Personal safety, violence, and home health.

A critical issue facing the health care industry today is the potential impact of community and interpersonal violence on home health care. The purposes of this study were to (1) serve as a source for understanding the personal safety risk issues facing home care staff in a large Midwest region and its surrounding rural areas; (2) provide an understanding of how perceived threats to personal safety may impact patient care and patient outcomes; (3) identify strategies for increasing the personal safety of direct care staff; and (4) identify organizational, educational, and procedural issues that impede or enhance staff safety. A triangulated qualitative design was used including focus groups, in-depth individual interviews, critical event narratives, and a participant self-report form. The study used a purposive sample consisting of 5 men and 56 women who were either administrators or direct care staff from 13 home health agencies. Seven major themes emerged: (1) unsafe conditions that direct care staff must face; (2) organizational and administrative issues that impede or promote the personal safety of staff; (3) ethical issues staff face daily; (4) protective factors associated with maintaining safety; (5) issues of gender, race, age, and experience; (6) education and training; and (7) the potential impact that staff's fear of interpersonal and community violence can have on patient care and patient outcomes.

Tandem arrayed ligation of expressed sequence tags (TALEST): a new method for generating global gene expression profiles.

We have developed a new and simple method for quantitatively analyzing global gene expression profiles from cells or tissues. The process, called TALEST, or tandem arrayed ligation of expressed sequence tags, employs an oligonucleotide adapter containing a type IIs restriction enzyme site to facilitate the generation of short (16 bp) ESTs of fixed position in the mRNA. These ESTs are flanked by GC-clamped punctuation sequences which render them resistant to thermal denaturation, allowing their concatenation into long arrays and subsequent recognition and analysis by high-throughput DNA sequencing. A major advantage of the TALEST technique is the avoidance of PCR in all stages of the process and hence the attendant sequence-specific amplification biases that are inherent in other gene expression profiling methods such as SAGE, Differential Display, AFLP, etc. which rely on PCR.

Isolation of fibroblast growth factor receptor binding sequences using evolved phage display libraries.

A fusion protein construct consisting of the short form of human fibroblast growth factor (FGFR) fused to the heavy chain of mouse IgG1 was used to screen four phage display libraries displaying 8, 13, 38 and 43 amino acids at the amino terminus of the bacteriophage M13 gene III minor coat protein. Phage with specific FGFR binding activity were isolated from the 13, 38 and 43 mer libraries. One of the highest affinity phage clones from the 13mer library was chosen to be further evolved by oligonucleotide saturation mutagenesis. We have isolated evolved sequences that have approximately 8 times the relative binding affinity of the parent sequence. The phage clones have a minimum consensus sequence of CR/SXLLXGAPFXXXXC, where X represents positions tolerant of several amino acids. A synthetic peptide based on this sequence specifically inhibits FGF from binding to its receptor in an in vitro ELISA.

Isolation of erythropoietin receptor agonist peptides using evolved phage libraries.

Cyclic peptides capable of activating the erythropoietin receptor (EPOR) were isolated from phage display libraries by screening with a novel EPOR-IgG fusion protein reagent. A parental clone ERB1 (EPO Receptor Binder 1) was first isolated from a phage display library displaying 38 random amino acids as an N-terminal fusion with the M13 minor capsid protein, pill. An evolved library was then produced from the parental sequence using an oligonucleotide saturation mutagenesis strategy which yielded EPOR binding sequences with 20 times the relative affinity of ERB1. Two synthetic peptides were constructed from these sequences both of which bind the EPO receptor in specific ELISA, and act as full agonists in EPO dependent cell proliferation assays. These peptides are 18 amino acids in length, disulfide-bonded, and have a minimum consensus sequence of CXXGWVGXCXXW, where X represents positions tolerant of several amino acids.

Expression and ligand binding assays of soluble cytokine receptor-immunoglobulin fusion proteins.

We have developed a cloning vector for the expression of type I cytokine receptor, NO, extracellular domain (ECD)-mouse IgG1 Fc fusion proteins. The vector has a versatile polylinker that allows in-frame cloning of the receptor ECD with the mouse IgG1 sequence to encode a receptor ECD-IgG1 fusion construct. The receptor-IgG1 fusion proteins are transiently expressed in useful amounts following transfection of the expression vector into COS7 cells and G418 selection. The mouse IgG1 portion of the fusion protein provides a universal handle for purification on an affinity matrix and detection by anti-mouse IgG antibodies in ELISA or Western blot formats. The expressed receptor ECD-IgG1 fusion proteins bind their cognate ligands. In order to demonstrate that the fusion proteins have similar ligand binding affinities as the native receptors, the affinity constants (Kd) for EPOR, TNFR, IL-4R, and IL-6R-IgG1 fusion proteins were measured by surface plasmon resonance and shown to be in good agreement with published values. The TNFR-IgG1 fusion protein was employed in a demonstration of a novel ELISA format for detecting cytokine receptor binding to cytokine.

Identification of novel human WW domain-containing proteins by cloning of ligand targets.

A recently described protein module consisting of 35-40 semiconserved residues, termed the WW domain, has been identified in a number of diverse proteins including dystrophin and Yes-associated protein (YAP). Two putative ligands of YAP, termed WBP-1 and WBP-2, have been found previously to contain several short peptide regions consisting of PPPPY residues (PY motif) that mediate binding to the WW domain of YAP. Although the function(s) of the WW domain remain to be elucidated, these observations strongly support a role for the WW domain in protein-protein interactions. Here we report the isolation of three novel human cDNAs encoding a total of nine WW domains, using a newly developed approach termed COLT (cloning of ligand targets), in which the rapid cloning of modular protein domains is accomplished by screening cDNA expression libraries with specific peptide ligands. Two of the new genes identified appear to be members of a family of proteins, including Rsp5 and Nedd-4, which have ubiquitin-protein ligase activity. In addition, we demonstrate that peptides corresponding to PY and PY-like motifs present in several known signaling or regulatory proteins, including RasGAP, AP-2, p53BP-2 (p53-binding protein-2), interleukin-6 receptor-alpha, chloride channel CLCN5, and epithelial sodium channel ENaC, can selectively bind to certain of these novel WW domains.

Cloning of ligand targets: systematic isolation of SH3 domain-containing proteins.

Based on the prevalence of modular protein domains, such as Src homology domain 3 and 2 (SH3 and SH2), among important signaling molecules, we have sought to identify new SH3 domain-containing proteins. However, modest sequence similarity among these domains restricts the use of DNA-based methods for this purpose. To circumvent this limitation, we have developed a functional screen that permits the rapid cloning of modular domains based on their ligand-binding activity. Using operationally defined SH3 ligands from combinatorial peptide libraries, we screened a series of mouse and human cDNA expression libraries. We found that 69 of the 74 clones isolated encode at least one SH3 domain. These clones encode 18 different SH3-containing proteins, 10 of which have not been described previously. The isolation of entire repertoires of modular domain-containing proteins will prove invaluable in genome analysis and in bringing new targets into drug discovery programs.

Construction and screening of M13 phage libraries displaying long random peptides.

We have constructed two phage display libraries expressing N-terminal pIII fusions in M13 composed of 37 and 43 random amino acid domains, respectively. The D38 library expresses 37 random amino acids with a central alanine residue, and the DC43 library contains 43 random amino acids with a central cysteine flanked by two glycine residues, giving the displayed peptide the potential to form disulfide loops of various sizes. We demonstrate that the majority of random sequences in both libraries are compatible in pentavalent display with phage viability. The M13 phage display vector itself has been engineered to contain a factor Xa protease cleavage site to provide an alternative to acid elution during affinity selection. An in-frame amber mutation has been inserted between the pIII cloning sites to allow for efficient selection against nonrecombinant phage in the library. These libraries have been panned against mAb 7E11-C5, which recognizes the prostate-specific membrane antigen (PSM). Isolated phage display a consensus sequence that is homologous to a region in the PSM molecule.

Tendamistat as a scaffold for conformationally constrained phage peptide libraries.

The alpha-amylase inhibitor Tendamistat (Hoe-467), a 74 amino acid beta-sheet protein from Streptomyces tendae has been expressed on the surface of the filamentous bacteriophage M13. Phage displaying Tendamistat inhibit the hydrolysis of starch by alpha-amylase, indicating that the displayed protein is functional. The displayed Tendamistat has been used as a molecular scaffold for the presentation of constrained random peptides. Two loops, comprising residues 38 to 40 and 60 to 65 of Tendamistat, were randomized using PCR mutagenesis. Libraries of approximately 10(8) different mutant Tendamistat molecules were tested for binding to monoclonal antibody A8, which recognizes endothelin. After three cycles of biopanning, phage were isolated that specifically bound the monoclonal antibody. Loop swapping and alanine replacement mutagenesis indicated that residues in the 60 to 65 loop are responsible for binding to the monoclonal antibody. This work demonstrates the use of relatively small non-antibody protein scaffolds for the presentation of constrained random peptide sequences to select for novel binding molecules.

Constrained peptide libraries as a tool for finding mimotopes.

The monoclonal antibody (mAb) KAA8 recognizes the peptide angiotensin II (AII). The KAA8 mAb was biopanned using two phage-displayed peptide libraries, one unconstrained, the other constrained by a disulfide bond. After several cycles of biopanning, both libraries showed enrichment for phage that bind KAA8. Phage isolated from the unconstrained library contain a consensus sequence that matches the sequence of AII. A consensus sequence was also identified from the constrained library that does not resemble the AII sequence, and represents a mimotope of AII. We have also demonstrated that monovalent phage display can be used to discriminate between modest and high-affinity binding peptides.

Intermediate filament formation by a yeast protein essential for organelle inheritance.

Intermediate filaments are abundant cytoskeletal components whose specific cellular functions are poorly understood. The Saccharomyces cerevisiae protein MDM1 displays structure and solubility properties that are similar to those of intermediate filament proteins of animal cells. Yeast cells that have a mutant form of MDM1 exhibit temperature-sensitive growth and defective transfer of nuclei and mitochondria to daughter cells during incubation at the nonpermissive temperature of 37 degrees C. The purified, wild-type MDM1 protein readily forms 10-nanometer-wide filaments at either 4 degrees C or 37 degrees C. In contrast, the purified, mutant protein forms filaments at 4 degrees C but fails to form such structures at 37 degrees C. These results suggest that intermediate filament proteins are universal components of eukaryotic cells.

Nuclear and mitochondrial inheritance in yeast depends on novel cytoplasmic structures defined by the MDM1 protein.

The mdml mutation causes temperature-sensitive growth and defective transfer of nuclei and mitochondria into developing buds of yeast cells at the nonpermissive temperature. The MDM1 gene was cloned by complementation, and its sequence revealed an open reading frame encoding a potential protein product of 51.5 kD. This protein displays amino acid sequence similarities to hamster vimentin and mouse epidermal keratin. Gene disruption demonstrated that MDM1 is essential for mitotic growth. Antibodies against the MDM1 protein recognized a 51-kD polypeptide that was localized by indirect immunofluorescence to a novel pattern of spots and punctate arrays distributed throughout the yeast cell cytoplasm. These structures disappeared after shifting mdm1 mutant cells to the nonpermissive temperature, although the cellular level of MDM1 protein was unchanged. Affinity-purified antibodies against MDM1 also specifically recognized intermediate filaments by indirect immunofluorescence of animal cells. These results suggest that novel cytoplasmic structures containing the MDM1 protein mediate organelle inheritance in yeast.

Temperature-sensitive yeast mutants defective in mitochondrial inheritance.

The distribution of mitochondria to daughter cells is an essential feature of mitotic cell growth, yet the molecular mechanisms facilitating this mitochondrial inheritance are unknown. We have isolated mutants of Saccharomyces cerevisiae that are temperature-sensitive for the transfer of mitochondria into a growing bud. Two of these mutants contain single, recessive, nuclear mutations, mdm1 and mdm2, that cause temperature-sensitive growth and aberrant mitochondrial distribution at the nonpermissive temperature. The absence of mitochondria from the buds of mutant cells was confirmed by indirect immunofluorescence microscopy and by transmission electron microscopy. The mdm1 lesion also retards nuclear division and prevents the transfer of nuclei into the buds. Cells containing the mdm2 mutation grown at the nonpermissive temperature sequentially form multiple buds, each receiving a nucleus but no mitochondria. Neither mdm1 or mdm2 affects the transfer of vacuolar material into the buds or causes apparent changes in the tubulin- or actin-based cytoskeletons. The mdm1 and mdm2 mutations are cell-cycle specific, displaying an execution point in late G1 or early S phase.

Absence of nocturnal plasma melatonin surge under long and short artificial photoperiods in the domestic sow.

The circadian plasma melatonin profile of German Landrace sows was determined at 1-h intervals for 31 h after exposure to each of four different artificial photoperiods. Under 12L:12D, melatonin concentrations in four sows during the light phase ranged from 22 +/- 5.9 to 96 +/- 25.1 pg/ml (mean +/- SEM). During the dark phase (total dark) the individual concentrations increased two- to fivefold over the peak individual light phase values in three sows. This nocturnal surge was of 3.8 h duration and peaked at 190, 294, and 546 pg/ml at 0100 h, which was 5.5 h after the onset of dark. The surge was abolished in these animals after exposure to 16L:8D and could not be reinstated by the subsequent exposure to 8L:16D. During the latter two photoperiods the mean concentrations were consistently less than the maximum mean value of 30.8 +/- 10 pg/ml during the dark phase and 55 +/- 21.9 pg/ml during the light phase. All the sows displayed regular estrous cycles during the study, and the day of the estrous cycle on which the samples were obtained under each photoperiod was not significantly correlated with the presence or absence of the nocturnal surge. A separate experiment using different animals confirmed the nocturnal surge under 12L:12D. In two out of four cycling sows the peak concentrations during the dark phase (122 and 110 pg/ml at 0400 and 0500 h) were two- to sixfold higher than those of the light phase. Absence of the nocturnal surge under long and short daylengths may be a factor contributing to the decline in reproductive performance during the summer and winter months.

Change in duration of elevated concentrations of melatonin is the major factor in photoperiod response of the tammar, Macropus eugenii.

In Exp. 1, 10 quiescent non-lactating tammars were exposed to 15L:9D (Days -41 to -1), 24L:0D (Days 0 to 14), 15L:9D (Days 15 to 34) and then to ambient increasing daylength from 13L:11D on Day 35. From Days 0 to 22 they received a s.c. injection of melatonin (400 ng/kg, N -5) on the arachis oil vehicle (N = 5) in the evening (19:30 h) 2.5 h before dark. Exposure to 24L:0D abolished the nocturnal plasma melatonin rise but this was reinstated by subsequent exposure to 15L:9D. Of 5 melatonin-treated tammars, 4 gave birth on Day 45, so had failed to respond to the melatonin injection alone but reactivated when this was combined with the endogenous melatonin rise during exposure to 15L:9D. Of 5 control tammars, 4 remained quiescent until reactivated by the decrease in daylength to 13L:11D, and gave birth significantly later (Day 63.7 +/- 2.2, mean +/- s.e.m., P less than 0.05). In Exp. 2, 6 tammars were exposed to 15L:9D (Days -15 to -1) and then to 12L:12D (Days 0 to 15) by extending the dark phase by 3 h in the morning. This extended the nocturnal melatonin rise by 2-3 h in the morning and all 6 tammars gave birth on Day 31.2 +/- 1.0. A transient pulse of peripheral plasma prolactin (81.5 +/- 31.0 ng/ml) was detected at dawn during 15L:9D in all 6 tammars but was not observed in any of them 5 days after exposure to 12L:12D. Together these results do not support the time of day hypothesis but indicate that increase in duration of the nocturnal melatonin rise mediates the effects of decreased daylength on reactivation of the corpus luteum, and that the first detectable result of this may be the abolition of a transient prolactin pulse at the end of the dark phase.

Seasonal changes in the circadian plasma melatonin profile of the tammar, Macropus eugenii.

The circadian plasma melatonin profile of a marsupial, the tammar, was determined at various stages of the annual reproductive cycle. At 6-14 days after each of the solstices and equinoxes, six females were exposed to a photoperiod equivalent to the natural day length at these times. Serial blood samples were taken 8 days later at 2-4-hourly intervals, and plasma melatonin concentrations were measured by radioimmunoassay. Melatonin concentrations were elevated during the dark phase of each photoperiod, and there were significant changes between the profiles in each season. The amplitude of the nocturnal rise was significantly higher (P less than 0.05) during the breeding season after the summer solstice (peak 259.5 +/- 26.8 pg/ml, mean +/- SEM) and autumnal equinox (287 +/- 53.2 pg/ml) compared to those during the nonbreeding season after the winter solstice (111.5 +/- 10.5 pg/ml) and vernal equinox (154.5 +/- 10.4 pg/ml). The duration of the nocturnal rise was significantly correlated (r=0.996, P less than 0.01) with the length of the dark phase and so was shortest after the summer solstice and longest after the winter solstice. Either of these changes in amplitude or duration might provide the photoperiodic information that regulates the annual reproductive cycle of the tammar.

Effect of pinealectomy on plasma melatonin, prolactin and progesterone concentrations during seasonal reproductive quiescence in the tammar, Macropus eugenii.

Non-lactating female tammars were pinealectomized (N = 5) or sham-operated (N = 6) in October, during the austral period of increasing daylength. After autopsy in February the completeness of pinealectomy was assessed by histological examination of the epithalamic region. Pinealectomy, but not the sham-operation, abolished the nocturnal rise in plasma melatonin but weekly plasma prolactin concentrations were similar in both groups from October to February. Plasma progesterone concentrations remained low (less than 160 pg/ml) in both groups until after the summer solstice in December, when there was a 2-4-fold increase (greater than 300 pg/ml) by late January, indicating reactivation of the quiescent corpus luteum. The mean date of birth or oestrus for the pinealectomized tammars was January 11 +/- 4.8 days (s.e.m.), and for the sham-operated tammars January 24 +/- 5.1 days. These results demonstrate that the pineal is not necessary for the maintenance of seasonal quiescence or to induce reactivation of the corpus luteum at the start of the normal breeding season after the summer solstice.