Development of the Nursing Community Apgar Questionnaire (NCAQ): a rural nurse recruitment and retention tool.

INTRODUCTION: Health professional shortages are a significant issue throughout the USA, particularly in rural communities. Filling nurse vacancies is a costly concern for many critical access hospitals (CAH), which serve as the primary source of health care for rural communities. CAHs and rural communities have strengths and weaknesses that affect their recruitment and retention of rural nurses. The purpose of this study was to develop a tool that rural communities and CAHs can utilize to assess their strengths and weaknesses related to nurse recruitment and retention. METHODS: The Nursing Community Apgar Questionnaire (NCAQ) was developed based on an extensive literature review, visits to multiple rural sites, and consultations with rural nurses, rural nurse administrators and content experts. RESULTS: A quantitative interview tool consisting of 50 factors that affect rural nurse recruitment and retention was developed. The tool allows participants to rate each factor in terms of advantage and importance level. The tool also includes three open-ended questions for qualitative analysis. CONCLUSIONS: The NCAQ was designed to identify rural communities' and CAHs' strengths and challenges related to rural nurse recruitment and retention. The NCAQ will be piloted and a database developed for CAHs to compare their results with those in the database. Furthermore, the NCAQ results may be utilized to prioritize resource allocation and tailor rural nurse recruitment and retention efforts to highlight a community's strengths. The NCAQ will function as a useful real-time tool for CAHs looking to assess and improve their rural nurse recruitment and retention practices and compare their results with those of their peers. Longitudinal results will allow CAHs and their communities to evaluate their progress over time. As the database grows in size, state, regional, and national results can be compared, trends may be discovered and best practices identified.

Cerebellar granule cells contain a membrane mitogen for cultured Schwann cells.

Proliferation of Schwann cells is one of the first events that occurs after contact with a growing axon. To further define the distribution and properties of this axonal mitogen, we have (a) cocultured cerebellar granule cells, which lack glial ensheathment in vivo with Schwann cells; and (b) exposed Schwann cell cultures to isolated granule cell membranes. Schwann cells cocultured with granule cells had a 30-fold increase in the labeling index over Schwann cells cultured alone, suggesting that the mitogen is located on the granule cell surface. Inhibition of granule cell proteoglycan synthesis caused a decrease in the granule cells' ability to stimulate Schwann cell proliferation. Membranes isolated from cerebellar granule cells when added to Schwann cell cultures caused a 45-fold stimulation in [3H]thymidine incorporation. The granule cell mitogenic signal was heat and trypsin sensitive and did not require lysosomal processing by Schwann cells to elicit its proliferative effect. The ability of granule cells and their isolated membranes to stimulate Schwann cell proliferation suggests that the mitogenic signal for Schwann cells is a ubiquitous factor present on all axons regardless of their ultimate state of glial ensheathment.

Transfection of neonatal rat Schwann cells with SV-40 large T antigen gene under control of the metallothionein promoter.

Secondary cultures of Schwann cells were transfected with a plasmid containing the SV-40 T antigen gene expressed under the control of the mouse metallothionein-I promoter. We used the calcium phosphate method for transfection and obtained a transfection efficiency of 0.01%. The colonies were cloned by limited dilution, and these cloned cell lines were carried in medium containing zinc chloride (100 microM). One cloned cell line, which has now been carried for 180 doublings, appears to have a transformed phenotype with a doubling time of 20 h. These cells express SV-40 T antigen while maintaining established Schwann cell properties (positive staining for 217c, Ran-2, A5E3, glial fibrillary acidic protein, presence of 2',3'-cyclic nucleotide phosphohydrolase [CNPase] activity, and the ability to synthesize sulfogalactosylceramide and mRNA for the myelin protein, P0). Removal of zinc chloride from the medium resulted in reduced expression of T antigen and a change in the appearance of the cells to a more bipolar shape, although they still did not exhibit contact inhibition and maintained a doubling time of 20 h. These cells now became Ran-2-negative and showed increases in CNPase activity and in their ability to synthesize sulfogalactosylceramide. The amount of P0 mRNA remained unchanged. Transfected Schwann cells, however, stopped dividing when they contacted either basal lamina or neurites and became bipolar in appearance. The Schwann cells in contact with the neurites then extended processes to wrap around bundles of neurites. Transfection with the SV-40 T antigen gene therefore provides a method for obtaining Schwann cell lines that continue to express properties associated with untransfected cells in culture and may be used to study axon-Schwann cell interaction.

Tumor-associated immunoglobulins in pulmonary carcinoma.

In view of the uncertainty of location and significance of immunoglobulin in tumors found by elution or rosette formation (as reported in the literature), the presence of IgG, IgM, and IgA in human carcinoma of the lung was studied by means of the peroxidase-antiperoxidase method. Surgically obtained specimens from patients with known survival times were used in this study. Membranous as well as cytoplasmic location of IgG was demonstrated more frequently than was that of IgA or IgM. The number of tumor cells carrying immunoglobulin varied greatly, even within a given case. Albumin could be demonstrated in tumor cells in 10 of 20 specimens, but there was poor correlation with immunoglobuin. In some instances, only the necrotic part of the tumor or the stroma was immunoreactive. The results are discussed and suggest that Fc receptors are not involved in the binding of immunoglobin by pulmonary carcinoma cells.

Glial fibrillary acidic protein synthesized in vitro using messenger RNA from a human glioma cell line.

Messenger RNA (mRNA) extracted from a continuous human glioma cell line grown in culture or as a solid tumor was translated in an mRNA-dependent reticulocyte lysate system. Translation products labeled with [35S]methionine were immunoprecipitated with antiserum specific for glial fibrillary acidic (GFA) protein, separated by one- and two-dimensional polyacrylamide gel electrophoresis and analyzed fluorographically. Immunoprecipitates from both cell culture and tumor mRNA translations had a molecular weight of 49,000 daltons, consistent with GFA protein extracted from human tissue. In two dimensions, the 49,000-dalton band resolved into two to three spots at pH 5.7-5.9, the isoelectric point of GFA protein. Minor lower molecular weight products were detected in fluorographs of heavily overloaded gels or in film exposed for extended periods of time. These data indicate that the GFA protein produced by this glioma cell line is chemically and immunologically similar to normal human GFA protein, which suggests that the primary phenotypic expression of GFA protein in this tumor cell line is not altered by the neoplastic process.

Characterization of an antiserum against an axolemma-enriched fraction.

An antiserum was raised to rat central nervous system (CNS) axolemma-enriched fractions (AEF), which showed no cross-reactivity with myelin proteins or liver microsomes yet gave an endpoint titer of 1:51 200 to CNS AEF by the enzyme-linked immunosorbent assay (ELISA). Immunochemical staining of electroblotted proteins from rat CNS and peripheral nervous system (PNS) AEFs separated by gel electrophoresis identified a major reactive band at 38.5 kD. CNS AEF also showed major immunoreactivity at 91 kD (+/- 3 kD) and a broad band from 110 kD to 130 kD. By immunoperoxidase staining the antiserum specifically recognized the axolemma of peripheral nerve and synaptic terminals in the CNS. The significance of the specificity is discussed with respect to anti-synaptosome antisera.

Effects of primary antiserum dilution on staining of "antigenrich" tissues with the peroxidase antiperoxidase technique.

The effect of primary antiserum dilution on staining results with the peroxidase antiperoxidase method were investigated using frozen sections of perfused rat cerebellum and optic nerve. Results comparable to formalin fixed and paraffin embedded tissue were attainable only when low antiserum concentrations were used. Optimal staining of antigen rich tissue, such as frozen sections, with the peroxidase antiperoxidase method required low antiserum concentrations apparently to minimize the binding of both antigen-binding fragments of the bridging antibody to the tissue bound antiserum. It appears that low antiserum concentration insures that sufficient bridge antibody molecules will be only singly bound and thus free to attach the peroxidase antiperoxidase complex.

Immunocytologic analyses of 10 nm intermediate filaments in the nervous system of Myxicola.

Antibodies prepared in rabbits against Myxicola infundibulum neurofilaments have been employed to stain neurofilaments immunohistochemically in intact Myxicola infundibulum nervous tissue. Paraffin-embedded and frozen sections (5--6 mu) were examined at the light microscopic level with Sternberger's peroxidase-antiperoxidase method, and Vibratome (20--40 mu) sections were studied at the ultrastructural level with Nakane's conjugated peroxidase method. The neurofilament antibody stained only neurons and axons at the light microscopic level. The staining pattern at the electron microscopic level corresponded to the neurofilaments within axons and neurons. Glial cells, which surround the axons, contain large bundles of filaments that resemble astrocytic filaments in mammalian astrocytes. These filaments do not stain with the anti-neurofilament antibody. Neurons, neurofilaments, glial cells, glial filaments, and nonnervous tissue showed no peroxidase staining when specific antiserum absorbed with neurofilaments was used. These structures were also unstained when antiserum to the glial fibrillary acidic protein of mammalian central nervous system astrocytes was substituted for the neurofilament antiserum. Therefore, in Myxicola infundibulum, the antigenic determinants of the neurofilament protein, as recognized immunohistochemically by anti-neurofilament protein antibodies, are not shared with those of glial filaments.

Morphogenic role for acetylcholinesterase in axonal outgrowth during neural development.

Acetylcholinesterase (AChE) is the enzyme that hydrolyzes the neurotransmitter acetylcholine at cholinergic synapses and neuromuscular junctions. However, results from our laboratory and others indicate that AChE has an extrasynaptic, noncholinergic role during neural development. This article is a review of our findings demonstrating the morphogenic role of AChE, using a neuronal cell culture model. We also discuss how these data suggest that AChE has a cell adhesive function during neural development. These results could have additional significance as AChE is the target enzyme of agricultural organophosphate and carbamate pesticides as well as the commonly used household organophosphate chlorpyrifos (Dursban). Prenatal exposure to these agents could have adverse effects on neural development by interfering with the morphogenic function of AChE.

Release of membrane-associated growth factors during neural injury.

The release of membrane-associated growth factors after neural injury may influence the outcome of the recovery. For example, for remyelination to occur after neural injury it is critical for the glial cell to proliferate prior to remyelination in both the PNS and CNS. In the CNS, the relative response of the oligodendrocytes and astroglia to growth factors mobilized during neural injury may play a role in the cellular dynamics of repair of neural injury or scarring and subsequent failure to repair neural injury. In support of this view, we have studied the mitotic potential and cell cycle kinetics of cultured adult oligodendrocytes and found that these adult cells respond only weakly to factors such as FGF which are known to be potent mitogens for neonatal cells. However, given the same dose of FGF, adult astrocytes are mitotically stimulated to a much greater degree than are the adult oligodendrocytes (Vick and De Vries, unpublished observations). Given the pathways which may be operative in the release of growth factors after injury, it has not escaped our attention that, provided the released factors are in equilibrium with easily accessible and peripheral body fluids, these released factors may serve as new markers for neural injury. Further experiments are in progress to explore this possibility.

Freeze fracture analysis of the axolemma of cultured dorsal root ganglion neurons in the absence of Schwann cells.

The distribution of intramembranous particles within the axolemma of cultured dorsal root ganglion neurons was determined by freeze-fracture microscopy. Utilizing culture conditions which eliminate Schwann cells, the particle distribution of the P-face, 735 +/- 119 microns2, and E-face, 100 +/- 39 microns2 resembled that of pre- and non-myelinated axons in vivo and no node-like E-face particle patching was seen. These results indicate that cultured neurite development is similar to that seen in vivo and that axons maintained in a glial-free environment do not develop nodal, E-face membrane specializations.

Glial fibrillary acidic protein synthesized in vitro using messenger RNA from jimpy mouse spinal cord.

Glial fibrillary acidic (GFA) protein was synthesized in vitro in a rabbit reticulocyte lysate system programmed with messenger RNA (mRNA) extracted from Jimpy mouse spinal cord. It was identical in molecular weight and charge to that synthesized from normal mouse mRNA and GFA protein extracted from normal mouse cord. These data suggest that the Jimpy mutation does not affect the primary phenotypic expression of GFA protein.

The subcellular localization of lipid in myelin-free axonal preparations.

Bovine myelin-free axonal preparations were subjected to a series of washes designed to partition membranes from other cellular components initially present in these preparations. These washes were composed entirely of membranous structures, essentially free of neurofilament protein subunits, and contained high specific activity of acetylcholinesterase, an axolemma-specific enzyme. The distribution of acetylcholinesterase in the washes paralleled the distribution of lipid and the lipid composition of these washes closely resembled that of bovine axolemma-enriched fractions. In addition, acetylcholinesterase, lipid and galactocerebroside were histo- and immunohistochemically localized on similar structures in the starting material. Our results demonstrate that some of the lipid in myelin-free axonal preparations may be accounted for by axolemma.

Evidence for the direct role of acetylcholinesterase in neurite outgrowth in primary dorsal root ganglion neurons.

Dorsal root ganglion (DRG) neurons show a transient peak expression of acetylcholinesterase (AChE) during periods of axonal outgrowth prior to synaptogenesis, suggesting that AChE has a non-enzymatic role during development. We have previously shown that perturbation of cell surface AChE in cultured embryonic rat DRG neurons results in decreased neurite outgrowth and neurite detachment. In this report, we demonstrate a direct correlation between endogenous AChE content and neurite outgrowth in primary DRG neurons. Adenoviral vectors were constructed using full-length rat AChE(T) cDNA in either the sense or antisense orientations to overexpress or knock down AChE expression, respectively. Treatment with the sense-expressing vector produced a 2.5-fold increase in AChE expression and a 2-fold increase in neurite length compared with either untreated or null virus-treated control cells. Conversely, treatment with the antisense-expressing vector reduced AChE expression by 40% and resulted in a reduction in neurite length of similar magnitude. We also observed that overexpression of AChE resulted in greater branching at the distal tips of each primary neurite as well as an increase in cell body size. These findings further indicate that AChE expressed on the axonal surface of developing DRG neurons may modulate their adhesive properties and thereby support axonal development.

Antisera to an axolemma-enriched fraction inhibit neurite outgrowth and destroy axons in vitro.

Antisera prepared to an axolemma-enriched fraction derived from rat brain inhibited neurite outgrowth and destroyed mature axons in spinal cord-dorsal root ganglia cultures. Similar antibody-mediated anti-axon effects may be important in some diseases of the human nervous system.

Inverse correlation of acetylcholinesterase (AChE) activity with the presence of neurofilament inclusions in dorsal root ganglion neurons cultured in the presence of a reversible inhibitor of AChE.

We have previously shown that treatment of cultured dorsal root ganglion neurons (DRGN) with a highly specific, reversible acetylcholinesterase (AChE) inhibitor, BW284c51, retards neuritic outgrowth in a dose dependent manner and is accompanied by the presence of abnormal, perikaryal neurofilament (NF) inclusions in approximately 40% of the cells. Since subpopulations of DRGN have been classified according to their levels of AChE activity, we have combined immunocytochemical and enzyme histochemical techniques to investigate a possible correlation between AChE activity and the presence of NF inclusion formation. Our results show that after inhibitor treatment, cells with low levels of AChE activity have a greater percentage of inclusions, with nearly 75% of cells with undetectable levels of AChE activity containing inclusions. In contrast, inclusions were present in only 3.2% of cells with high levels of AChE activity. This inverse relationship between AChE activity and the presence of NF inclusions supports our previous observations that this enzyme may have extra-synaptic functions which could affect neuronal development and regeneration.

Morphometric analysis of the developing optic nerve of the F1 heterotic mouse and its parental strains.

During the period of active myelination, the CNS of the F1 hybrid mouse, derived from DBA/2J (D2) and C57BL/6J (B6) parental strains, displays levels of myelin-specific markers which are greater than in either parent. This so-called hypermyelination has been attributed to hybrid vigor or heterosis. Morphometric comparison of the optic nerves of F1 and parental strains revealed that the F1 contains larger myelinated axons and fewer premyelinated axons. These observations suggest that, compared with its parental strains, the F1 hybrid shows an early onset and accelerated rate of myelination.

Myelinated dorsal root ganglion cultures activate both the alternative and classical pathways of complement.

We used rat myelinated dorsal root ganglion (MDRG) cultures to study antibody and complement-mediated mechanisms of peripheral demyelinating diseases. Heat inactivated serum from a patient (LT) with peripheral neuropathy and a monoclonal IgM reactive with myelin-associated glycoprotein (anti-MAG) and sulfated glucuronosyl glycolipids (anti-SGGL) was used as an antibody source. Incubation of whole human serum (WHS) or WHS and anti-SGGL with MDRGs resulted in reduction of classical and alternative pathway hemolytic activities and the development of abnormal myelin sheaths. Incubation of MDRG cultures with C2-deficient serum showed activation of the alternative complement pathway. Classical pathway hemolytic activity was reduced when Factor B-depleted serum was incubated with MDRG cultures. The rat MDRG culture system provides a good model system of a peripheral nerve and has therefore been used by several investigators to study antibody and complement-mediated demyelination associated with peripheral neuropathies. However, our studies indicate a high degree of complement activation and membrane disruption of cultures incubated with WHS.

A nuclear matrix protein stabilized by lead exposure: current knowledge and future prospects.

p32/6.3, a low-abundance, highly conserved nuclear protein, is a target for lead. Very few low abundance nuclear proteins have been described and no others have been associated with lead. Its wide distribution and conservation indicate a fundamental nuclear role. Further, it increases many fold in grey matter of brain and spinal cord during the neonatal period; there are no other identified nuclear proteins which serve as markers for this period of nervous system development. There are several links between lead and p32/6.3. It is a major component of lead-induced intranuclear inclusion bodies from the kidney. Its accumulation in kidney is a relatively early event in the process of lead intoxication. Exposure to lead increases p32/6.3 in mouse neuroblastoma 2a cells within one day, blocking its degradation almost completely. These observations suggest that lead either structurally alters p32/6.3 or inhibits a protease for which p32/6.3 is a substrate. In these lead-treated cells nuclear envelope invaginations and small nuclear bodies increase. The possible involvement of lead and p32/6.3 with the formation and movement of nuclear bodies is discussed.

Immunoperoxidase localization of treponema pallidum: its use in formaldehyde-fixed and paraffin-embedded tissue sections.

The spirochete, Treponema pallidum, the causative agent of syphilis, has been successfully localized in formaldehydefixed and paraffin-embedded tissue sections using rabbit anti-T pallidum antiserum with two immunoperoxidase techniques. These techniques, the indirect peroxidase-labeled antibody method and the peroxidase-antiperoxidase (PAP) method are compared for sensitivity and degree of nonspecific staining. Both offer substantial advantages over conventional silver-impregnation techniques, but the indirect peroxidase-labeled antibody method seems better, based on the intensity of staining and the simplicity of procedure.