Heterozygosity for the mouse Apex gene results in phenotypes associated with oxidative stress.

Apurinic/apyrimidinic endonuclease is a key enzyme in the process of base excision repair, required for the repair of spontaneous base damage that arises as a result of oxidative damage to DNA. In mice, this endonuclease is coded by the Apex gene, disruption of which is incompatible with embryonic life. Here we confirm the embryonic lethality of Apex-null mice and report the phenotypic characterization of mice that are heterozygous mutants for the Apex gene (Apex+/-). We show that Apex heterozygous mutant cells and animals are abnormally sensitive to increased oxidative stress. Additionally, such animals manifest elevated levels of oxidative stress markers in serum, and we show that dietary supplementation with antioxidants restores these to normal levels. Apex+/- embryos and pups manifest reduced survival that can also be partially rescued by dietary supplementation with antioxidants. These results are consistent with a proposed role for this enzyme in protection against the deleterious effects of oxidative stress and raise the possibility that humans with heterozygous mutations in the homologous HAP1 gene may be at increased risk for the phenotypic consequences of oxidative stress in cells.

Caring in forensic nursing. Expanding the holistic model.

Nurses have practiced forensic nursing for many years without formal recognition of the domain as a discipline for specialized study. Forensic nursing expands the traditional concept of holism to include the medical-legal aspects of potential or actual client problems. The American Nurses Association (ANA) and the International Association of Forensic Nurses (IAFN) recently have designated forensic nursing as a unique specialty. Recognition of forensic nursing as a specialty area of practice within nursing mandates that the specialty define and explicate its major conceptual base. A concept germane to the practice of forensic nursing is that of caring. Caring as a central concept to the role of this specialty is discussed, and the implications for its inclusion in a curriculum preparing forensic nurses are explored.

Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation.

Neurons containing the neuropeptide orexin (hypocretin) are located exclusively in the lateral hypothalamus and send axons to numerous regions throughout the central nervous system, including the major nuclei implicated in sleep regulation. Here, we report that, by behavioral and electroencephalographic criteria, orexin knockout mice exhibit a phenotype strikingly similar to human narcolepsy patients, as well as canarc-1 mutant dogs, the only known monogenic model of narcolepsy. Moreover, modafinil, an anti-narcoleptic drug with ill-defined mechanisms of action, activates orexin-containing neurons. We propose that orexin regulates sleep/wakefulness states, and that orexin knockout mice are a model of human narcolepsy, a disorder characterized primarily by rapid eye movement (REM) sleep dysregulation.

Adipose expression of the phosphoenolpyruvate carboxykinase promoter requires peroxisome proliferator-activated receptor gamma and 9-cis-retinoic acid receptor binding to an adipocyte-specific enhancer in vivo.

A putative adipocyte-specific enhancer has been mapped to approximately 1 kilobase pair upstream of the cytosolic phosphoenolpyruvate carboxykinase (PEPCK) gene. In the present study, we used transgenic mice to identify and characterize the 413-base pair (bp) region between -1242 and -828 bp as a bona fide adipocyte-specific enhancer in vivo. This enhancer functioned most efficiently in the context of the PEPCK promoter. The nuclear receptors peroxisome proliferator-activated receptor gamma (PPARgamma) and 9-cis-retinoic acid receptor (RXR) are required for enhancer function in vivo because: 1) a 3-bp mutation in the PPARgamma-/RXR-binding element centered at -992 bp, PCK2, completely abolished transgene expression in adipose tissue; and 2) electrophoretic mobility supershift experiments with specific antibodies indicated that PPARgamma and RXR are the only factors in adipocyte nuclear extracts which bind PCK2. In contrast, a second PPARgamma/RXR-binding element centered at -446 bp, PCK1, is not involved in adipocyte specificity because inactivation of this site did not affect transgene expression. Moreover, electrophoretic mobility shift experiments indicated that, unlike PCK2, PCK1 is not selective for PPARgamma/RXR binding. To characterize the enhancer further, the rat and human PEPCK 5'-flanking DNA sequences were compared by computer and found to have significant similarities in the enhancer region. This high level of conservation suggests that additional transcription factors are probably involved in enhancer function. A putative human PCK2 element was identified by this sequence comparison. The human and rat PCK2 elements bound PPARgamma/RXR with the same affinities. This work provides the first in vivo evidence that the binding of PPARgamma to its target sequences is absolutely required for adipocyte-specific gene expression.

Identification and management of pregnancy-related low back pain.

Back pain is a common complaint of women during pregnancy. It is frequently dismissed as trivial and inevitable. This article gives an overview of recent research on pregnancy-related back pain that documents the impact of this pain on women's lives, during and beyond the childbearing year. It argues for a more active approach to the prevention and management of back pain during pregnancy. Two common back pain types, lumbar pain and posterior pelvic pain, are described and basic management techniques for the woman and her primary caregiver are suggested. Red flag findings that necessitate specialist referral are also highlighted, as are suggestions for further research.

Normal luminal bacteria, especially Bacteroides species, mediate chronic colitis, gastritis, and arthritis in HLA-B27/human beta2 microglobulin transgenic rats.

Genetic and environmental factors are important in the pathogenesis of clinical and experimental chronic intestinal inflammation. We investigated the influence of normal luminal bacteria and several groups of selected bacterial strains on spontaneous gastrointestinal and systemic inflammation in HLA-B27 transgenic rats. Rats maintained germfree for 3-9 mo were compared with littermates conventionalized with specific pathogen-free bacteria. Subsequently, germfree transgenic rats were colonized with groups of five to eight bacteria that were either facultative or strictly anaerobic. Transgenic germfree rats had no gastroduodenitis, colitis, or arthritis, but developed epididymitis and dermatitis to the same degree as conventionalized rats. Colonic proinflammatory cytokine expression was increased in transgenic conventionalized rats but was undetectable in germfree and nontransgenic rats. Colitis progressively increased over the first 4 wk of bacterial exposure, then plateaued. Only transgenic rats colonized with defined bacterial cocktails which contained Bacteroides spp. had colitis and gastritis. Normal luminal bacteria predictably and uniformly induce chronic colonic, gastric and systemic inflammation in B27 transgenic F344 rats, but all bacterial species do not have equal activities.

Gonadal steroid hormones and hypothalamic opioid circuitry.

Endogenous opioid peptides derived from several gene families are localized within hypothalamic regions known to be involved in the regulation of reproduction. For example, the proenkephalin gene products, met- and leu-enkephalin, and the proopiomelanocortin (POMC) gene product, beta-endorphin, are found in the rat medial preoptic area (MPOA). Moreover, the expression of these peptides and their receptors varies across the estrous cycle in the female rat. We have examined the gonadal steroid regulation of mu-opiate receptors and opioid peptides in the MPOA, and POMC mRNA expression in neurons that innervate the MPOA. mu-Opiate receptors in the MPOA are sexually dimorphic and gonadal steroid hormone-dependent. Hormonal priming of ovariectomized rats with estrogen and progesterone (P) upregulates MPOA mu-receptors 27, but not 3, hr after P treatment. Inhibition of protein synthesis during the first 6 hr after P prevents receptor upregulation. The density of beta-endorphin fibers in the MPOA also increases following hormone treatment, and POMC mRNA expression in neurons that innervate the MPOA is induced by hormone treatment beginning 13 hr after P treatment. This delayed response might be ubiquitous among POMC neurons, as those innervating the median eminence also exhibit increased POMC mRNA expression along a similar time course. The results suggest that hormonal feedback regulates opioid peptides which act at mu-receptors in the MPOA to influence reproductive behavior and cyclicity. These opioid functions represent an important component in the complex regulatory processes which control reproduction.

Soft drugs--XVI. Design, evaluation and transdermal penetration of novel soft anticholinergics based on methatropine.

Atropine has been reported to produce unwanted systemic side effects on topical administration into the eye. The same problem could arise when atropine is used topically as a suppressant of eccrine sweating. In this study, the principles of soft drug design were applied to methatropine. A hypothetical carboxylate metabolite of methatropine was reactivated by esterification with cyclic and alicyclic alcohols to yield a series of compounds (3a-g). In vitro evaluation by guinea pig ileum assay indicated that the compounds are potent anticholinergics and the lead carboxylate metabolite is about 60 times less potent than the most active compound of the series. The activity was found to decrease with the increasing side chain length. The n-octanol/water partition coefficients were found to be directly dependent on the chain length for the compounds made with straight chain alcohols. The transdermal permeability coefficients across the hairless mice skin were found to be directly dependent on the partition coefficients. The soft drugs are found to metabolize extensively during the penetration process compared to the unmetabolizable nature of methatropine. The soft drugs reported in this study will probably be able to elicit a local action at the site of application but will probably be metabolized rapidly in the systemic circulation, thereby avoiding the systemic side effects with a consequent increase in the therapeutic index.

Regional dependence of morphine-induced mu-opiate receptor down-regulation in perinatal rat brain.

The effect of perinatal morphine administration was examined in various brain regions using in vitro receptor autoradiography. Morphine was administered by continuous s.c. infusion of 10 mg/kg per day; brains of offspring were examined at five days of age. Morphine exposure reduced mu-receptor binding density in the preoptic area of hypothalamus, but not in the primary somatosensory cortex. mu-Receptor density was greater in the medial preoptic area of females than males, and in superficial layers of cortex in males than females. The results suggest that morphine has selective regional effects on mu-receptor ontogeny in rat brain.

Competitive (AP7) and non-competitive (MK-801) NMDA receptor antagonists differentially alter glucose utilization in rat cortex.

The effects of D,L-2-amino-7-phosphonoheptanoic acid (AP7), a competitive N-methyl-D-aspartate (NMDA) receptor antagonist, and MK-801, a non-competitive NMDA receptor antagonist, on regional brain metabolism were studied in unanesthetized, freely moving rats by using the quantitative [14C]2-deoxyglucose autoradiographic procedure. AP7 (338 or 901 mg/kg) produced a dose-dependent decrease of metabolic activity throughout most of the regions studied including sensory, motor, and limbic cortices. In contrast, MK-801 (0.1 or 1.0 mg/kg) resulted in a dose-dependent decrease of metabolic activity in sensory cortices, and an increase in limbic regions such as the hippocampal stratum lacunosum moleculare and entorhinal cortex. MK-801 also produced a biphasic response in agranular motor cortex, whereby the low dose increased while the high dose decreased labeling. In addition, MK-801 produced heterogeneous effects on regional cerebral metabolism in sensory cortices. Metabolic activity decreased in layer IV relative to layer Va following MK-801 treatment in primary somatosensory (SI) and visual (VI) cortices, suggesting a shift in activity from afferent fibers innervating layer IV to those innervating layer Va. MK-801 administration also decreased metabolic activity in granular SI relative to dysgranular SI, and in VI relative to secondary visual cortex (VII), thus providing a relative sparing of activity in dysgranular SI and VII. Thus, the non-competitive NMDA receptor antagonist suppressed activity from extrinsic neocortical sources, enhancing relative intracortical activity and stimulating limbic regions, while the competitive NMDA antagonist depressed metabolic activity in all cortical regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Serotonin, but not dopamine, metabolites are increased in selected brain regions of subordinate male rats in a colony environment.

Subordinate male laboratory rats maintained in mixed-sex groups in a Visible Burrow System habitat show a complex pattern of stress-related changes including enhanced defensive behavior, early mortality and increased voluntary ethanol consumption. Analysis of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) levels indicated that 5-HT levels do not differ between colony subordinates, colony dominants, and singly-housed control animals. However, 5-HIAA levels were higher in subordinates than either dominants or control animals in the preoptic area, amygdala, hippocampus, and spinal cord, and, were higher than dominants only, in entorhinal cortex. Subordinates' regional 5-HIAA/5-HT ratios were reliably higher than those of dominant or control animals in midbrain and spinal cord and reliably higher than dominants only, in hypothalamus. Dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) levels and DA/DOPAC ratios were affected neither in hypothalamus nor midbrain. These findings suggest that a consistent increase of 5-HIAA levels in selected brain regions of subordinate rats may represent a biological substrate for a well-characterized pattern of alterations in defensive behaviors for these animals.

Novel developmental specificity in the nervous system of transgenic animals expressing growth hormone fusion genes.

The development of methods for introducing foreign genes into the germ line of mice provides an approach for studying mechanisms underlying inducible and developmental gene regulation. Transgenic animals expressing foreign genes have thus been used to test models of the role played by specific DNA sequences in determining cell-specific expression. Results from these experiments suggest that tissue-specific expression is the consequence of a cis-acting regulatory sequence. However, these results do not exclude the possibility that cell-specific expression of some genes might be 'coded' by combinations of regulatory elements. We have previously described the production of transgenic mice from eggs microinjected with metallothionein-I/growth hormone (MGH) fusion genes, and now demonstrate that the juxtaposition of sequences from two different genes can be deciphered by cells to generate novel tissue specificities. Although expression of the endogenous metallothionein and growth hormone genes has not been detected in neuronal cells, transgenic mice clearly express an MGH fusion gene in a restricted subset of neurones. These results suggest a model in which tissue-specific patterns of expression of certain genes are determined by combinations of cis-acting regulatory sequences.

Isolation of human intestinal neurotensin.

The biologically active peptide neurotensin (NT) has been isolated from fresh postmortem human small intestine and its identity with bovine hypothalamic and intestinal neurotensin has been established. Purification was achieved by gel filtration and two ion exchange chromatography steps; material was detected by radioimmunoassay (RIA). A substance was obtained that had integral molar ratios of amino acids and eluted in a single peak during reverse-phase high pressure liquid chromatography (HPLC). This material had an amino acid composition and COOH-terminal sequence identical with those of bovine NT. Human and bovine neurotensin gave rise to the same fragments when treated with papain; they were indistinguishable in RIAs using three different region-specific antisera and in their hypotensive effect on anesthetized rats. Using mucosal scrapings obtained immediately post-mortem from four subjects, the concentration of immunoreactive neurotensin was found to increase from duodenum to distal ileum, in agreement with results obtained in other mammalian species.

Inhibition of gastric acid secretion by pirenzepine (LS 519) in man.

Pirenzepine is a new tricyclic drug used in the treatment of peptic ulcer disease. The effect of two doses of pirenzepine (25 mg twice daily and 50 mg thrice daily) was examined in ten healthy volunteers during basal acid secretion and under stimulation with two doses of pentagastrin (0.166 microgram/kg . h and 1 microgram/kg.h given as continuous intravenous infusion). Serum drug concentrations were determined by radioimmunoassay, and parallel studies of the salivary function were performed. Pirenzepine, 25 mg twice daily, reduced basal acid output by 50% and 55%, respectively, and inhibited stimulated acid output by 31% and 26%, respectively. The higher dose of pirenzepine, 50 mg thrice daily, augmented the effect insignificantly despite markedly increased serum drug levels. The recommended therapeutic dose of 25 mg twice daily gave no salivary inhibition. Pirenzepine may have an anticholinergic effect on the parietal cell, although systemic side effects were not seen. Pirenzepine does not competitively inhibit pentagastrin-stimulated acid secretion.

Clinical follow-up: treatment and outcome of functional headache patients treated with biofeedback.

Sixteen diagnosed functional headache patients treated with biofeedback techniques of frontalis EMG feedback and/or peripheral temperature feedback, with an average headache history of 18.2 years, were evaluated at an average interval of 37 weeks posttreatment. On the average, patients showed further improvement in their headache status as measured by average daily headache pain scores in contrast with termination values. Data were obtained via structured interviews conducted by an individual not involved in patient's treatment. Patients on the average showed declines in frontalis EMG during the follow-up sessions, but not increases in temperature as had been expected. The unclear role played by increasing peripheral temperature in follow-up of migraine patients is discussed.