Translation elongation factor eEF1A2 is essential for post-weaning survival in mice.

Translation elongation factor eEF1A, formerly known as EF-1 alpha, exists as two variant forms; eEF1A1, which is almost ubiquitously expressed, and eEF1A2, whose expression is restricted to muscle and brain at the level of whole tissues. Expression analysis of these genes has been complicated by a general lack of availability of antibodies that specifically recognize each variant form. Wasted mice (wst/wst) have a 15.8-kilobase deletion that abolishes activity of eEF1A2, but before this study it was unknown whether the deletion also affected neighboring genes. We have generated a panel of anti-peptide antibodies and used them to show that eEF1A2 is expressed at high levels in specific cell types in tissues previously thought not to express this variant, such as pancreatic islet cells and enteroendocrine cells in colon crypts. Expression of eEF1A1 and eEF1A2 is shown to be generally mutually exclusive, and we relate the expression pattern of eEF1A2 to the phenotype seen in wasted mice. We then carried out a series of transgenic experiments to establish whether the expression of other genes is affected by the deletion in wasted mice. We show that aspects of the phenotype such as motor neuron degeneration relate precisely to the relative expression of eEF1A1 and eEF1A2, whereas the immune system abnormalities are likely to result from a stress response. We conclude that loss of eEF1A2 function is solely responsible for the abnormalities seen in these mice.

Mechanical vibration in neonatal transport: a randomized study of different mattresses.

OBJECTIVE: To test the hypothesis that a gel mattress is most effective in attenuating mechanical vibration in neonatal transport, we performed a randomized block study of four mattress combinations (none, foam, gel, gel on foam) using mannequins and an ambulance traveling on fixed routes (city, highway). STUDY DESIGN: Mechanical vibration was assessed by measuring vertical accelerations at two locations: the forehead of a 2000-gm mannequin and the transport incubator base. From time histories of these accelerations, root mean square (RMS) values and power spectral density functions were calculated. The effect of the mattress on the transmission of vibration was determined from ratios of the RMS values at the two locations. An RMS ratio of < 1.0 indicates attenuation, whereas a ratio of > 1.0 indicates accentuation of vibration. From the power spectral density functions, the natural frequency of the system was determined for each mattress combination in relation to the natural frequencies of the ambulance. To determine the effect of the weight of the mannequin on vibration, additional measurements were performed using a 300-gm mannequin. RESULTS: All the observed RMS ratios were > 1. The highest ratios were observed on the city route in the absence of the gel mattress. The gel mattress, used alone or with the foam mattress, in contrast to foam or no mattress, shifted the natural frequency of the system away from the natural frequencies of the ambulance, avoiding a large amplification of vibration. A decrease in the weight of the mannequin caused the gel mattress to be less effective in attenuating vibration. CONCLUSION: A gel mattress, used alone or with a foam mattress, results in the least accentuation of vibration, but vibration in ambulance transport is not attenuated by any of the mattress combinations. The hazard of vibration may be particularly relevant when transporting extremely low birth weight neonates. These findings indicate a need for study and design of more effective devices that can reduce the vibratory stress.

Continuing education for registered nurses in the Highlands and Western Isles of Scotland.

This paper focuses on an educational approach used in the Highlands and Western Isles of Scotland in the continuing education of registered nurses. It includes a rationale for the educational approach used by this author and her colleagues, and a discussion of the approach itself. The discussion includes reference to concepts borrowed from learning theorists that underpin the approach, and case study material to illustrate how it works in practice. It is argued that the educational approach in this paper can potentially lead to meaningful learning. Meaningful learning is defined in this context. Finally, the outcomes of the educational approach are highlighted, with a discussion of its value to educators elsewhere.

Local cyclosporine immunotherapy of heart transplants in rats enhances survival.

High-dose systemic cyclosporine (CyA) administration frequently results in severe side effects. To evaluate a means of limiting the adverse effects of CyA, we implanted CyA-collagen matrices (0.2 or 1 mg/kg/day released) around the cardiac homografts at the time of rat heterotopic (neck) heart transplantation. Control animals received empty collagen (nondrug) matrix implants. A fourth group received CyA matrix (1 mg/kg/day released), implanted in a distal subdermal leg pouch at the time of heart transplantation. Rejection was determined by the absence of contraction in the transplanted heart. No animal received any other immunosuppression. Parallel groups of animals had whole blood, heart, and kidney CyA levels measured on the sixth posttransplant day. Local immunosuppression with high-dose CyA in a controlled-release matrix resulted in a significant survival advantage (mean survival time, 17.1 days; control, 6.9 days; p less than 0.001). The lower dose of CyA also demonstrated significant survival benefits (10.1 days), with clinically negligible blood CyA levels and very low kidney CyA levels. Both doses of epicardial local release CyA were well absorbed locally, resulting in very high CyA levels in cardiac tissue. Local immunotherapy of transplanted hearts with CyA was shown to be an effective means of preventing rejection. If this technology can be developed, this approach may prove advantageous clinically, both in extending transplantation and in minimizing systemic side effects of immunosuppression.

Ultrastructural characterization of two new human endometrial carcinoma cell lines and normal human endometrial epithelial cells cultured on extracellular matrix.

Two new lines of human endometrial carcinoma (HEC) cells, one from an adenocarcinoma and one from a highly metastatic serous papillary carcinoma, were established in culture. Structural and morphologic properties of these cells at early passage were compared with those of cultured normal human endometrial epithelial (NHEE) cells. For these studies, cells were grown on a conventional plastic surface or on an extracellular matrix substrate (Matrigel), and examined by transmission electron microscopy and immunofluorescent light microscopy. The HEC cells appeared morphologically similar on plastic and Matrigel, whereas the NHEE cells showed significantly greater epithelial morphologic differentiation on Matrigel than on plastic. On extracellular matrix, the morphologic differences observed between HEC cells and NHEE cells were primarily of an architectural nature, which may be in part explained by differences between NHEE and HEC cells in the arrangement of actin microfilaments and cytokeratin intermediate filaments. Furthermore, HEC cells displayed extensive networks of vimentin intermediate filaments, which were absent from the NHEE cells. These observations support the hypothesis that architectural deregulation is a prominent feature of endometrial carcinoma, and that cytoskeletal alterations may uncouple HEC cell ultrastructural morphology from the influence of extracellular matrix.

Expression of transforming growth factor beta 1 by human endometrial carcinoma cell lines: inverse correlation with effects on growth rate and morphology.

We examined the effects of transforming growth factor beta 1 (TGF-beta 1) on various aspects of the cell biology of human endometrial carcinoma (HEC) cell lines in vitro, as well as the expression of TGF-beta 1 mRNA by these cell lines. Cell lines from eight HEC tumors, representing a variety of histological subtypes, were studied in order to test the generality of conclusions regarding the effects of TGF-beta 1 on this particular tumor cell type. The growth of five HEC cell lines was inhibited by TGF-beta 1 (10 ng/ml), while growth of three cell lines was not inhibited. The effects on growth correlated with morphological alterations induced by TGF-beta 1; the cell lines with inhibited growth displayed a larger, flatter, more contact-inhibited phenotype, while the cell lines whose growth ws not inhibited showed few discernible morphological alterations in response to TGF-beta 1. Northern analysis of TGF-beta 1 mRNA levels revealed that the three HEC cell lines unresponsive to TGF-beta 1 treatment expressed relatively large amounts of TGF-beta 1. Correspondingly, the five HEC cell lines which responded to TGF-beta 1 with growth and morphological changes expressed much lower levels of TGF-beta 1 mRNA. These results suggest that the sensitivity of human HEC cell lines to TGF-beta 1 is variable and that this sensitivity is inversely correlated with the level of expression of TGF-beta 1.

Genetic and cellular basis of multistep carcinogenesis.

Experimental evidence indicates that cancer development is a multistep process, and that multiple genetic changes are required before a normal cell becomes fully neoplastic. These genetic changes involve oncogenes, tumor suppressor genes, and possibly senescence genes. From studies in vivo using several different animal models, the stages are broadly defined as initiation, progression, and clearly involve both genetic and epigenetic events. Studies in vitro using cell culture systems have allowed the multistep process to be dissected in greater detail at both the cellular and molecular genetic level. In the Syrian hamster embryo cell culture model, neoplastic progression requires four heritable changes, involving activation of two oncogenes and loss of two tumor suppressor genes. Like the experimental systems, a limited number of studies of human tumors suggest that the multistep paradigm is also applicable, and that similar genetic events are involved in the development of cancer in humans.

Establishment and characterization of a human cell line from a serous papillary endometrial carcinoma.

The cell line SPEC-1, derived from a human serous papillary endometrial carcinoma (SPEC), has been established and repetitively subcultured for over 18 months. SPEC is a clinically aggressive histologic variant of endometrial adenocarcinoma with a significantly poorer prognosis. The SPEC cells exhibit morphologic and ultrastructural characteristics of transformed epithelial cells. The cells were further characterized with regard to growth kinetics, histochemistry, karyotype, and tumorigenicity. These studies indicate that several properties of the SPEC cells in culture contrast markedly with those of both typical endometrial adenocarcinoma cell lines and normal endometrial epithelia, as described in the literature. The most significant of these differences concern cytogenetic and ultrastructural features. The implications of these unique characteristics are discussed with regard to the relationship they may have to the unusually aggressive biological behavior of this tumor cell type in vivo. This SPEC cell line should prove useful in future studies designed to determine important factors in the biological behavior of human tumor cells.

Local controlled release of 1-hydroxyethylidene diphosphonate using silicone-rubber matrices. Effects of sterilization on in vitro release and in vivo efficacy.

Calcification (CALC) is the most frequent cause of the clinical failure of bioprosthetic heart valves (BHVs). Controlled release of disodium ethanehydroxydiphosphonate (EHDP) has been demonstrated to inhibit subdermal BHV calcification at effective low local doses, avoiding adverse effects. However, the eventual circulatory use of controlled release EHDP necessitates addressing several critical issues that may affect efficacy. These include the effects of sterilization on EHDP release and the efficacy of sustained release matrices containing CaEHDP, which is less soluble than NaEHDP. The effects of CaEHDP-NaEHDP incorporation and steam sterilization on controlled release of EHDP from silicone-rubber matrices was studied both in vitro and in vivo using a rat subdermal model and sheep tricuspid valve replacements. Autoclaved EHDP matrices (20% wt/wt) released 88.9% +/- 7.84 of contained drug after 140 days in vitro, compared with control (87.6% +/- 10.3 cumulative release). Autoclaved EHDP matrices completely inhibited BHV CALC in 60 day rat subdermal implants (8.84 +/- 3.68 micrograms Ca++/mg tissue), comparable to nonsterilized EHDP-loaded matrices (7.06 +/- 2.00 micrograms Ca++/mg tissue). Nontreated CALC levels were 183 +/- 7.60 micrograms Ca++/mg tissue. Na-CaEHDP co-incorporation into silicone rubber matrices markedly prolonged controlled release with the 1:1 Na-CaEHDP mixture demonstrating an extrapolated release duration of approximately 20 years, assuming the total amount of dispersed drug was released. Data from tricuspid valve replacements in sheep demonstrate erratic control calcification (41.3 +/- 14.9 micrograms Ca++/mg tissue), but complete suppression of BHV calcification with Na2EHDP controlled release (5.74 +/- 1.35 micrograms Ca++/mg tissue) after 150 days.

Prostaglandin H synthase-catalyzed metabolism and DNA binding of 2-naphthylamine.

The oxidation of the bladder carcinogen 2-naphthylamine (2-NA) by prostaglandin H synthase (PHS) in vitro was examined. Oxygen uptake studies of 2-NA oxidation in the presence of glutathione, as well as extensive product analysis data, are consistent with a one-electron mechanism of 2-NA oxidation by PHS. The formation of 2-nitrosonaphthalene is not observed under any condition. Metabolism studies with a purified PHS preparation confirm that 2-NA oxidation is dependent upon the peroxidase activity of the enzyme complex, and that a variety of organic hydroperoxides may support the reaction. Horseradish peroxidase oxidizes 2-NA to the same products but, depending on pH, in very different proportions from those obtained with PHS. Oxidation of 2-NA by a one-electron chemical oxidant results in a product profile similar to that obtained in the enzymatic systems. The above data are consistent with a one-electron mechanism of 2-NA oxidation by PHS. The metabolism data provide evidence for the formation of two types of potentially reactive electrophiles: 2-imino-1-naphthoquinone and a free radical species. We further examine the time course of covalent binding of [3H]2-NA products to DNA in vitro, and compare this with the reaction of authentic [3H]2-amino-1-naphthol (2-A-1-N) product(s) with DNA and protein. A significant amount of the PHS-catalyzed binding of 2-NA to DNA is derived from a short-lived intermediate; furthermore, the time course of binding is very rapid. Conversely, the binding to DNA of 2-A-1-N (presumably in the form of 2-imino-1-naphthoquinone) occurs to a lower extent and is not time dependent under the conditions studied. 2-A-1-N binds to protein, however, at a rapid rate and to three orders of magnitude greater extent than to DNA. The PHS-catalyzed binding of 2-NA to DNA was studied under several conditions; binding was shown conclusively to result from the peroxidase activity of the PHS complex. In addition, greater levels of binding were observed at pH 5.0 than at pH 7.6, and when catalyzed by horseradish peroxidase/H2O2 rather than PHS. These are conditions under which 2-A-1-N formation is negligible or nonexistent. These results demonstrate that in the PHS system, a reactive product(s) in addition to 2-A-1-N is generated which binds to DNA, and that this product is probably a free radical.(ABSTRACT TRUNCATED AT 400 WORDS)

Metabolism of aromatic amines by prostaglandin H synthase.

The metabolism of aromatic amines by the peroxidase activity of prostaglandin H synthase (PHS) has been studied in this laboratory by use of two model compounds, the carcinogenic primary amine 2-aminofluorene (2-AF) and the substituted amine aminopyrine (AP). 2-AF is oxidized by PHS to 2, 2-azobisfluorene, 2-aminodifluorenylamine, 2-nitrofluorene, polymeric material, and products covalently bound to macromolecules. In the presence of phenolic compounds, 2-AF oxidation results in the formation of amine/phenol adducts. The data are consistent with a one-electron mechanism of 2-AF oxidation by PHS; furthermore, an N-hydroxy intermediate is not involved in 2-AF metabolism by PHS. PHS also catalyzes the binding of 2-AF to DNA in vitro. Unique 2-AF/DNA adducts were isolated and are distinct from the N-(deoxyguanosin-8-yl)-2-AF adduct formed from the reaction of N-hydroxy-2-AF with DNA. These new adducts represent a marker unique to peroxidative activation of 2-AF. AP is oxidized by the peroxidase activity of PHS to the cation radical, with one molecule of hydroperoxy fatty acid reduced for every two molecules of AP free radical formed. The decay of the AP radical follows second order kinetics, supporting the proposed mechanism in which the AP radical disproportionates to an iminium cation, followed by hydrolysis of this species to the demethylated amine and formaldehyde. In the presence of glutathione, the cation radical is reduced to the parent amine, resulting in the formation of the glutathione thiyl radical. It thus appears that both primary and substituted aromatic amines may undergo one-electron oxidation by PHS.

The cytochrome P-450 monooxygenase system of rabbit bladder mucosa: enzyme components and isozyme 5-dependent metabolism of 2-aminofluorene.

The microsomal fraction prepared from the mucosa of rabbit bladder was analyzed for the presence of enzymes and activities associated with the cytochrome P-450-dependent monooxygenase system. Reduced nicotinamide adenine dinucleotide phosphate:cytochrome P-450 reductase (315 units/mg protein), reduced nicotinamide adenine dinucleotide:cytochrome b5 reductase (920 units/mg protein), cytochrome P-450 (0.22 nmol/mg protein), and cytochrome b5 (0.31 nmol/mg protein) were present in the microsomal preparation. Individual isozymes of cytochrome P-450, forms 2, 5, and 6, but not form 4, were detected by immunochemical methods. Treatment of rabbits with either phenobarbital or 2,3,7,8-tetrachlorodibenzo-p-dioxin did not alter the concentrations of these isozymes in the bladder preparation. Monooxygenase activities (pmol product/min/protein) in the bladder microsomal fraction were observed for benzphetamine N-demethylation (290), 7-ethoxyresorufin O-deethylation (29), 7-ethoxycoumarin O-deethylation (28), benzo(a)pyrene hydroxylation (10), and 2-aminofluorene hydroxylation (1400). The metabolism of 2-aminofluorene was determined by high performance liquid chromatography and scintillation counting; two products, 2-nitrosofluorene and 2,2'-azoxybisfluorene, were identified by chromatographic retention times, ultraviolet-visible spectroscopy, and mass spectrometry. Two additional metabolites were tentatively identified as N-hydroxy-2-aminofluorene and a ring-hydroxylated product. The metabolism of 2-aminofluorene was inhibited by antibodies to cytochrome P-450 form 5 or to reduced nicotinamide adenine dinucleotide phosphate:cytochrome P-450 reductase and by carbon monoxide (CO:O2, 4:1), but not by antibodies to cytochrome P-450 form 2. Acetylation of 2-aminofluorene in the presence of ethyl acetate (and deacetylation of 2-acetylaminofluorene) mediated by an enzyme sensitive to inhibition by either paraoxon or sodium fluoride was also observed.