase substrate. We report here studies of the noncoding exons of p36, which combined with our earlier studies of the coding exons, allow us to conclude that the murine p36 gene is 34 kb in length with 14 exons. Comparison of the genes coding for mouse and human p36 (annexin II) and mouse, rat and human p35 (annexin I) and pigeon cp35 (an annexin I-related protein) shows strong genomic structural conservation supporting the hypothesis that these genes had a common ancestor. Both human and murine p36 mRNAs were found to be alternatively spliced in their 5' noncoding region. In both cases exon 2 is a cassette exon, which is present in a small fraction of p36 mRNAs. In type 1 mouse p36 mRNA the first noncoding 44 base exon 1 is joined to exon 3, the first of the 12 coding exons. In type 2 mRNA a 70 base noncoding exon (exon 2) is inserted between exon 1 and exon 3. Type 1 mRNA was present in all cell types studied as revealed by Northern analysis and primer extension, whereas type 2 mRNA could only be detected by RACE or PCR, indicating that it is of very low abundance. The major transcription start site of the mouse p36 gene was mapped by primer extension to be 61 bp upstream of the AUG initiation codon, which corresponds to type 1 mRNA, The murine p36 gene enhancer/promoter region contains a putative TATA box and several other potential regulatory sequences. The two alternatively-spliced human p36 mRNAs differ by the presence or absence of a noncoding 81 base exon (exon 2) inserted after exon 1, with exon 2-containing mRNAs representing approximately 10% of total p36 mRNA. The 300 bp spanning the promoter and exons 1-3 of the human and murine p36 genes show strong sequence homology immediately before and after the major transcription start site except in the region corresponding to exon 2, where homology is more limited.

Perceived barriers to getting published: results of the JANAC survey.

In order to identify strategies to promote the publication of ANAC members' knowledge and expertise, the authors conducted a survey of JANAC readers. Twenty-four respondents, almost all clinicians, identified 25 areas of interest they want to develop for publication. Barriers to getting published fell into four categories: lack of time, writing skills, self-confidence, and motivation or ideas. Suggestions for facilitating scholarship focused mainly on collaboration with writing experts and learning through educational workshops. The results of this study can serve as a guide in the development of services to readers interested in their own development as scholars.

Evaluating interventions for fear of contagion.

Fear of contagion, an anxious response to the perceived threat of catching HIV, has been shown to lessen with contact with persons with AIDS. Two different continuing educational offerings presented a panel of persons living with HIV as an intervention to decrease fear of contagion. Presession and postsession questionnaires demonstrated a significant decrease in levels of fear in the one-day program (n = 39), but not in the six-week course (n = 14). Willingness to care for those with HIV was unchanged in either offering. The study provides evidence for the effectiveness of exposure to persons living with HIV to decrease fear of contagion, even in a sample that had low fear scores initially.

Contributing factors to fear of HIV contagion in registered nurses.

This study tested the relationships of homophobia, fear of the unknown, fear of death, and fear of punishment as predictors of fear of HIV contagion. Knowledge of transmission and emotional involvement with a person at risk for HIV were hypothesized as decreasing fear among 114 randomly-selected RNs. Results supported significant relationships for all predictors except fear of death. Homophobia, lack of knowledge, lack of emotional involvement, and fear of the unknown predicted 57 percent of the variance in fear of contagion.

Cultivating scholarship in the clinical setting.

Many clinical nursing departments have added scholarly activity as a goal, expecting staff development personnel to implement the needed programs for research utilization, professional presentations, and clinical investigations. In this article, the author provides specific strategies for starting a research program and nurturing its development, addressing the emotional needs as well as the knowledge and resource needs of the learner. These strategies provide a blueprint for building the research utilization, initiation, and publication productivity in a clinical setting.