Absence of opioid stress-induced analgesia in mice lacking beta-endorphin by site-directed mutagenesis.

A physiological role for beta-endorphin in endogenous pain inhibition was investigated by targeted mutagenesis of the proopiomelanocortin gene in mouse embryonic stem cells. The tyrosine codon at position 179 of the proopiomelanocortin gene was converted to a premature translational stop codon. The resulting transgenic mice display no overt developmental or behavioral alterations and have a normally functioning hypothalamic-pituitary-adrenal axis. Homozygous transgenic mice with a selective deficiency of beta-endorphin exhibit normal analgesia in response to morphine, indicating the presence of functional mu-opiate receptors. However, these mice lack the opioid (naloxone reversible) analgesia induced by mild swim stress. Mutant mice also display significantly greater nonopioid analgesia in response to cold water swim stress compared with controls and display paradoxical naloxone-induced analgesia. These changes may reflect compensatory upregulation of alternative pain inhibitory mechanisms.

Adenylyl cyclase inhibition and altered G protein subunit expression and ADP-ribosylation patterns in tissues and cells from Gi2 alpha-/-mice.

The inhibition of alpha i2-/- mouse cardiac isoproterenol-stimulated adenylyl cyclase (AC; EC 4.6.1.1) activity by carbachol and that of alpha i2-/- adipocyte AC by phenylisopropyladenosine (PIA), prostaglandin E2, and nicotinic acid were partially, but not completely, inhibited. While the inhibition of cardiac AC was affected in all alpha i2-/- animals tested, only 50% of the alpha i2-/- animals showed an impaired inhibition of adipocyte AC, indicative of a partial penetrance of this phenotype. In agreement with previous results, the data show that Gi2 mediates hormonal inhibition of AC and that Gi3 and/or Gi1 is capable of doing the same but with a lower efficacy. Disruption of the alpha i2 gene affected about equally the actions of all the receptors studied, indicating that none of them exhibits a striking specificity for one type of Gi over another and that receptors are likely to he selective rather than specific in their interaction with functionally homologous G proteins (e.g., Gi1, Gi2, Gi3). Western analysis of G protein subunit levels in simian virus 40-transformed primary embryonic fibroblasts from alpha i2+/+ and alpha i2-/- animals showed that alpha i2 accounts for about 50% of the immunopositive G protein alpha subunits and that loss of the alpha i2 is accompanied by a parallel reduction in G beta 35 and G beta 36 subunits and by a 30-50% increase in alpha i3. This suggests that G beta-gamma levels may be regulated passively through differential rates of turnover in their free vs. trimeric states. The existence of compensatory increase(s) in alpha i subunit expression raises the possibility that the lack of effect of a missing alpha i2 on AC inhibition in adipocytes of some alpha i2-/- animals may be the reflection of a more pronounced compensatory expression of alpha i3 and/or alpha i1.

Accelerated evolution and Muller's rachet in endosymbiotic bacteria.

Many bacteria live only within animal cells and infect hosts through cytoplasmic inheritance. These endosymbiotic lineages show distinctive population structure, with small population size and effectively no recombination. As a result, endosymbionts are expected to accumulate mildly deleterious mutations. If these constitute a substantial proportion of new mutations, endosymbionts will show (i) faster sequence evolution and (ii) a possible shift in base composition reflecting mutational bias. Analyses of 16S rDNA of five independently derived endosymbiont clades show, in every case, faster evolution in endosymbionts than in free-living relatives. For aphid endosymbionts (genus Buchnera), coding genes exhibit accelerated evolution and unusually low ratios of synonymous to nonsynonymous substitutions compared to ratios for the same genes for enterics. This concentration of the rate increase in nonsynonymous substitutions is expected under the hypothesis of increased fixation of deleterious mutations. Polypeptides for all Buchnera genes analyzed have accumulated amino acids with codon families rich in A+T, supporting the hypothesis that substitutions are deleterious in terms of polypeptide function. These observations are best explained as the result of Muller's ratchet within small asexual populations, combined with mutational bias. In light of this explanation, two observations reported earlier for Buchnera, the apparent loss of a repair gene and the overproduction of a chaperonin, may reflect compensatory evolution. An alternative hypothesis, involving selection on genomic base composition, is contradicted by the observation that the speedup is concentrated at nonsynonymous sites.

Preserved speech abilities and compensation following prefrontal damage.

Lesions to left frontal cortex in humans produce speech production impairments (nonfluent aphasia). These impairments vary from subject to subject and performance on certain speech production tasks can be relatively preserved in some patients. A possible explanation for preservation of function under these circumstances is that areas outside left prefrontal cortex are used to compensate for the injured brain area. We report here a direct demonstration of preserved language function in a stroke patient (LF1) apparently due to the activation of a compensatory brain pathway. We used functional brain imaging with positron emission tomography (PET) as a basis for this study.

Muller's ratchet decreases fitness of a DNA-based microbe.

Muller proposed that an asexual organism will inevitably accumulate deleterious mutations, resulting in an increase of the mutational load and an inexorable, ratchet-like, loss of the least mutated class [Muller, H.J. (1964) Mutat. Res. 1, 2-9]. The operation of Muller's ratchet on real populations has been experimentally demonstrated only in RNA viruses. However, these cases are exceptional in that the mutation rates of the RNA viruses are extremely high. We have examined whether Muller's ratchet operates in Salmonella typhimurium, a DNA-based organism with a more typical genomic mutation rate. Cells were grown asexually under conditions expected to result in high genetic drift, and the increase in mutational load was determined. S. typhimurium accumulated mutations under these conditions such that after 1700 generations, 1% of the 444 lineages tested had suffered an obvious loss of fitness, as determined by decreased growth rate. These results suggest that in the absence of sex and with high genetic drift, genetic mechanisms, such as back or compensatory mutations, cannot compensate for the accumulation of deleterious mutations. In addition, we measured the appearance of auxotrophs, which allowed us to calculate an average spontaneous mutation rate of approximately 0.3-1.5 x 10(-9) mutations per base pair per generation. This rate is measured for the largest genetic target studied so far, a collection of about 200 genes.

Ovulation efficiency is reduced in mice that lack plasminogen activator gene function: functional redundancy among physiological plasminogen activators.

Several lines of indirect evidence suggest that plasminogen activation plays a crucial role in degradation of the follicular wall during ovulation. However, single-deficient mice lacking tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), or PA inhibitor type 1(PAI-1) gene function were recently found to have normal reproduction, although mice with a combined deficiency of tPA and uPA were significantly less fertile. To investigate whether the reduced fertility of mice lacking PA gene function is due to a reduced ovulation mechanism, we have determined the ovulation efficiency in 25-day-old mice during gonadotropin-induced ovulation. Our results reveal that ovulation efficiency is normal in mice with a single deficiency of tPA or uPA but reduced by 26% in mice lacking both physiological PAs. This result suggests that plasminogen activation plays a role in ovulatory response, although neither tPA nor uPA individually or in combination is obligatory for ovulation. The loss of an individual PA seems to be functionally complemented by the remaining PA but this compensation does not appear to involve any compensatory up-regulation. Our data imply that a functionally redundant mechanism for plasmin formation operates during gonadotropin-induced ovulation and that PAs together with other proteases generate the proteolytic activity required for follicular wall degradation.

Maintenance of pre-mRNA secondary structure by epistatic selection.

Linkage disequilibrium between polymorphisms in a natural population may result from various evolutionary forces, including random genetic drift due to sampling of gametes during reproduction, restricted migration between subpopulations in a subdivided population, or epistatic selection. In this report, we present evidence that the majority of significant linkage disequilibria observed in introns of the alcohol dehydrogenase locus (Adh) of Drosophila pseudoobscura are due to epistatic selection maintaining secondary structure of precursor mRNA (pre-mRNA). Based on phylogenetic-comparative analysis and a likelihood approach, we propose secondary structure models of Adh pre-mRNA for the regions of the adult intron and intron 2 where clustering of linkage disequilibria has been observed. Furthermore, we applied the likelihood ratio test to the phylogenetically predicted secondary structure in intron 1. In contrast to the other two structures, polymorphisms associated with the more conserved stem-loop structure of intron 1 are in low frequency, and linkage disequilibria have not been observed. These findings are qualitatively consistent with a model of compensatory fitness interactions. This model assumes that mutations disrupting pairing in a secondary structural element are individually deleterious if they destabilize a functionally important structure; a second "compensatory" mutation, however, may restabilize the structure and restore fitness.

Erythropoiesis and globin gene expression in mice lacking the transcription factor NF-E2.

Previous studies in transgenic mice and cultured cells have indicated that the major enhancer function for erythroid cell expression of the globin genes is provided by the heterodimeric basic-leucine zipper transcription factor NF-E2. Globin gene expression within cultured mouse erythroleukemia cells is highly dependent on NF-E2. To examine the requirement for this factor in vivo, we used homologous recombination in embryonic stem cells to generate mice lacking the hematopoietic-specific subunit, p45 NF-E2. The most dramatic aspect of the homozygous mutant mice was an absence of circulating platelets, which led to the death of most animals due to hemorrhage. In contrast, the effect of loss of NF-E2 on the erythroid lineage was surprisingly mild. Although neonates exhibited severe anemia and dysmorphic red-cell changes, probably compounded by concomitant bleeding, surviving adults exhibited only mild changes consistent with a small decrease in the hemoglobin content per cell. p45 NF-E2-null mice responded to anemia with compensatory reticulocytosis and splenomegaly. Globin chain synthesis was balanced, and switching from fetal to adult globins progressed normally. Although these findings are consistent with the substitution of NF-E2 function in vivo by one or more compensating proteins, gel shift assays using nuclear extracts from p45 NF-E2-null mice failed to reveal novel complexes formed on an NF-E2 binding site. Thus, regulation of globin gene transcription through NF-E2 binding sites in vivo is more complex than has been previously appreciated.

Hippocampal long-term depression and depotentiation are defective in mice carrying a targeted disruption of the gene encoding the RI beta subunit of cAMP-dependent protein kinase.

The cAMP-dependent protein kinase (PKA) has been shown to play an important role in long-term potentiation (LTP) in the hippocampus, but little is known about the function of PKA in long-term depression (LTD). We have combined pharmacologic and genetic approaches to demonstrate that PKA activity is required for both homosynaptic LTD and depotentiation and that a specific neuronal isoform of type I regulatory subunit (RI beta) is essential. Mice carrying a null mutation in the gene encoding RI beta were established by use of gene targeting in embryonic stem cells. Hippocampal slices from mutant mice show a severe deficit in LTD and depotentiation at the Schaffer collateral-CA1 synapse. This defect is also evident at the lateral perforant path-dentate granule cell synapse in RI beta mutant mice. Despite a compensatory increase in the related RI alpha protein and a lack of detectable changes in total PKA activity, the hippocampal function in these mice is not rescued, suggesting a unique role for RI beta. Since the late phase of CA1 LTP also requires PKA but is normal in RI beta mutant mice, our data further suggest that different forms of synaptic plasticity are likely to employ different combinations of regulatory and catalytic subunits.

Cerebrovascular alterations in mice lacking neuronal nitric oxide synthase gene expression.

Nitric oxide (NO) is known to mediate increases in regional cerebral blood flow elicited by CO2 inhalation. In mice with deletion of the gene for neuronal NO synthase (NOS), CO2 inhalation augments cerebral blood flow to the same extent as in wild-type mice. However, unlike wild-type mice, the increased flow in mutants is not blocked by the NOS inhibition, N omega-nitro-L-arginine, and CO2 exposure fails to increase brain levels of cGMP. Topical acetylcholine elicits vasodilation in the mutants which is blocked by N omega-nitro-L-arginine, indicating normal functioning of endothelial NOS. Moreover, immunohistochemical staining for endothelial NOS is normal in the mutants. Thus, following loss of neuronal NOS, the cerebral circulatory response is maintained by a compensatory system not involving NO.

Mice lacking extracellular superoxide dismutase are more sensitive to hyperoxia.

Extracellular superoxide dismutase (EC-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) is a secreted Cu- and Zn-containing tetrameric glycoprotein, the bulk of which is bound to heparan sulfate proteoglycans in the interstitium of tissues. To test the function of EC-SOD in vivo, mice carrying a targeted disruption of the EC-SOD gene were generated. The EC-SOD null mutant mice develop normally and remain healthy until at least 14 months of age. No compensatory induction of other SOD isoenzymes or other antioxidant enzymes was observed. When stressed by exposure to > 99% oxygen, the EC-SOD null mutant mice display a considerable reduction in survival time compared to wild-type mice and an earlier onset of severe lung edema. These findings suggest that while under normal physiological conditions other antioxidant systems may substitute for the loss of EC-SOD; when the animal is stressed these systems are unable to provide adequate protection.

Identification of human brain regions underlying responses to resistive inspiratory loading with functional magnetic resonance imaging.

Compensatory ventilatory responses to increased inspiratory loading are essential for adequate breathing regulation in a number of pulmonary diseases; however, the human brain sites mediating such responses are unknown. Midsagittal and axial images were acquired in 11 healthy volunteers during unloaded and loaded (30 cmH2O; 1 cmH2O = 98 Pa) inspiratory breathing, by using functional magnetic resonance imaging (fMRI) strategies (1.5-tesla MR; repetition time, 72 msec; echo time, 45 msec; flip angle, 30 degrees; field of view, 26 cm; slice thickness, 5 mm; number of excitations, 1; matrix, 128 x 256). Digital image subtractions and region of interest analyses revealed significantly increased fMRI signal intensity in discrete areas of the ventral and dorsal pons, interpeduncular nucleus, basal forebrain, putamen, and cerebellar regions. Upon load withdrawal, certain regions displayed a rapid fMRI signal off-transient, while in others, a slower fMRI signal decay emerged. Sustained loading elicited slow decreases in fMRI signal across activated regions, while second application of an identical load resulted in smaller signal increases compared to initial signal responses (P < 0.001). A moderate inspiratory load is associated with consistent regional activation of discrete brain locations; certain of these regions have been implicated in mediation of loaded breathing in animal models. We speculate that temporal changes in fMRI signal may indicate respiratory after-discharge and/or habituation phenomena.

Organismal duplication, inclusive fitness theory, and altruism: understanding the evolution of endosperm and the angiosperm reproductive syndrome.

For almost a century, events relating to the evolutionary origin of endosperm, a unique embryo-nourishing tissue that is essential to the reproductive process in flowering plants, have remained a mystery. Integration of recent advances in phylogenetic reconstruction, comparative reproductive biology, and genetic theory can be used to elucidate the evolutionary events and forces associated with the establishment of endosperm. Endosperm is shown to be derived from one of two embryos formed during a rudimentary process of "double fertilization" that evolved in the ancestors of angiosperms. Acquisition of embryo-nourishing behavior (with accompanying loss of individual fitness) by this supernumerary fertilization product was dependent upon compensatory gains in the inclusive fitness of related embryos. The result of the loss of individual fitness by one of the two original products of double fertilization was the establishment of endosperm, a highly modified embryo/organism that reproduces cryptically through behavior that enhances the fitness of its associated embryo within a seed. Finally, although triploid endosperm remains a synapomorphy of angiosperms, inclusive fitness analysis demonstrates that the embryo-nourishing properties of endosperm initially evolved in a diploid condition.

Male/Female Manifestations of Narcissistic Personality and Behavioral Disorders.

Specifically, this paper will address the following topics : 1. The history of psychoanalytic thinking onnarcissism will be discussed, leading up to more recent ideas on the narcissistic personality disorder. 2. Drawing on historical and current ideas, an integrated definition of the narcissistic personality disorder will be presented and elaborated upon, including an examination of differing male/female narcissistic compensatory styles. 3. To foster an understanding of the development of narcissistic defenses and of differing gender styles of defense, various theories relating to gender differences in the narcissistic personality disorder will be explored: self/object relations theories, Kohut's theory on narcissism, psychosexual development theories, behavioral manifestation theories, and bodily development theories.4. The role of preoedipal development, as it relates to the formation of the male/female narcissistic personality disorder, will be examined. This section of the paper will propose that, in the narcissistic personality disorder, a pathological arrest occurred during the second or third year of life in response to trauma experienced at that time. The degree and timing of the trauma and the degree of structuralization preceding the trauma all contribute to the rigidity of the narcissistic disorder and the severity of the pathology. 5. The role of oedipal development in the male/female narcissistic personality disorder will be discussed. This discussion will address the intrapsychic configurations which arise during the oedipal period, after narcissistic defenses have solidified during the preoedipal years. While narcissism can be seen as a developmental line, with narcissistic defenses arising at any time during development, this paper will focus primarily on defenses which arise during the separation/individuation phases of development.

El rol del conocimiento y de las habilidades intelectuales generales en la adquisición del aprendizaje complejo

El objetivo principal del presente trabajo es examinar la relación existente entre la habilidad intelectual y la habilidad de organización del conocimiento dentro de un modelo más general sobre los factores explicativos de la adquisición de conocimientos y habilidades. Se trata de comprobar si se produce un efecto compensatorio entre la habilidad intelectual general y la habilidad para organizar el conocimiento, así como establecer si el aprendizaje de los sujetos que obtienen un mayor conocimiento en un dominio particular viene predicho/explicado por las mismas variables que explican el aprendizaje de los sujetos que obtienen un menor conocimiento. Los resultados, en una muestra de estudiantes universitarios, indican que no se produce un efecto de interacción entre ambos factores, sino más bien un efecto aditivo. No obstante, sí se observa que el efecto de la inteligencia no es el mismo en los distintos niveles de conocimiento adquirido.