Genetic variation in 12S and 16S mitochondrial rDNA genes of four geographically isolated populations of Gulf Coast ticks (Acari: Ixodidae).

Single-strand conformation polymorphism (SSCP) analysis was examined in a 303-bp region of the 16S and 12S mitochondrial rDNA genes to study haplotype frequencies among populations of Gulf Coast ticks collected from Refugio Co., TX, Payne Co., OK, and two sites in Osage Co., KS. Seven haplotypes were identified from the 16S rDNA gene fragment, whereas only two haplotypes were detected from the 12S fragment. Only the results from the 16S rDNA fragment are discussed. Haplotype diversity was greatest in Kansas (site 1), where three of the four haplotypes detected were unique to this site. All Gulf Coast tick populations shared the fourth haplotype. Two haplotypes were determined for Texas and Oklahoma populations, one of which appeared only in Texas, whereas the other was shared. Nei's haplotype diversity (h) indicated that the Texas population was relatively homogeneous (15%), whereas the remaining populations were heterogeneous (42-59%), although the Bonferroni confidence interval found no significant differences (P < 0.05). Nucleotide sequencing of the seven haplotypes and subsequent phylogenetic analysis using neighbor joining showed a monophyletic relationship among these haplotypes. One haplotype, shared by both Oklahoma and Kansas (site 2), was basal to the remaining haplotypes and formed a distinct clade. Two haplotypes, both from Kansas (site 1), formed a unique clade, whereas the remaining four haplotypes were unresolved polytomies.

Chimeric Mos1 and piggyBac transposases result in site-directed integration.

Genetic transformation systems based on Mos1 and piggyBac transposable elements are used to achieve stable chromosomal integration. However, integration sites are randomly distributed in the genome and transgene expression can be influenced by position effects. We developed a novel technology that utilizes chimeric transposases to direct integration into specific sites on a target DNA molecule. The Gal4 DNA binding domain was fused to the NH(2) terminus of the Mos1 and piggyBac transposases and a target plasmid was created that contained upstream activating sequences (UAS), to which the Gal4 DBD binds with high affinity. The transpositional activity of the Gal4-Mos1 transposase was 12.7-fold higher compared to controls where the Gal4-UAS interaction was absent and 96% of the recovered transposition products were identical, with integration occurring at the same TA site. In a parallel experiment, a Gal4-piggyBac transposase resulted in an 11.6-fold increase in transpositional activity compared to controls, with 67% of the integrations occurring at a single TTAA site. This technology has the potential to minimize nonspecific integration events that may result in insertional mutagenesis and reduced fitness. Site-directed integration will be advantageous to the manipulation of genomes, study of gene function, and for the development of gene therapy techniques.

Entomopathogenic Fungi: New Insights into Host-Pathogen Interactions.

Although many insects successfully live in dangerous environments exposed to diverse communities of microbes, they are often exploited and killed by specialist pathogens. Studies of host-pathogen interactions (HPI) provide valuable insights into the dynamics of the highly aggressive coevolutionary arms race between entomopathogenic fungi (EPF) and their arthropod hosts. The host defenses are designed to exclude the pathogen or mitigate the damage inflicted while the pathogen responds with immune evasion and utilization of host resources. EPF neutralize their immediate surroundings on the insect integument and benefit from the physiochemical properties of the cuticle and its compounds that exclude competing microbes. EPF also exhibit adaptations aimed at minimizing trauma that can be deleterious to both host and pathogen (eg, melanization of hemolymph), form narrow penetration pegs that alleviate host dehydration and produce blastospores that lack immunogenic sugars/enzymes but facilitate rapid assimilation of hemolymph nutrients. In response, insects deploy an extensive armory of hemocytes and macromolecules, such as lectins and phenoloxidase, that repel, immobilize, and kill EPF. New evidence suggests that immune bioactives work synergistically (eg, lysozyme with antimicrobial peptides) to combat infections. Some proteins, including transferrin and apolipophorin III, also demonstrate multifunctional properties, participating in metabolism, homeostasis, and pathogen recognition. This review discusses the molecular intricacies of these HPI, highlighting the interplay between immunity, stress management, and metabolism. Increased knowledge in this area could enhance the efficacy of EPF, ensuring their future in integrated pest management programs.

Promoter-directed expression of recombinant fire-fly luciferase in the salivary glands of Hermes-transformed Aedes aegypti.

Molecular genetic analyses of biological properties characteristic of insect vectors of disease, such as hematophagy and competence for pathogens, require the ability to isolate and characterize genes involved in these processes. We have been working to develop molecular approaches for studying the promoter function of genes that are expressed specifically in the adult salivary glands of the yellow fever mosquito, Aedes aegypti. Genomic DNA fragments containing cis-acting promoter elements from the Maltase-like I (MalI) and Apyrase (Apy) genes were cloned so as to direct the expression of the reporter gene, luciferase (luc). The function of the promoters was assayed transiently in cultured insect cells and by germ-line transformation of Ae. aegypti. MalI and Apy DNA fragments consisting of at least 650 nucleotides (nt) of DNA immediately adjacent to the 5'-end of the initiation codon of the mosquito genes directed constitutive expression of the luc reporter gene in cultured cells. When introduced into Ae. aegypti chromosomes, approximately 1.5 kilobases (kb) of each promoter were able to direct the predicted developmental-, sex- and tissue-specific expression of the reporter gene in patterns identical to those determined for the respective endogenous genes.

The 5' regulatory region from the Drosophila pseudoobscura hsp82 gene results in a high level of reporter gene expression in Lucilia cuprina embryos.

We have previously examined the efficiency of two Drosophila melanogaster promoters to enable reporter gene expression in embryos of the Australian sheep blowfly, Lucilia cuprina. Both the hsp70 heat-shock promoter and the actin5C promoter resulted in low levels of expression of a reporter gene in these embryos. In this study, the D. pseudoobscura hsp82 promoter (phsp82) was tested for its ability to direct the expression of the Escherichia coli chloramphenicol acetyltransferase-encoding gene (cat). We report that the level of CAT activity in L. cuprina embryos was comparable to that obtained with the same construct in D. melanogaster, indicating that phsp82 functions efficiently in this non-drosophilid insect. The results suggest that phsp82 may be utilised in other non-drosophilid insects in which poor expression levels are obtained from constructs containing the hsp70 or actin5C promoters.

An eye color gene for the detection of transgenic non-drosophilid insects.

A genetic marker for identifying transgenic Musca domestica by changes in eye color is described. The Drosophila melanogaster tryptophan oxygenase gene, vermilion (v), was tested for its ability to genetically complement the mutant tryptophan oxygenase gene in houseflies homozygous for green (ge). The v cDNA, placed under the control of the hsp82 promoter of D. pseudoobscura was transiently expressed in M. domestica embryos homozygous for the tryptophan oxygenase gene, ge, resulting in the rescue of adult eye color. The use of a gene from D. melanogaster to complement an eye color mutant in Musca provides the opportunity to develop a gene vector system for M. domestica and a select group of other non-drosophilid insects in which homologous mutations exist.

Purified mariner (Mos1) transposase catalyzes the integration of marked elements into the germ-line of the yellow fever mosquito, Aedes aegypti.

Derivatives of the mariner transposable element, Mos1, from Drosophila mauritiana, can integrate into the germ-line of the yellow fever mosquito, Aedes aegypti. Previously, the transposase required to mobilize Mos1 was provided in trans by a helper plasmid expressing the enzyme under the control of the D. psuedoobscura heat-shock protein 82 promoter. Here we tested whether purified recombinant Mos1 transposase could increase the recovery of Ae. aegypti transformants. Mos1 transposase was injected into white-eyed, kh(w)/kh(w), Ae. aegypti embryos with a Mos1 donor plasmid containing a copy of the wild-type allele of the D. melanogaster cinnabar gene. Transformed mosquitoes were recognized by partial restoration of eye color in the G(1) animals and confirmed by Southern analyses of genomic DNA. At Mos1 transposase concentrations approaching 100 nM, the rate of germ-line transformants arising from independent insertions in G(0) animals was elevated 2-fold compared to that seen in experiments with helper plasmids. Furthermore, the recovery of total G(1) transformants was increased 7.5-fold over the frequency seen with co-injected helper plasmid. Southern blot analyses and gene amplification experiments confirmed the integration of the transposons into the mosquito genome, although not all integrations were of the expected cut-and-paste type transposition. The increased frequency of germ-line integrations obtained with purified transposase will facilitate the generation of Mos1 transgenic mosquitoes and the application of transgenic approaches to the biology of this important vector of multiple pathogens.

Predictive validity of a screen for partner violence against women.

BACKGROUND: While public health leaders recommend screening for partner violence, the predictive value of this practice is unknown. The purpose of this study was to test the ability of a brief three-question violence screen to predict violence against women in the ensuing months. METHODS: We conducted a prospective cohort study of adult women participating in the Colorado Behavioral Risk Factor Surveillance System (BRFSS), a population-based, random-digit-dialing telephone survey. During 8 monthly cohorts, 695 women participated in the BRFSS; 409 women participated in follow-up telephone interviews approximately 4 months later. Violent events during the follow-up period, measured using a modified 28-item Conflict Tactics Scale, were compared between women who initially screened positive and those who screened negative. RESULTS: Among BRFSS respondents, 8.4% (95% confidence interval [CI]=6.3%-10.5%) had an initial positive screen. During the follow-up period, women who screened positive were 46.5 times (5.4-405) more likely to experience severe physical violence, 11.7 times (5.0- 27.3) more likely to experience physical violence, 3.6 (2.4-5.2) times more likely to experience verbal aggression, and 2.5 times (1.2-5.1) more likely to experience sexual coercion. In a multivariate model, separation from one's spouse and a positive screen were significant independent predictors of physical violence. CONCLUSIONS: A brief violence screen identifies a subset of women at high risk for verbal, physical, and sexual partner abuse over the following 4 months. Women with a positive screen who are separated from their spouse are at highest risk.

Femoral osteotomy in Perthes' disease. Results at maturity.

Upper femoral osteotomy is a recognised treatment for selected patients with Perthes' disease. The results of this procedure were investigated at skeletal maturity in 44 patients (48 hips). The indication for operation was Catterall group II, III, and IV hips with 'head-at-risk' signs. Harris and Iowa scores were calculated clinically, and each hip was assigned radiographically to one of the five Stulberg classes, its initial Catterall grading checked and other relevant indices measured. Results showed excellent clinical function. Shortening was present in 14 hips (29%) and a positive Trendelenburg's sign was seen in 12 (25%). On radiographic assessment 58% of hips were Stulberg class I or II, with a good prognosis. The results of femoral osteotomy were better than those for conservatively treated hips in all age groups except those under five years.

Early mobilisation of Colles' fractures. A prospective trial.

The results of a prospective controlled trial of early mobilisation of Colles' fractures in the elderly are presented. Early mobilisation produced less pain and a stronger grip. It did not lead to any greater loss of reduction of the fracture. However, there was no significant improvement in the final range of movement of the wrist. Immobilisation of the wrist for six weeks in plaster is extremely inconvenient for the elderly living alone and the patients greatly appreciated the reduction of this period of time to a minimum.

Biodegradable pin fixation of elbow fractures in children. A randomised trial.

A prospective randomised clinical trial was undertaken to compare biodegradable polyglycolic acid pins with standard Kirschner wires used to fix displaced elbow fractures in children. Twenty-four children were enrolled in the trial; 14 had fractures of the lateral condyle of the humerus, eight of the medial epicondyle and two had olecranon fractures. Eleven fractures were fixed with Kirschner wires and 13 with polyglycolic acid pins. Fracture union with full function occurred in all cases within six months. Kirschner wires caused problems including infection in three cases, soft-tissue ossification in one and they required removal under general anaesthesia in nine cases. No such complications occurred with polyglycolic acid pins but one patient in this group developed avascular necrosis and premature fusion of the medial epicondyle.

Controlling malaria transmission with genetically-engineered, Plasmodium-resistant mosquitoes: milestones in a model system.

We are developing transgenic mosquitoes resistant to malaria parasites to test the hypothesis that genetically-engineered mosquitoes can be used to block the transmission of the parasites. We are developing and testing many of the necessary methodologies with the avian malaria parasite, Plasmodium gallinaceum, and its laboratory vector, Aedes aegypti, in anticipation of engaging the technical challenges presented by the malaria parasite, P. falciparum, and its major African vector, Anopheles gambiae. Transformation technology will be used to insert into the mosquito a synthetic gene for resistance to P. gallinaceum. The resistance gene will consist of a promoter of a mosquito gene controlling the expression of an effector protein that interferes with parasite development and/or infectivity. Mosquito genes whose promoter sequences are capable of sex- and tissue-specific expression of exogenous coding sequences have been identified, and stable transformation of the mosquito has been developed. We now are developing the expressed effector portion of the synthetic gene that will interfere with the transmission of the parasites. Mouse monoclonal antibodies that recognize the circumsporozoite protein of P. gallinaceum block sporozoite invasion of mosquito salivary glands, as well as abrogate the infectivity of sporozoites to a vertebrate host, the chicken, Gallus gallus, and block sporozoite invasion and development in susceptible cell lines in vitro. Using the genes encoding these antibodies, we propose to clone and express single-chain antibody constructs (scFv) that will serve as the effector portion of the gene that interferes with transmission of P. gallinaceum sporozoites.

Brachial plexus injury: a survey of incidence and referral pattern.

The aim of this study was to establish the incidence and the pattern of referral of injuries to the brachial plexus for a single calendar year: 1987. A questionnaire was sent to every orthopaedic consultant and referral centre in the United Kingdom. Replies were received from 402 surgeons. It is estimated that in 1987 there were at least 336 patients with traumatic injuries and 137 patients with other problems related to the plexus. Only 145 patients were seen at a referral centre. The anatomical nature, mode of injury and methods of treatment were analysed for both the referred and unreferred patients. We found that injuries to the brachial plexus are far more common than is generally appreciated, but only a minority of patients are referred for specialist evaluation and treatment.

The long term result of digital nerve repair in no-man's land.

30 isolated lesions of digital nerves which had been repaired by microsurgical techniques were examined in 27 patients. The average length of follow-up was 25 months (range: 12-48 months). Patients were assessed subjectively using a visual analogue scale and examined for light touch, pain, two-point discrimination, electrical conductance and sensory threshold. Although 37% of fingers regained normal two-point discrimination, only 27% of patients graded their overall result as excellent and 40% complained of persistent hyperaesthesia for up to two years. None felt that their finger had regained normal sensation. All patients undergoing digital nerve repair should be warned that hyperaesthesia may persist for several years and that an adult will never regain normal sensation.

Utilizing narrative inquiry to evaluate a nursing doctorate program professional residency.

Because the University of Colorado (CU) School of Nursing Nursing Doctorate (ND) Program initiated an innovative nursing educational reform, emerging program evaluation challenges were addressed to ensure successful implementation and program quality. The study's purpose was to evaluate the effectiveness of the ND professional clinical residency (fourth and final year) from the students' perspectives. Therefore, this evaluation was exploratory and inductive to focus on the primary questions: "How does one become an ND nurse during the residency?" (process) and "What is an ND nurse?" (outcome). Additionally, an explanation of how interactive processes affected residency experiences was addressed. The narrative inquiry framework made available a special access to the human experiences of time, order, and change during the residency process. Ten students in the first CU ND Program residency participated. Narrative data for qualitative analysis were obtained from students' monthly written vignettes and verbal sharing of their clinical experiences during conferences. Vignette formats directed students to describe significant residency experiences and share reflections on the events. One finding suggested that students' formative progression through the residency occurred in four phases similar to cognitive development theories. Additional findings confirmed students' growth toward and summative attainment of ND outcome behaviors, including holistic clinical proficiency, client advocacy, and promotion of professional growth for colleagues.

Doctoral education in nursing: evaluation of a nontraditional program option.

Selected outcomes of an intensive, summer-only doctoral program in nursing were assessed using quantitative and qualitative data obtained from program-specific questionnaires and Educational Testing Service (ETS) Graduate Program Self-Assessment (GPSA) student and faculty questionnaires. Students evaluated the overall program as successful, satisfying, and stimulating, but also as stressful. Educational interventions to ameliorate student stress did not impact student evaluations of reported stress levels. Comparisons of summer-only and regular academic year students showed no differences in demographics, although differences between groups were found in long-term goals. Overall ETS questionnaire data were consistent with the Holzemer and Chambers (1986) baseline data for schools of nursing and showed no unusual patterns or trends. Summer-only and regular program student ratings were similar on all ETS scales.

The Caring Efficacy Scale: nurses' self-reports of caring in practice settings.

The Caring Efficacy Scale (CES) assesses belief in one's ability to express a caring orientation and to develop caring relationships with clients or patients. This article describes the development of this Likert-type self-report scale to assess nurses' caring efficacy. It also discusses a preliminary reliability and validity study assessing the caring efficacy of novice and experienced nurses from three nursing programs: baccalaureate, nursing doctorate, and master's. The CES was found to have high internal consistency, and it possessed significant positive relationships with a measure of clinical competence.

Synaptic plasticity in the molluscan peripheral nervous system: physiology and role for peptides.

The plasticity of a synapse in the molluscan peripheral nervous system was examined under a variety of experimental, physiological, and pharmacological conditions. These studies employed the isolated salivary glands and attached buccal ganglia of the freshwater snail Helisoma. Action potentials evoked in buccal neuron 4 normally evoke a large excitatory postsynaptic potential (EPSP) which drives an action potential in gland secretory cells. In order to measure modulation of the EPSP, action potential generation in gland cells was prevented by bathing the preparation in low calcium, high magnesium salines. The relationship between the gland EPSP amplitude and specific physiological properties of neuron 4 was analyzed. In common with some central molluscan synapses, the EPSP was found to be strongly influenced by the membrane potential of neuron 4. Specifically, its amplitude was reduced by hyperpolarization of the neuron 4 soma. The relationship between EPSP amplitude and somatic potential of neuron 4 was linear in the range from resting potential (-47 +/- 6mV) to -100 mV. Furthermore, the EPSP amplitude was directly proportional to the action potential half-width of neuron 4. In order to evaluate the possible physiological role of this action potential/EPSP relationship, we examined whether gland EPSPs are modulated during the spike broadening that occurs in both spontaneous burst activity and imposed impulse trains. The preceding action potential/EPSP relationship was maintained under both of these conditions, i.e., EPSP magnitude increased as spikes broadened during bursts or trains. The peptidergic modulation of neuroglandular transmission was also examined. The molluscan peptide SCPB was found to depolarize neuron 4 and an increase in EPSP amplitude was concomitantly observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Vertical subluxation of the patella: an unusual feature of multiple epiphyseal dysplasia.

Abnormally shaped, usually double-layered, patellae are recognized as occurring in multiple epiphyseal dysplasia. The case described is unusual in that an associated defect appeared in the femur and resulted in mechanical symptoms.

An angiostatic agent in treatment of a recurrent aneurysmal bone cyst.

Healing of an aggressive recurrent aneurysmal bone cyst of the distal humerus was induced by oral dexamethasone, an angiostatic agent. The severe pain and tenderness resolved rapidly. The soft tissue swelling subsided, and a thick layer of bone formed around the cyst. Complete healing followed saucerization and curettage of the remaining vascular tissue in the center of the cyst. We suggest that angiostatic agents be included in the nonoperative methods of treatment of aggressive aneurysmal bone cysts.