Induction of stress response proteins and experimental renal ischemia/reperfusion.

BACKGROUND: The induction of stress response (heat shock) proteins (HSPs) is a highly conserved response that protects many cell types from diverse physiological and environmental stressors. We tested the hypothesis that the induction of HSPs is protective in experimental renal ischemia/reperfusion injury. METHODS: The effect of prior heat stress was examined in a rat model of renal ischemia. Postischemic renal function, histopathology, myeloperoxidase activity, and mortality were determined in hyperthermia and sham hyperthermia groups. RESULTS: HSP84, HSP70, and HSP22 mRNA were increased after eight minutes but not four minutes of hyperthermia. The induction of HSP84 and HSP70 was blocked by pretreatment with quercetin. Improvement in renal function, mortality, and histologic abnormalities was seen with eight minutes of hyperthermia six hours before ischemia. Protection was dependent on the timing of ischemia relative to heat stress and was not observed when HSPs were not induced. Postischemic increases in renal myeloperoxidase activity were markedly attenuated in the hyperthermia compared with the sham hyperthermia group. CONCLUSION: Endogenous protective mechanisms may be important in renal ischemia/reperfusion injury.

Overexpression of SERCA2b in the heart leads to an increase in sarcoplasmic reticulum calcium transport function and increased cardiac contractility.

The sarcoplasmic reticulum calcium ATPase SERCA2b is an alternate isoform encoded by the SERCA2 gene. SERCA2b is expressed ubiquitously and has a higher Ca(2+) affinity compared with SERCA2a. We made transgenic mice that overexpress the rat SERCA2b cDNA in the heart. SERCA2b mRNA level was approximately approximately 20-fold higher than endogenous SERCA2b mRNA in transgenic hearts. SERCA2b protein was increased 8-10-fold in the heart, whereas SERCA2a mRNA/protein level remained unchanged. Confocal microscopy showed that SERCA2b is localized preferentially around the T-tubules of the SR, whereas SERCA2a isoform is distributed both transversely and longitudinally in the SR membrane. Calcium-dependent calcium uptake measurements showed that the maximal velocity of Ca(2+) uptake was not changed, but the apparent pump affinity for Ca(2+) (K(0.5)) was increased in SERCA2b transgenic mice (0.199 +/- 0.011 micrometer) compared with wild-type control mice (0.269 +/- 0.012 micrometer, p < 0.01). Work-performing heart preparations showed that SERCA2b transgenic hearts had a higher rates of contraction and relaxation, shorter time to peak pressure and half-time for relaxation than wild-type hearts. These data show that SERCA2b is associated in a subcompartment within the sarcoplasmic reticulum of cardiac myocytes. Overexpression of SERCA2b leads to an increase in SR calcium transport function and increased cardiac contractility, suggesting that SERCA2b plays a highly specialized role in regulating the beat-to-beat contraction of the heart.

Functional analysis of human FEN1 in Saccharomyces cerevisiae and its role in genome stability.

The flap endonuclease, FEN1, is an evolutionarily conserved component of DNA replication from archaebacteria to humans. Based on in vitro results, it processes Okazaki fragments during replication and is involved in base excision repair. FEN1 removes the last primer ribonucleotide on the lagging strand and it cleaves a 5' flap that may result from strand displacement during replication or during base excision repair. Its biological importance has been revealed largely through studies in the yeast Saccharomyces cerevisiae where deletion of the homologous gene RAD27 results in genome instability and mutagen sensitivity. While the in vivo function of Rad27 has been well characterized through genetic and biochemical approaches, little is understood about the in vivo functions of human FEN1. Guided by our recent results with yeast RAD27, we explored the function of human FEN1 in yeast. We found that the human FEN1 protein complements a yeast rad27 null mutant for a variety of defects including mutagen sensitivity, genetic instability and the synthetic lethal interactions of a rad27 rad51 and a rad27 pol3-01 mutant. Furthermore, a mutant form of FEN1 lacking nuclease function exhibits dominant-negative effects on cell growth and genome instability similar to those seen with the homologous yeast rad27 mutation. This genetic impact is stronger when the human and yeast PCNA-binding domains are exchanged. These data indicate that the human FEN1 and yeast Rad27 proteins act on the same substrate in vivo. Our study defines a sensitive yeast system for the identification and characterization of mutations in FEN1.

Pseudo-pseudoaneurysm of the left ventricle.

Left ventricular pseudoaneurysms are an uncommon complication of myocardial infarction and need urgent surgical repair. Though it is critical that they be accurately identified, pseudoaneurysms are occasionally misdiagnosed. We report an abnormality which may be mislabeled a pseudoaneurysm which we term a pseudo-pseudoaneurysm. The approach to accurate diagnosis of pseudoaneurysms is discussed.

Anti-CD3-activated killer T cells: Interleukin-6 modulates the induction of major histocompatibility complex-unrestricted cytotoxicity and the expression of genes coding for cytotoxic effector molecules.

We have investigated the role of interleukin-6 (IL-6) in the induction of major histocompatibility complex (MHC)-unrestricted cytotoxicity, as well as granzyme B, perforin, and Fas ligand gene expression, following mouse T lymphocyte activation with anti-CD3 monoclonal antibody (mAb). The generation of anti-CD3-activated killer-T (AK-T) cells was inhibited when anti-IL-6 neutralizing mAb was added at initiation of culture but not 24 h later, indicating that IL-6 is involved at an early stage of AK-T cell development. However, AK-T cell induction in the presence of exogenous IL-6 did not result in enhanced cytotoxicity, suggesting that saturating levels of IL-6 are normally synthesized in AK-T cell cultures. The inhibitory effect of IL-6 neutralization on AK-T cell generation could not be attributed to a defect in AK-T cell proliferation or to an inability of AK-T cells to recognize and adhere to P815 tumor target cells. However, IL-2 synthesis and CD25 expression were downregulated in AK-T cell cultures performed in the presence of anti-IL-6 mAb. In addition, IL-6 neutralization resulted in decreased expression of granzyme B and perforin, but not Fas ligand, mRNA. Exogenous IL-2 (50 U/ml) added at initiation of culture completely reversed the inhibitory effect of anti-IL-6 mAb on AK-T cell development, restoring CD25 expression and tumoricidal activity, as well as granzyme B and perforin mRNA expression, to control levels. We conclude that IL-6 modulates AK-T cell induction through an IL-2-dependent mechanism.

Animal caries models for evaluating fluoride dentifrices.

To determine the ability of animal caries models to evaluate the cariostatic potential of fluoride dentifrices, we examined four different coronal caries models. These currently used rat caries models were: Francis' hypomineralized area model, Gaffar's CARA rat model, the Connecticut model, and the Indiana model. Available data indicated that all of these models were capable of detecting significant cariostatic benefits from clinically proven fluoride dentifrices. For the most part, these animal models were also able to demonstrate a response to increasing concentrations of fluoride, particularly if the fluoride was provided in an ionic rather than a complexed form. Data were also presented which indicated the potential for animal caries models to be used to predict the clinical benefits of non-fluoride systems as well as the utility of fluoride systems for the prevention of root-surface caries. It is concluded that animal caries models play a significant role in the development and evaluation of fluoride and other systems for the clinical prevention of dental caries.

Regulation of endothelin 1 gene by fluid shear stress is transcriptionally mediated and independent of protein kinase C and cAMP.

Fluid shear stress induces a number of morphological and functional changes in vascular endothelium, including a rapid and significant down-regulation of endothelin 1 (ET-1) mRNA and peptide release in bovine aortic endothelial cells. We show here that both the cell alignment and ET-1 down-regulation depend on on-going protein synthesis, and that the latter is the result of a decrease in transcription, as shown by nuclear run-off assay, and not the result of changes in ET-1 mRNA half-life. The treatment of endothelial cells with either phorbol 12-myristate 13-acetate (100 nM) to activate protein kinase C (PKC) or forskolin (10 microM) to stimulate adenylate cyclase sharply decreased ET-1 mRNA. However, the phorbol-induced ET-1 decrease was, unlike the shear-induced down-regulation, independent of active protein synthesis. Physiological shear stress (20 dynes/cm2) did not significantly activate PKC, as assessed by PKC translocation and enzymatic activity assay and failed to increase intracellular cAMP content. Furthermore treatment with calphostin C (1 microM) did not prevent the shear-induced down-regulation of ET-1. DNA transfection experiments suggest that the shear stress-responsive element of the ET-1 gene is contained in the sequence between -2.5 kb and -2.9 kb of the 5'-upstream region. Neither the transcription factor AP-1 binding site nor the GATA-2-factor binding site, necessary for the basal level of transcription of ET-1 gene, is sufficient to confer shear-responsiveness to the reporter gene. These results suggest that shear stress regulates the transcription of the ET-1 gene via an upstream cis element by a distinct mechanism not dependent on the PKC or cAMP pathways.

Use of the Suicide Probability Scale (SPS) with adolescents.

This study examined the utility of the Suicide Probability Scale (SPS) by comparing results obtained with a sample of 217 normal adolescents (M = 16.2 years) to the findings obtained in Cull and Gill's (1988) standardization effort. The present sample scored significantly higher than Cull and Gill's normative sample in SPS item, subscale, and total scores. In addition, the SPS generally failed to discriminate between the present, adolescent sample and Cull and Gill's inpatient psychiatric sample. Moreover, item-subscale correlations obtained for the present sample often differed from those reported by Cull and Gill, and factor analysis of SPS item scores failed to replicate Cull and Gill's four-factor solution. Findings suggest the need for caution when using the SPS to assess adolescent suicide potential and underscore the need for additional research regarding the instrument's efficacy in distinguishing between normal and suicidal adolescents.

Dimensions of everyday memory in young adulthood.

This paper reports the findings of two studies on everyday memory in young adulthood. In Study 1, 387 male and female college students (18-22 years old) completed the 25-item Cognitive Failures Questionnaire (CFQ; Broadbent, Cooper, Fitzgerald & Parkes, 1982). Principal components analysis yielded five internally consistent factors: distractibility; misdirected actions; spatial/kinaesthetic memory; interpersonal intelligence; and memory for names. Further, each of these dimensions was interpretable within an information-processing framework. Study 2 examined the relation of the five everyday memory dimensions obtained in Study 1 to measures of working memory and traditional intelligence in a separate sample of 32 college students. Findings obtained in Study 2 suggest that attentional processes may be important components of the everyday memory construct.

"I've got a lot to do and i don't think i'll have the time": Gender differences in late adolescents' narratives of the future.

The present study examined gender differences in late adolescents' future narratives.Thirty-nine male and 43 female late adolescents (M=20.01 years) completed 90-minute individual interviews assessing dimensional and thematic aspects of the future narrative as well as psychological profile characteristics (abstract reasoning, psychological distress, self-concept, and self-esteem). As predicted, gender differences emerged in the anticipation and projected timing of adulthood transition events. More females than males anticipated marriage and parenthood; females also anticipated younger ages at marriage and parenthood than males. Examination of adolescents' narratives of the life course beyond the adulthood transition revealed greater extensionoverall among males than females. No gender differences in extension or densitywere obtained for the anticipated occupational domain, and no gender differences were obtained in extension in the family domain. Female adolescents, however, anticipated more events in the family domain than did males. The findings are discussed in terms of the implicit theories of adulthood that inform adolescents' future narratives.

Cytoarchitectural relationships between [3H]ouabain binding and mRNA for isoforms of the sodium pump catalytic subunit in rat brain.

We examined the cell type-specific expression of the alpha 1, alpha 2, and alpha 3 subunits of the sodium pump in rat brain using in situ hybridization and [3H]ouabain autoradiography. These techniques allowed us to colocalize mRNA and functional alpha 2/alpha 3 pumps on adjacent sections. The perikarya of many neurons possessed high levels of alpha 1 and/or alpha 3 transcripts, while alpha 2 mRNA appeared to be present in only a few neuronal types. [3H]Ouabain binding in general paralleled the distribution of alpha 3 mRNA-positive neurons. The regional variation of alpha 1 and alpha 3 transcripts was complex and varied. Large neurons of the olfactory bulb and piriform cortex expressed high levels of alpha 3 transcripts, but low levels of alpha 1 mRNA. In frontal cortex, neurons of layers II-III were enriched in alpha 1 mRNA, while those in layer V exhibited high levels of alpha 3 transcripts. In the hippocampus, principal neurons expressed all three alpha subunit mRNAs. CA subfield pyramidal neurons exhibited a high alpha 3/alpha 1 ratio, while dentate granule cells and hilar pyramidal neurons expressed approximately equal levels of alpha 1 and alpha 3. In the cerebellum, Purkinje and Golgi cells were rich in alpha 3 mRNA, while the granule cells appeared to express only alpha 1 transcripts. The distribution of functional sodium pump protein, as localized by [3H]ouabain binding, was highest in the neuropil of the hippocampus and cerebral cortex, and lowest over perikarya and white matter. [3H]ouabain did not bind to alpha 1 pump units, as confirmed by the complete absence of labeling over the choroid plexus, a tissue expressing only alpha 1 mRNA. In the cerebellum, regions of dense [3H]ouabain binding were localized to the granule cell layer, the inner third of the molecular layer in the basket region, and the deep cerebellar nuclei. Surprisingly, the dense neuropil in the outer 2/3 of the molecular layer lacked high [3H]ouabain binding. Thus, functional alpha 3 sodium pump units appear distributed to the axon terminals and not to apical dendrites of Purkinje, Golgi and basket cells. A similar pattern of increased [3H]ouabain binding in axonal but not dendritic fields of alpha 3-enriched neurons was present in the cerebral cortex and the hippocampus. Considering that many alpha 3-enriched neurons are of the Golgi I type with long axons, the alpha 3 isoform may be preferentially directed into axons to function in presynaptic membranes.

Patterns of affectivity in the transition to adolescence.

The present paper reports the findings of a factor analytic investigation of adolescent affect in a cross-sectional sample of 483 male and female fifth through ninth graders. Following the ESM method, adolescents carried electronic pagers and self-report booklets for one week. Students were paged seven times daily and completed self-report forms after each signal describing their moods and feelings. Principal components analysis yielded two internally consistent factors which were virtually identical to the positive and negative affect dimensions described in the literature on adult emotion. Contrary to findings reported in the developmental literature, arousal did not emerge as a dimension of adolescent affect. Further, the bipolar structure obtained for the entire sample, positive and negative affect, consistently emerged in separate analyses of adolescent gender and school groups. MANOVA analysis of estimated positive and negative affect scores indicated that while the underlying dimensions of adolescent affect were comparable to those observed for adults, variation along those dimensions may have been related to the social transitions (e.g., schooling) which punctuate adolescence.

Great expectations: Constructions of the life course during adolescence.

Despite numerous demographic analyses of the sequencing of life events during the transition to adulthood (Kett, 1977; Modell et al., 1976), little is known of the psychological expectancies that adolescents bring to this and other aspects of the life course experience. This research was conducted to determine whether the futures anticipated by high school and college students correspond to the demographic trends previously observed for comparable age groups (i.e., projected vs. actual age at marriage). The findings indicate that, with increasing age, adolescents acquire a shared life course perspective that is at once richer and more differentiated than that implied by previous demographic investigations. This shared life course perspective was manifest in two ways: (1) increased agreement or concordance in the types of future events anticipated, and (2) increased variability in the ages at which future events were expected to occur. These findings underscore the need to complement demographic analyses of the life course with psychological investigations of the expectancies that adolescents bring to the adulthood transition.

Expression of multiple Na+,K+-adenosine triphosphatase isoform genes in human hematopoietic cells. Behavior of the novel A3 isoform during induced maturation of HL60 cells.

Multiple isoenzymes of the Na+,K+-ATPase (alpha, alpha+, and alpha 3) have been identified by molecular cloning (Shull, G. E., J. Greeb, and J. B. Lingrel. 1986. Biochemistry. 25:8125-8132; and Schneider, J. W., R. W. Mercer, and E. J. Benz, Jr. 1987. Clin. Res. 35:585A. [Abstr.]). At least one of these, the alpha 3 chain, represents a novel form for which protein products and enzymatic activities are just beginning to be defined in rodents. We have recently demonstrated that expression of alpha 3 is largely confined to neuromuscular tissues of fetal and adult rats (Schneider, J. W., R. W. Mercer, M. Gilmore-Hebert, M. F. Utset, C. Lai, A. Greene, and E. J. Benz, Jr. 1988. Proc. Natl. Acad. Sci. USA. 85:284-288). We now report that certain human leukemia cell lines including HL60, HEL, and Molt 4 express mRNA for both alpha and alpha 3 isoforms of Na+,K+-ATPase; mRNA was not detected in several other cell lines, including K562 and U937; no cell lines expressed alpha+ mRNA. In uninduced HL60 cells, alpha 3 mRNA comprised 20-30% of total Na+,K+-ATPase mRNA. Furthermore, in HL60 and HEL cells, both alpha and alpha 3 mRNA declined after induction of maturation by DMSO, retinoic acid, or hemin. However, the reduction in alpha 3 mRNA was far more dramatic. alpha 3 mRNA virtually disappeared, but alpha mRNA declined by only approximately 50%. In contrast, when maturation of HL60 cells along the monocyte/macrophage lineage was induced by exposure to phorbol esters, alpha 3 mRNA remained abundant. Moreover, mRNA for the beta subunit of the Na+,K+-ATPase increased dramatically. Our results demonstrate that the alpha 3 isoform, formerly thought to be confined to neuromuscular tissues, is expressed in restricted lineages of hematopoietic origin. These leukemia cell lines should provide a useful model for analyzing regulation of the alpha 3 isoform gene and characterization of alpha 3 isoform activities.

Future-time perspective in adolescence: The present of things future revisited.

Several theorists have suggested that the observed changes in adolescent future-time perspective are due to the emergence of formal-operations reasoning [e. g., T. J. Cottle and S. Klineberg (1974),The Present of Things Future, Free Press-Macmillan, New York; P. Fraisse (1963),The Psychology of Time, Harper & Row, New York; H. Hartmann (1958),Ego Psychology and the Problem of Adaptation, International Universities Press, New York; J. Piaget (1968),Six Psychological Studies, Vintage Book, New York]. Using a cross-sectional sample of 60 Caucasian adolescents, the present study was designed to examine this hypostatized interrelation. Data obtained through individual interviews provide only limited support for a cognitive hypothesis. As predicted, older students showed greater future extension and the more cognitively advanced students proved better able to project a set of events into the distant future. However, neither the older, nor the more cognitively advanced, students projected a greater number or a more consistent set of future events than did their respective counterparts. Moreover, analysis of the types of events projected obtained significance only for grade level. The findings are discussed from a contextualist perspective, within which consideration is given to the influence of experiential and life-span status factors.