Identification of partial loss of function p53 gene mutations utilizing a yeast-based functional assay

Missense mutations within the central DNA binding region of p53 are the most prevalent mutations found in human cancer. Numerous studies indicate that ‘hot-spot’ p53 mutants (which comprise ∼30% of human p53 gene mutations) are largely devoid of transcriptional activity. However, a growing body of evidence indicates that some non-hot-spot p53 mutants retain some degree of transcriptional activity in vivo, particularly against strong p53 binding sites. We have modified a previously described yeast-based p53 functional assay to readily identify such partial loss of function p53 mutants. We demonstrate the utility of this modified p53 functional assay using a diverse panel of p53 mutants.

Adeno-associated viral vector-mediated gene transfer results in long-term enzymatic and functional correction in multiple organs of Fabry mice

Fabry disease is a lysosomal storage disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-gal A). This enzyme deficiency leads to impaired catabolism of α-galactosyl-terminal lipids such as globotriaosylceramide (Gb3). Patients develop painful neuropathy and vascular occlusions that progressively lead to cardiovascular, cerebrovascular, and renal dysfunction and early death. Although enzyme replacement therapy and bone marrow transplantation have shown promise in the murine analog of Fabry disease, gene therapy holds a strong potential for treating this disease in humans. Delivery of the normal α-gal A gene (cDNA) into a depot organ such as liver may be sufficient to elicit corrective circulating levels of the deficient enzyme. To investigate this possibility, a recombinant adeno-associated viral vector encoding human α-gal A (rAAV-AGA) was constructed and injected into the hepatic portal vein of Fabry mice. Two weeks postinjection, α-gal A activity in the livers of rAAV-AGA-injected Fabry mice was 20–35% of that of the normal mice. The transduced animals continued to show higher α-gal A levels in liver and other tissues compared with the untouched Fabry controls as long as 6 months after treatment. In parallel to the elevated enzyme levels, we see significant reductions in Gb3 levels to near normal at 2 and 5 weeks posttreatment. The lower Gb3 levels continued in liver, spleen, and heart, up to 25 weeks with no significant immune response to the virus or α-gal A. Also, no signs of liver toxicity occurred after the rAAV-AGA administration. These findings suggest that an AAV-mediated gene transfer may be useful for the treatment of Fabry disease and possibly other metabolic disorders.

Neural fate of seen and unseen faces in visuospatial neglect: A combined event-related functional MRI and event-related potential study

To compare neural activity produced by visual events that escape or reach conscious awareness, we used event-related MRI and evoked potentials in a patient who had neglect and extinction after focal right parietal damage, but intact visual fields. This neurological disorder entails a loss of awareness for stimuli in the field contralateral to a brain lesion when stimuli are simultaneously presented on the ipsilateral side, even though early visual areas may be intact, and single contralateral stimuli may still be perceived. Functional MRI and event-related potential study were performed during a task where faces or shapes appeared in the right, left, or both fields. Unilateral stimuli produced normal responses in V1 and extrastriate areas. In bilateral events, left faces that were not perceived still activated right V1 and inferior temporal cortex and evoked nonsignificantly reduced N1 potentials, with preserved face-specific negative potentials at 170 ms. When left faces were perceived, the same stimuli produced greater activity in a distributed network of areas including right V1 and cuneus, bilateral fusiform gyri, and left parietal cortex. Also, effective connectivity between visual, parietal, and frontal areas increased during perception of faces. These results suggest that activity can occur in V1 and ventral temporal cortex without awareness, whereas coupling with dorsal parietal and frontal areas may be critical for such activity to afford conscious perception.

Cyclic AMP Increases Cell Surface Expression of Functional Na,K-ATPase Units in Mammalian Cortical Collecting Duct Principal Cells

Cyclic AMP (cAMP) stimulates the transport of Na+ and Na,K-ATPase activity in the renal cortical collecting duct (CCD). The aim of this study was to investigate the mechanism whereby cAMP stimulates the Na,K-ATPase activity in microdissected rat CCDs and cultured mouse mpkCCDc14 collecting duct cells. db-cAMP (10−3 M) stimulated by 2-fold the activity of Na,K-ATPase from rat CCDs as well as the ouabain-sensitive component of 86Rb+ uptake by rat CCDs (1.7-fold) and cultured mouse CCD cells (1.5-fold). Pretreatment of rat CCDs with saponin increased the total Na,K-ATPase activity without further stimulation by db-cAMP. Western blotting performed after a biotinylation procedure revealed that db-cAMP increased the amount of Na,K-ATPase at the cell surface in both intact rat CCDs (1.7-fold) and cultured cells (1.3-fold), and that this increase was not related to changes in Na,K-ATPase internalization. Brefeldin A and low temperature (20°C) prevented both the db-cAMP-dependent increase in cell surface expression and activity of Na,K-ATPase in both intact rat CCDs and cultured cells. Pretreatment with the intracellular Ca2+ chelator bis-(o-aminophenoxy)-N,N,N′,N′-tetraacetic acid also blunted the increment in cell surface expression and activity of Na,K-ATPase caused by db-cAMP. In conclusion, these results strongly suggest that the cAMP-dependent stimulation of Na,K-ATPase activity in CCD results from the translocation of active pump units from an intracellular compartment to the plasma membrane.

Medial prefrontal cortex and self-referential mental activity: Relation to a default mode of brain function

Medial prefrontal cortex (MPFC) is among those brain regions having the highest baseline metabolic activity at rest and one that exhibits decreases from this baseline across a wide variety of goal-directed behaviors in functional imaging studies. This high metabolic rate and this behavior suggest the existence of an organized mode of default brain function, elements of which may be either attenuated or enhanced. Extant data suggest that these MPFC regions may contribute to the neural instantiation of aspects of the multifaceted “self.” We explore this important concept by targeting and manipulating elements of MPFC default state activity. In this functional magnetic resonance imaging (fMRI) study, subjects made two judgments, one self-referential, the other not, in response to affectively normed pictures: pleasant vs. unpleasant (an internally cued condition, ICC) and indoors vs. outdoors (an externally cued condition, ECC). The ICC was preferentially associated with activity increases along the dorsal MPFC. These increases were accompanied by decreases in both active task conditions in ventral MPFC. These results support the view that dorsal and ventral MPFC are differentially influenced by attentiondemanding tasks and explicitly self-referential tasks. The presence of self-referential mental activity appears to be associated with increases from the baseline in dorsal MPFC. Reductions in ventral MPFC occurred consistent with the fact that attention-demanding tasks attenuate emotional processing. We posit that both self-referential mental activity and emotional processing represent elements of the default state as represented by activity in MPFC. We suggest that a useful way to explore the neurobiology of the self is to explore the nature of default state activity.

Characterization of uracil-DNA glycosylase activity from Trypanosoma cruzi and its stimulation by AP endonuclease

The intracellular pathogen Trypanosoma cruzi is the etiological agent of Chagas’ disease. We have isolated a full-length cDNA encoding uracil-DNA glycosylase (UDGase), a key enzyme involved in DNA repair, from this organism. The deduced protein sequence is highly conserved at the C-terminus of the molecule and shares key residues involved in binding or catalysis with most of the UDGases described so far, while the N-terminal part is highly variable. The gene is single copy and is located on a chromosome of ∼1.9 Mb. A His-tagged recombinant protein was overexpressed, purified and used to raise polyclonal antibodies. Western blot analysis revealed the existence of a single UDGase species in parasite extracts. Using a specific ethidium bromide fluorescence assay, recombinant T.cruzi UDGase was shown to specifically excise uracil from DNA. The addition of both Leishmania major AP endonuclease and exonuclease III, the major AP endonuclease from Escherichia coli, produces stimulation of UDGase activity. This activation is specific for AP endonuclease and suggests functional communication between the two enzymes.

Nucleotide excision repair in rat male germ cells: low level of repair in intact cells contrasts with high dual incision activity in vitro

The acquisition of genotoxin-induced mutations in the mammalian germline is detrimental to the stable transfer of genomic information. In somatic cells, nucleotide excision repair (NER) is a major pathway to counteract the mutagenic effects of DNA damage. Two NER subpathways have been identified, global genome repair (GGR) and transcription-coupled repair (TCR). In contrast to somatic cells, little is known regarding the expression of these pathways in germ cells. To address this basic question, we have studied NER in rat spermatogenic cells in crude cell suspension, in enriched cell stages and within seminiferous tubules after exposure to UV or N-acetoxy-2-acetylaminofluorene. Surprisingly, repair in spermatogenic cells was inefficient in the genome overall and in transcriptionally active genes indicating non-functional GGR and TCR. In contrast, extracts from early/mid pachytene cells displayed dual incision activity in vitro as high as extracts from somatic cells, demonstrating that the proteins involved in incision are present and functional in premeiotic cells. However, incision activities of extracts from diplotene cells and round spermatids were low, indicating a stage-dependent expression of incision activity. We hypothesize that sequestering of NER proteins by mispaired regions in DNA involved in synapsis and recombination may underlie the lack of NER activity in premeiotic cells.

Biomechanical activation of vascular endothelium as a determinant of its functional phenotype

One of the striking features of vascular endothelium, the single-cell-thick lining of the cardiovascular system, is its phenotypic plasticity. Various pathophysiologic factors, such as cytokines, growth factors, hormones, and metabolic products, can modulate its functional phenotype in health and disease. In addition to these humoral stimuli, endothelial cells respond to their biomechanical environment, although the functional implications of this biomechanical paradigm of activation have not been fully explored. Here we describe a high-throughput genomic analysis of modulation of gene expression observed in cultured human endothelial cells exposed to two well defined biomechanical stimuli—a steady laminar shear stress and a turbulent shear stress of equivalent spatial and temporal average intensity. Comparison of the transcriptional activity of 11,397 unique genes revealed distinctive patterns of up- and down-regulation associated with each type of stimulus. Cluster analyses of transcriptional profiling data were coupled with other molecular and cell biological techniques to examine whether these global patterns of biomechanical activation are translated into distinct functional phenotypes. Confocal immunofluorescence microscopy of structural and contractile proteins revealed the formation of a complex apical cytoskeleton in response to laminar shear stress. Cell cycle analysis documented different effects of laminar and turbulent shear stresses on cell proliferation. Thus, endothelial cells have the capacity to discriminate among specific biomechanical forces and to translate these input stimuli into distinctive phenotypes. The demonstration that hemodynamically derived stimuli can be strong modulators of endothelial gene expression has important implications for our understanding of the mechanisms of vascular homeostasis and atherogenesis.

Ca2+-binding activity of a COOH-terminal fragment of the Drosophila BK channel involved in Ca2+-dependent activation

Mutational and biophysical analysis suggests that an intracellular COOH-terminal domain of the large conductance Ca2+-activated K+ channel (BK channel) contains Ca2+-binding site(s) that are allosterically coupled to channel opening. However the structural basis of Ca2+ binding to BK channels is unknown. To pursue this question, we overexpressed the COOH-terminal 280 residues of the Drosophila slowpoke BK channel (Dslo-C280) as a FLAG- and His6-tagged protein in Escherichia coli. We purified Dslo-C280 in soluble form and used a 45Ca2+-overlay protein blot assay to detect Ca2+ binding. Dslo-C280 exhibits specific binding of 45Ca2+ in comparison with various control proteins and known EF-hand Ca2+-binding proteins. A mutation (D5N5) of Dslo-C280, in which five consecutive Asp residues of the “Ca-bowl” motif are changed to Asn, reduces 45Ca2+-binding activity by 56%. By electrophysiological assay, the corresponding D5N5 mutant of the Drosophila BK channel expressed in HEK293 cells exhibits lower Ca2+ sensitivity for activation and a shift of ≈+80 mV in the midpoint voltage for activation. This effect is associated with a decrease in the Hill coefficient (N) for activation by Ca2+ and a reduction in apparent Ca2+ affinity, suggesting the loss of one Ca2+-binding site per monomer. These results demonstrate a functional correlation between Ca2+ binding to a specific region of the BK protein and Ca2+-dependent activation, thus providing a biochemical approach to study this process.

Can medial temporal lobe regions distinguish true from false? An event-related functional MRI study of veridical and illusory recognition memory

To investigate the types of memory traces recovered by the medial temporal lobe (MTL), neural activity during veridical and illusory recognition was measured with the use of functional MRI (fMRI). Twelve healthy young adults watched a videotape segment in which two speakers alternatively presented lists of associated words, and then the subjects performed a recognition test including words presented in the study lists (True items), new words closely related to studied words (False items), and new unrelated words (New items). The main finding was a dissociation between two MTL regions: whereas the hippocampus was similarly activated for True and False items, suggesting the recovery of semantic information, the parahippocampal gyrus was more activated for True than for False items, suggesting the recovery of perceptual information. The study also yielded a dissociation between two prefrontal cortex (PFC) regions: whereas bilateral dorsolateral PFC was more activated for True and False items than for New items, possibly reflecting monitoring of retrieved information, left ventrolateral PFC was more activated for New than for True and False items, possibly reflecting semantic processing. Precuneus and lateral parietal regions were more activated for True and False than for New items. Orbitofrontal cortex and cerebellar regions were more activated for False than for True items. In conclusion, the results suggest that activity in anterior MTL regions does not distinguish True from False, whereas activity in posterior MTL regions does.

Functional disorders of the sympathetic nervous system in mice lacking the α1B subunit (Cav 2.2) of N-type calcium channels

N-type voltage-dependent Ca2+ channels (VDCCs), predominantly localized in the nervous system, have been considered to play an essential role in a variety of neuronal functions, including neurotransmitter release at sympathetic nerve terminals. As a direct approach to elucidating the physiological significance of N-type VDCCs, we have generated mice genetically deficient in the α1B subunit (Cav 2.2). The α1B-deficient null mice, surprisingly, have a normal life span and are free from apparent behavioral defects. A complete and selective elimination of N-type currents, sensitive to ω-conotoxin GVIA, was observed without significant changes in the activity of other VDCC types in neuronal preparations of mutant mice. The baroreflex response, mediated by the sympathetic nervous system, was markedly reduced after bilateral carotid occlusion. In isolated left atria prepared from N-type-deficient mice, the positive inotropic responses to electrical sympathetic neuronal stimulation were dramatically decreased compared with those of normal mice. In contrast, parasympathetic nervous activity in the mutant mice was nearly identical to that of wild-type mice. Interestingly, the mutant mice showed sustained elevation of heart rate and blood pressure. These results provide direct evidence that N-type VDCCs are indispensable for the function of the sympathetic nervous system in circulatory regulation and indicate that N-type VDCC-deficient mice will be a useful model for studying disorders attributable to sympathetic nerve dysfunction.

Cerebral energetics and the glycogen shunt: Neurochemical basis of functional imaging

Positron-emission tomography and functional MRS imaging signals can be analyzed to derive neurophysiological values of cerebral blood flow or volume and cerebral metabolic consumption rates of glucose (CMRGlc) or oxygen (CMRO2). Under basal physiological conditions in the adult mammalian brain, glucose oxidation is nearly complete so that the oxygen-to-glucose index (OGI), given by the ratio of CMRO2/CMRGlc, is close to the stoichiometric value of 6. However, a survey of functional imaging data suggests that the OGI is activity dependent, moving further below the oxidative value of 6 as activity is increased. Brain lactate concentrations also increase with stimulation. These results had led to the concept that brain activation is supported by anaerobic glucose metabolism, which was inconsistent with basal glucose oxidation. These differences are resolved here by a proposed model of glucose energetics, in which a fraction of glucose is cycled through the cerebral glycogen pool, a fraction that increases with degree of brain activation. The “glycogen shunt,” although energetically less efficient than glycolysis, is followed because of its ability to supply glial energy in milliseconds for rapid neurotransmitter clearance, as a consequence of which OGI is lowered and lactate is increased. The value of OGI observed is consistent with passive lactate efflux, driven by the observed lactate concentration, for the few experiments with complete data. Although the OGI changes during activation, the energies required per neurotransmitter release (neuronal) and clearance (glial) are constant over a wide range of brain activity.

Requirement of the nicotinic acetylcholine receptor β2 subunit for the anatomical and functional development of the visual system

In the mammalian visual system the formation of eye-specific layers at the thalamic level depends on retinal waves of spontaneous activity, which rely on nicotinic acetylcholine receptor activation. We found that in mutant mice lacking the β2 subunit of the neuronal nicotinic receptor, but not in mice lacking the α4 subunit, retinofugal projections do not segregate into eye-specific areas, both in the dorso-lateral geniculate nucleus and in the superior colliculus. Moreover, β2−/− mice show an expansion of the binocular subfield of the primary visual cortex and a decrease in visual acuity at the cortical level but not in the retina. We conclude that the β2 subunit of the nicotinic acetylcholine receptor is necessary for the anatomical and functional development of the visual system.

Behind the scenes of functional brain imaging: A historical and physiological perspective

At the forefront of cognitive neuroscience research in normal humans are the new techniques of functional brain imaging: positron emission tomography and magnetic resonance imaging. The signal used by positron emission tomography is based on the fact that changes in the cellular activity of the brain of normal, awake humans and laboratory animals are accompanied almost invariably by changes in local blood flow. This robust, empirical relationship has fascinated scientists for well over a hundred years. Because the changes in blood flow are accompanied by lesser changes in oxygen consumption, local changes in brain oxygen content occur at the sites of activation and provide the basis for the signal used by magnetic resonance imaging. The biological basis for these signals is now an area of intense research stimulated by the interest in these tools for cognitive neuroscience research.

Event-related functional MRI: Past, present, and future

The past two decades have seen an enormous growth in the field of human brain mapping. Investigators have extensively exploited techniques such as positron emission tomography and MRI to map patterns of brain activity based on changes in cerebral hemodynamics. However, until recently, most studies have investigated equilibrium changes in blood flow measured over time periods upward of 1 min. The advent of high-speed MRI methods, capable of imaging the entire brain with a temporal resolution of a few seconds, allows for brain mapping based on more transient aspects of the hemodynamic response. Today it is now possible to map changes in cerebrovascular parameters essentially in real time, conferring the ability to observe changes in brain state that occur over time periods of seconds. Furthermore, because robust hemodynamic alterations are detectable after neuronal stimuli lasting only a few tens of milliseconds, a new class of task paradigms designed to measure regional responses to single sensory or cognitive events can now be studied. Such “event related” functional MRI should provide for fundamentally new ways to interrogate brain function, and allow for the direct comparison and ultimately integration of data acquired by using more traditional behavioral and electrophysiological methods.