Oxidative and nitrative injury in periventricular leukomalacia: a review.

Periventricular leukomalacia (PVL) is the major substrate of cerebral palsy in survivors of prematurity. Its pathogenesis is complex and likely involves ischemia/reperfusion in the critically ill premature infant, with impaired regulation of cerebral blood flow, as well as inflammatory mechanisms associated with maternal and/or fetal infection. During the peak period of vulnerability for PVL, developing oligodendrocytes (OLs) predominate in the white matter. We hypothesize that free radical injury to the developing OLs underlies, in part, the pathogenesis of PVL and the hypomyelination seen in long-term survivors. In human PVL, free radical injury is supported by evidence of oxidative and nitrative stress with markers to lipid peroxidation and nitrotyrosine, respectively. Evidence in normal human cerebral white matter suggests an underlying vulnerability of the premature infant to free radical injury resulting from a developmental mismatch of antioxidant enzymes (AOE) and subsequent imbalance in oxidant metabolism. In vitro studies using rodent OLs suggest that maturational susceptibility to reactive oxygen species is dependent, not only on levels of individual AOE, but also on specific interactions between these enzymes. Rodent in vitro data further suggest 2 mechanisms of nitric oxide damage: one involving the direct effect of nitric oxide on OL mitochondrial integrity and function, and the other involving an activation of microglia and subsequent release of reactive nitrogen species. The latter mechanism, while important in rodent studies, remains to be determined in the pathogenesis of human PVL. These observations together expand our knowledge of the role that free radical injury plays in the pathogenesis of PVL, and may contribute to the eventual development of therapeutic strategies to alleviate the burden of oxidative and nitrative injury in the premature infant at risk for PVL.

Selective protection by stably transfected human ALDH3A1 (but not human ALDH1A1) against toxicity of aliphatic aldehydes in V79 cells.

Toxic medium chain length alkanals, alkenals, and 4-hydroxyalkenals that are generated during lipid peroxidation are potential substrates for aldehyde dehydrogenase (ALDH) isoforms. We have developed transgenic cell lines to examine the potential for either human ALDH1A1 or ALDH3A1 to protect against damage mediated by these toxic aldehydes. Using crude cytosols from stably transfected cell lines, these aldehydes were confirmed to be excellent substrates for ALDH3A1, but were poorly oxidized by ALDH1A1. Expression of ALDH3A1 by stable transfection in V79 cells conferred a high level of protection against growth inhibition by the medium-chain length aldehyde substrates with highest substrate activity, including hexanal, trans-2-hexenal, trans-2-octenal, trans-2-nonenal, and 4-hydroxy-2-nonenal (HNE). This was reflected in a parallel ability of ALDH3A1 to prevent depletion of glutathione by these aldehydes. Expression of hALDH3 completely blocked the potent induction of apoptosis by HNE in both V79 cells and in a RAW 264.7 murine macrophage cell line, consistent with the observed total prevention of HNE-protein adduct formation. Structure-activity studies indicated that the rank order of potency for the contributions of HNE functional groups to toxicity was aldehyde >/=C2=C3 double bond>>C4-hydroxyl group. Oxidation of the aldehyde moiety of HNE to a carboxyl by ALDH3A1 expressed in stably transfected cell lines drastically reduced its potency for growth inhibition and apoptosis induction. In contrast, ALDH1A1 expression provided only moderate protection against trans-2-nonenal (t2NE), and none against the other six-nine carbon aldehydes. Neither ALDH1A1 nor ALDH3A1 conferred any protection against acrolein, acetaldehyde, or chloroacetaldehyde. A small degree of protection against malondialdehyde was afforded by ALDH1A1, but not ALDH3A1. Paradoxically, cells expressing ALDH3A1 were 1.5-fold more sensitive to benzaldehyde toxicity than control V79 cells. These studies demonstrate that expression of class 3 ALDH, but not class 1 ALDH, can be an important determinant of cellular resistance to toxicity mediated by aldehydes of intermediate chain length that are produced during lipid peroxidation.

Apoptosis in RAW 264.7 cells exposed to 4-hydroxy-2-nonenal: dependence on cytochrome C release but not p53 accumulation.

The toxic reactive aldehyde lipid peroxidation byproduct 4-hydroxy-2-nonenal (HNE) is thought to be a major contributor to oxidant stress-mediated cell injury. HNE induced apoptosis in RAW 264.7 murine macrophage cells in a dose-dependent manner within 6-8 h after exposure. Expression of the antiapoptotic protein Bcl-2 in stably transfected RAW 264.7 cells prevented HNE-induced internucleosomal DNA fragmentation and apoptosis, and these cells resume growth after a temporary (24-48 h) growth delay. While parental RAW 264.7 cells released mitochondrial cytochrome c within 3 h after HNE exposure, expression of Bcl-2 prevented cytochrome c release. In control cells, p53 protein levels peaked at 6-9 h after HNE exposure and then declined, while in Bcl-2 expressing cells, p53 levels were maximal at 6-9 h and remained elevated up to 96 h. Expression of SV40 large T-antigen, which forms a stable complex with p53 protein, via stable transfection-blocked transactivation of the p53-regulated gene p21(WAF1/CIP1), but did not affect induction of apoptosis by HNE, suggesting that p53 function is not important in HNE-induced apoptosis. These results suggest that cytochrome c release, but not p53 accumulation, plays an essential role in HNE-induced apoptosis in RAW 264.7 cells.

Structure-activity relationships for growth inhibition and induction of apoptosis by 4-hydroxy-2-nonenal in raw 264.7 cells.

4-Hydroxy-2-nonenal (HNE) is a highly reactive lipid aldehyde byproduct of the peroxidation of cellular membranes. The structure of HNE features three functional groups, a C1 aldehyde, a C2==C3 double bond, and a C4- hydroxyl group, each of which may contribute to the toxicity of the compound. In addition, the length of the aliphatic chain may influence toxic potency by altering lipophilicity. Using analogous compounds that lacked one or more of the structural moieties, the role of each of these structural motifs in the cytotoxicity of HNE was examined in a mouse alveolar macrophage cell line (RAW 264.7) by a cell survival and growth assay. The importance of these functional groups in the potency of HNE for induction of apoptosis was also examined. The rank order of effects on toxicity was C1---aldehyde >/= C2==C3 double bond >> C4---hydroxyl, with parallel results in both the survival/growth inhibition and apoptosis induction assays. The chain length also influenced toxicity in a series of alpha,beta-unsaturated alkenyl aldehydes, with increasing chain length yielding increasing toxicity. To confirm the importance of the aldehyde moiety, and to examine the role of metabolic detoxification in cellular defenses against HNE toxicity, a RAW 264.7 cell line overexpressing human aldehyde dehydrogenase-3 (hALDH3) was generated. This cell line exhibited nearly complete protection against HNE-protein adduct formation as well as HNE-induced apoptosis. These results illustrate the comparative significance of key structural features of HNE in relation to its potent toxicity and induction of apoptosis.

Chemoprotective functions of glutathione S-transferases in cell lines induced to express specific isozymes by stable transfection.

The authors have shown that expression of mGSTM1-1 or hGSTP1-1 in MCF-7 cells protects against DNA alkylation by 4-nitroquinoline-1-oxide (NQO) in an isozyme-specific manner and is commensurate with relative specific activity. Expression of GSTs also conferred protection against both DNA strand breaks and sister-chromatid exchange induced by NQO. Interestingly, GST expression did not protect against NQO cytotoxicity in transfected MCF-7 cell lines, although resistance to NQO cytotoxicity was observed in a T47D pi transfectant line, expressing much higher specific activity of the transfected hGSTP1-1. However, high level expression of hGSTP1-1 or mGSTM1-1 in V79 transfectants did not confer resistance to cytotoxicity, indicating that expression of GST alone is not sufficient. The authors have also shown protection against AFB1 in cell lines expressing transfected rat CYP2B1 (V79MZr2B1) and transfected mGST-Yc (mGSTA3-3). Protection was observed against both alkylation of DNA (3-fold) by [3H]AFB1 and against AFB1 cytotoxicity (7-fold). Similarly, V79MZr1A1 cells that express CYP1A1 and either transfected human or murine GSTP1-1 (< 5000 mIU/mg, CDNB) exhibited > 70% decrease in covalent labeling of total nucleic acids by [3H]BPDE. However, no protection against the cytotoxicity of BPDE was conferred by expression of hGSTP1-1. Overall, these results indicate that in some (NQO or BPDE), but not all (AFB1) cases, protection by GST expression against DNA damage is more effective than protection against cytotoxicity. In addition, there is evidence to indicate that additional factor(s) other than high GST isozyme expression level and good substrate efficacy affect the degree of protection against cytotoxicity of reactive electrophiles. This includes the differential protection against NQO cytotoxicity in T47D pi, but not V79 Xh pi-33 cells and also the recent studies which showed that expression of the MRP GS-X conjugate efflux transporter confers synergistic protection against NQO cytotoxicity when co-expressed with transfected human GSTP1-1 in MCF-7 cells. Thus, protective efficacy conferred by GST expression can vary with different cellular targets and/or experimental end-points, as well as with variations in relative specific activity or in different cellular phenotypic contexts.

Structure and folding of nascent polypeptide chains during protein translocation in the endoplasmic reticulum.

To investigate the role of protein folding and chaperone-nascent chain interactions in translocation across the endoplasmic reticulum membrane, the translocation of wild type and mutant forms of preprolactin were studied in vivo and in vitro. The preprolactin mutant studied contains an 18-amino acid substitution at the amino terminus of the mature protein, eliminating a disulfide-bonded loop domain. In COS-7 cells, mutant prolactin accumulated in the endoplasmic reticulum as stable protein-protein and disulfide-bonded aggregates, whereas wild type prolactin was efficiently secreted. In vitro, wild type and mutant preprolactin translocated with equal efficiency although both translation products were recovered as heterogeneous aggregates. Studies with translocation intermediates indicated that aggregation occurred co-translationally. To evaluate the contribution of lumenal chaperones to translocation and folding, in vitro studies were performed with native and reconstituted, chaperone-deficient membranes. The absence of lumenal chaperones was associated with a decrease in translocation efficiency and pronounced aggregation of the translation products. These studies suggest that chaperone-nascent chain interactions significantly enhance translocation and indicate that in the absence of such interactions, aggregation can serve as the predominant in vitro protein folding end point. The ramifications of these observations on investigations into the mechanism of translocation are discussed.

Changes in gonadal weights, serum lipids, and glucose during maturation in the juvenile northern bobwhite quail (Colinus virginianus).

Changes in serum lipids, serum glucose, and gonadal development during maturation of Colinus virginianus were determined in two trials. In Trial 1, sex, BW, and serum concentrations of total cholesterol, high, low, and very low density lipoprotein cholesterol, triglycerides, and glucose were determined every 2 d from 2 to 14 d and weekly from 21 to 49 d. Relative ovary or testis weights between 14 and 49 d were determined weekly. Body weight increased and all measured serum constituents changed in males and females between 2 and 49 d; however, relative testis weight decreased between 14 and 49 d. In Trial 2, sex, BW, cholesterol, and relative ovary and testis weights were determined in birds at 56 and 63 d of age. The distinctness of external sexual phenotype and its relationship to BW, cholesterol, and relative gonadal weight were also determined at 56 and 63 d. Body weight increased and serum cholesterol decreased in both sexes between 56 and 63 d, but had no association with the distinctness of external sexual phenotype. It was concluded that the development of external sexual identity in male and female Northern Bobwhite quail prior to sexual maturity was not related to changes in BW, gonad weight, serum lipids, or serum glucose before 63 d of age.

Mechanism of resistance of human immunodeficiency virus type 1 to 2',3'-dideoxyinosine.

A molecular clone containing the wild-type reverse transcriptase (RT) coding region of human immunodeficiency virus type 1 (HIV-1) was constructed, and site-directed mutagenesis was used to introduce mutations--Leu74-->Val (L74V), T215Y, and the combination L74V/T215Y--into the RT coding region. The proteins were purified by immunoaffinity chromatography. Assays were performed with mutant and wild-type RT to determine substrate and inhibitor specificity. All three mutant enzymes catalyzed the incorporation of substrate 2'-deoxynucleoside 5'-triphosphates (dNTPs) as efficiently as wild-type HIV-1 RT. Small changes were observed in the Km values for dNTPs with all three mutant enzymes, while more significant changes were noted in sensitivity to nucleoside 5'-triphosphate analogues that inhibit the enzyme activity. Results suggest that altered substrate recognition by the HIV-1 RT is involved in the mechanism of resistance.

Patterns of suicide among hospitalized mentally disordered offenders.

The case records of 29 mentally disordered offenders who committed suicide in a state hospital were reviewed. Information was collected on demographic and clinical characteristics; time, method and location of the suicides; and the presence of suicide indicators. Comparisons were made between the characteristics of the suicide patients and those of the current hospital population. Major findings were: 80 percent of the suicides were committed by psychotic patients and 66 percent were by patients with diagnoses which included paranoia; 48 percent of the suicide patients had been charged with or convicted of murder or attempted murder; most suicides occurred in individual patient rooms; nearly 50 percent of the patients had a history of suicide attempts; and other common indicators of suicide risk were often masked by psychotic symptomatology. Several recommendations for identifying and managing potentially suicidal patients in this population are presented.

Predominant site of flow limitation and mechanisms of postexertional asthma.

To determine if a relationship existed between the site of airway obstruction and the mechanisms of exercise-induced asthma, we studied the predominant site of flow limitation, as determined by the mid-vital capacity ratios of maximal expiratory flow with air (Vmax air) and 80% helium-20% oxygen (Vmax He-O2), before and after physical exertion in 12 asthmatics. These observations were then related to the effects seen after vagal blockade and inhibition of mediator release. Five subjects increased Vmax He-O2/Vmax air ratios suggesting that the predominant site of flow limitation was in large airways. This group had their postexercise bronchospasm abolished by pretreatment with an anticholinergic agent. Seven subjects decreased their flow ratios indicating predominant small airway obstruction. Anticholinergic agents, although producing bronchodilation, did not alter their bronchospastic response to exercise. However, pretreatment with disodium cromoglycate did significantly diminish the response of this group. Thus the airway response to exercise in asthmatics is heterogeneous in terms of predominant site of flow limitation and this factor appears to relate to mechanisms.

On line determination of lung volumes by plethysmography and digital computer.

This report describes a method of obtaining lung volumes on line with a minicomputer and whole body plethysmograph. The results of this approach were compared with standard methodology for a wide range of values and found to be in good agreement. This system can be readily implemented using standard hardware and laboratory techniques. Its advantages are its accuracy ,rapidity of data analysis, and retrieval; through the latter it allows for an assessment of such factors as variability of subject performance.

Comparison of arm versus leg work in induction of acute episodes of asthma.

The severity of exercise-induced asthma varies with the type of exercise performed. To determine whether such variation could be attributed to the use of different muscle groups, we exercised arms separately from legs using a bicycle ergometer. First, arms were exercised to exhaustion, then legs were exercised at the same load for the same duration. Arm work resulted in greater ventilation, heart rate, hydrogen ion concentration, and airway obstruction than did leg work. Later, legs were exercised to exhaustion using a load more than twice that of the arm work. Both the exhausting leg work and exhausting arm work resulted in significant bronchospasm and acidosis, whereas the nonexhausting leg work did not. These data suggest that, in arm and/or leg exercise, the relationship of work load to muscle mass is a determinant of airway obstruction.

An assessment of the pulmonary response to exercise in asthma and an analysis of the factors influencing it.

The purposes of this study were to determine (1) whether an exercise stimulus could be repeatedly applied to a group of asthmatics and normal control subjects with reproducible metabolic and ventilatory consequences; (2) the effect of this stimulus on multiple aspects of pulmonary mechanics in both groups; (3) the degree of within- and between-day variation in response and the factors influencing it; and (4) the effects of pretreatment with disodium cromoglycate. Airway resistance, specific conductance, total lung capacity and its subdivisions, and forced expiratory volumes and flow rates were measured in 21 asthmatics and 8 normal control subjects before and after treadmill exercise. Minutes ventilation, tidal volume, repiratory frequency, oxygen consumption, carbon dioxide production, heart rate, and end-tidal carbon dioxide tensions were measured during exercise and recovery. The asthmatics were studied twice datly on 2 separate days. Disodium cromoglycate was administered to the asthmatics before the fourth trial. The control subjects were studied twice on the same day without any interventions. There was no difference between exercise trials as measured by any of the gas exchange variables and there were no within-day differences in baseline pulmonary mechanics in either group. In contrast to the control group, all of the asthmatics had increasing airway obstruction after the exercise challenge. There were no between -day differences in the baseline data or response to exercise in the asthmatics except that the mechanical response was less after disodium cromoglycate, which suggests that mediator release played a part. Although as a group the stimulus and response were reproducible, when data of each trial were related to the type and degree of baseline dysfunction there was a direct relationship between pre-existing obstruction and magnitude of response. This suggests that exercise-induced asthma is not an all-or-none event, but rather a continuum of responses profoundly influenced by the pre-challenge state of the airways.

Effect of fish and poultry byproduct meal on the small intestine and gizzard of broilers.

Commercial mixed-sex broiler chicks were used in two experiments to study the effects of various levels of Peruvian fish meal (PFM), menhaden fish meal (MFM) and poulty byproduct meal (PBPM) on the small intestine and gizzard. Intestinal and gizzard lesions were observed in broilers receiving finisher diets containing 7% or 12% PFM. The broilers receiving diets containing 12% MFM or 12% PBPM had intestinal and gizzard lesion scores that were not significantly different from those of the control broilers. The severity of intestinal lesions was greater than that of gizzard lesions in most groups. The causative agent in the PFM has not been identified.