Extracellular calcium sensed by a novel cation channel in hippocampal neurons

Extracellular concentrations of Ca²⁺ change rapidly and transiently in the brain during excitatory synaptic activity. To test whether such changes in Ca²⁺ can play a signaling role we examined the effects of rapidly lowering Ca²⁺ on the excitability of acutely isolated CA1 and cultured hippocampal neurons. Reducing Ca²⁺ excited and depolarized neurons by activating a previously undescribed nonselective cation channel. This channel had a single-channel conductance of 36 pS, and its frequency of opening was inversely proportional to the concentration of Ca²⁺. The inhibition of gating of this channel was sensitive to ionic strength but independent of membrane potential. The ability of this channel to sense Ca²⁺ provides a novel mechanism whereby neurons can respond to alterations in the extracellular concentration of this key signaling ion.     

A fusion pore phenotype in mast cells of the ruby-eye mouse

Using patch-clamp capacitance and amperometric techniques, we have identified an exocytotic phenotype that affects the function of the fusion pore, the molecular structure that connects the lumen of a secretory vesicle with the extracellular environment during exocytosis. Direct observation of individual exocytotic events in mast cells from the ruby-eye mouse (ru/ru) showed a 3-fold increase in the fraction and duration of transient fusion events with respect to wild-type mice. The fraction of the total fusion events that were transient increased from 0.22 ± 0.02 (wild type) to 0.65 ± 0.02 (ru/ru), and the average duration of these events increased from 418 ± 32 ms (wild type) to 1207 ± 89 ms (ru/ru). We also show that this phenotype can reduce and delay an evoked secretory response by causing the fusion of vesicles that have been previously emptied by repeated cycles of transient fusion. The exocytotic phenotype that we describe here may be a cause of diseases like platelet storage pool deficiency and prolonged bleeding times for which the ruby-eye mouse serves as an animal model. Furthermore, the identification of the gene causing the fusion pore phenotype reported here will illuminate the molecular mechanisms regulating exocytotic fusion.     

Impaired long-term potentiation induction in dentate gyrus of calretinin-deficient mice

Calretinin (Cr) is a Ca²⁺ binding protein present in various populations of neurons distributed in the central and peripheral nervous systems. We have generated Cr-deficient (Cr^−/−) mice by gene targeting and have investigated the associated phenotype. Cr^−/− mice were viable, and a large number of morphological, biochemical, and behavioral parameters were found unaffected. In the normal mouse hippocampus, Cr is expressed in a widely distributed subset of GABAergic interneurons and in hilar mossy cells of the dentate gyrus. Because both types of cells are part of local pathways innervating dentate granule cells and/or pyramidal neurons, we have explored in Cr^−/− mice the synaptic transmission between the perforant pathway and granule cells and at the Schaffer commissural input to CA1 pyramidal neurons. Cr^−/− mice showed no alteration in basal synaptic transmission, but long-term potentiation (LTP) was impaired in the dentate gyrus. Normal LTP could be restored in the presence of the GABA_A receptor antagonist bicuculline, suggesting that in Cr^−/− dentate gyrus an excess of γ-aminobutyric acid (GABA) release interferes with LTP induction. Synaptic transmission and LTP were normal in CA1 area, which contains only few Cr-positive GABAergic interneurons. Cr^−/− mice performed normally in spatial memory task. These results suggest that expression of Cr contributes to the control of synaptic plasticity in mouse dentate gyrus by indirectly regulating the activity of GABAergic interneurons, and that Cr^−/− mice represent a useful tool to understand the role of dentate LTP in learning and memory.     

Stoichiometric coupling of brain glucose metabolism and glutamatergic neuronal activity

To determine the relationship between cerebral Glc metabolism and glutamatergic neuronal function, we used ¹³C NMR spectroscopy to measure, simultaneously, the rates of the tricarboxylic acid cycle and Gln synthesis in the rat cortex in vivo. From these measurements, we calculated the rates of oxidative Glc metabolism and glutamate–neurotransmitter cycling between neurons and astrocytes (a quantitative measure of glutamatergic neuronal activity). By measuring the rates of the tricarboxylic acid cycle and Gln synthesis over a range of synaptic activity, we have determined the stoichiometry between oxidative Glc metabolism and glutamate–neurotransmitter cycling in the cortex to be close to 1:1. This finding indicates that the majority of cortical energy production supports functional (synaptic) glutamatergic neuronal activity. Another implication of this result is that brain activation studies, which map cortical oxidative Glc metabolism, provide a quantitative measure of synaptic glutamate release.     

Modulation of an early step in the secretory machinery in hippocampal nerve terminals

In hippocampal neurons, neurotransmitter release can be regulated by protein kinase A (PKA) through a direct action on the secretory machinery. To identify the site of PKA modulation, we have taken advantage of the ability of the neurotoxin Botulinum A to cleave the synaptic protein SNAP-25. Cleavage of this protein decreases the Ca²⁺ responsiveness of the secretory machinery by partially uncoupling Ca²⁺-sensing from fusion per se. This is expressed as a shift toward higher Ca²⁺ levels of the Ca²⁺ to neurotransmitter release relationship and as a perturbation of synaptic delay under conditions where secretion induced by the Ca²⁺-independent secretagogue ruthenium red is unimpaired. We find that SNAP-25 cleavage also perturbs PKA-dependent modulation of secretion; facilitation of ruthenium red-evoked neurotransmitter release by the adenylyl cyclase activator forskolin is blocked completely after Botulinum toxin A action. Together with our observation that forskolin modifies the Ca²⁺ to neurotransmitter release relationship, our results suggest that SNAP-25 acts as a functional linker between Ca²⁺ detection and fusion and that PKA modulates an early step in the secretory machinery related to calcium sensing to facilitate synaptic transmission.     

Readily releasable pool size changes associated with long term depression

We have estimated, for hippocampal neurons in culture, the size of the autaptic readily releasable pool before and after stimulation of the sort that produces culture long term depression (LTD). This stimulation protocol causes a decrease in the pool size that is proportional to the depression of synaptic currents. To determine if depression in this system is synapse specific rather than general, we have also monitored synaptic transmission between pairs of cultured hippocampal neurons that are autaptically and reciprocally interconnected. We find that the change in synaptic strength is restricted to the synapses on the target neuron that were active during LTD induction. When viewed from the perspective of the presynaptic neuron, however, synapse specificity is partial rather than complete: synapses active during induction that were not on the target neuron were partially depressed.     

Characterization of tumor necrosis factor-deficient mice

Although tumor necrosis factor (TNF) initially came to prominence because of its anti-tumor activity, most attention is now focused on its proinflammatory actions. TNF appears to play a critical role in both early and late events involved in inflammation, from localizing the noxious agent and amplifying the cellular and mediator responses at the local site and systemically, to editing (e.g., apoptosis) injured cells or effete immune cells and repairing inflammatory damage. We have generated mice deficient in TNF (TNF^−/− mice) and have begun to examine the multiple functions attributed to TNF. TNF^−/− mice develop normally and have no gross structural or morphological abnormalities. As predicted, they are highly susceptible to challenge with an infectious agent (Candida albicans), are resistant to the lethality of minute doses of lipopolysaccharide (LPS) following D-galactosamine treatment, have a deficiency in granuloma development, and do not form germinal centers after immunization. Phagocytic activity of macrophages appears relatively normal, as do T cell functions, as measured by proliferation, cytokine release, and cytotoxicity. B cell response to thymus-independent antigens is normal, but the Ig response to thymus-dependent antigen is reduced. Surprisingly, cytokine production induced by LPS appears essentially intact, with the exception of reduced colony-stimulating factor activity. Other unexpected findings coming from our initial analysis are as follows. (i) TNF has low toxicity in TNF^−/− mice. (ii) TNF^−/− mice show an anomalous late response to heat-killed Corynebacterium parvum. In contrast to the prompt response (granuloma formation, hepatosplenomegaly) and subsequent resolution phase in C. parvum-injected TNF^+/+ mice, similarly treated TNF^−/− mice show little or no initial response, but then develop a vigorous, disorganized inflammatory response leading to death. These results suggest that TNF has an essential homeostatic role in limiting the extent and duration of an inflammatory process—i.e., an anti-inflammatory function. (iii) In contrast to the expectation that TNF^+/+ mice and TNF^+/− mice would have identical phenotypes, TNF^+/− mice showed increased susceptibility to high-dose LPS lethality, increased susceptibility to Candida challenge, and delayed resolution of the C. parvum-induced inflammatory process, indicating a strong gene dose requirement for different actions of TNF.     

Cosmic microwave background theory

A long-standing goal of theorists has been to constrain cosmological parameters that define the structure formation theory from cosmic microwave background (CMB) anisotropy experiments and large-scale structure (LSS) observations. The status and future promise of this enterprise is described. Current band-powers in -space are consistent with a ΔT flat in frequency and broadly follow inflation-based expectations. That the levels are ~(10⁻⁵)² provides strong support for the gravitational instability theory, while the Far Infrared Absolute Spectrophotometer (FIRAS) constraints on energy injection rule out cosmic explosions as a dominant source of LSS. Band-powers at 100 suggest that the universe could not have re-ionized too early. To get the LSS of Cosmic Background Explorer (COBE)-normalized fluctuations right provides encouraging support that the initial fluctuation spectrum was not far off the scale invariant form that inflation models prefer: e.g., for tilted Λ cold dark matter sequences of fixed 13-Gyr age (with the Hubble constant H₀ marginalized), n_s = 1.17 ± 0.3 for Differential Microwave Radiometer (DMR) only; 1.15 ± 0.08 for DMR plus the SK95 experiment; 1.00 ± 0.04 for DMR plus all smaller angle experiments; 1.00 ± 0.05 when LSS constraints are included as well. The CMB alone currently gives weak constraints on Λ and moderate constraints on Ω_tot, but theoretical forecasts of future long duration balloon and satellite experiments are shown which predict percent-level accuracy among a large fraction of the 10+ parameters characterizing the cosmic structure formation theory, at least if it is an inflation variant.     

Brain monoamine oxidase A inhibition in cigarette smokers

Several studies have documented a strong association between smoking and depression. Because cigarette smoke has been reported to inhibit monoamine oxidase (MAO) A in vitro and in animals and because MAO A inhibitors are effective antidepressants, we tested the hypothesis that MAO A would be reduced in the brain of cigarette smokers. We compared brain MAO A in 15 nonsmokers and 16 current smokers with [¹¹C]clorgyline and positron emission tomography (PET). Four of the nonsmokers were also treated with the antidepressant MAO inhibitor drug, tranylcypromine (10 mg/day for 3 days) after the baseline PET scan and then rescanned to assess the sensitivity of [¹¹C]clorgyline binding to MAO inhibition. MAO A levels were quantified by using the model term λk₃which is a function of brain MAO A concentration. Smokers had significantly lower brain MAO A than nonsmokers in all brain regions examined (average reduction, 28%). The mean λk₃ values for the whole brain were 0.18 ± 0.04 and 0.13 ± 0.03 cc_brain (ml_plasma)⁻¹ min⁻¹ for nonsmokers and smokers, respectively; P < 0.0003). Tranylcypromine treatment reduced λk₃ by an average of 58% for the different brain regions. Our results show that tobacco smoke exposure is associated with a marked reduction in brain MAO A, and this reduction is about half of that produced by a brief treatment with tranylcypromine. This suggests that MAO A inhibition needs to be considered as a potential contributing variable in the high rate of smoking in depression and in the development of more effective strategies for smoking cessation.     

Altered short-term synaptic plasticity and reduced muscle strength in mice with impaired regulation of presynaptic CaV2.1 Ca2+ channels

Facilitation and inactivation of P/Q-type calcium (Ca²⁺) currents through the regulation of voltage-gated Ca²⁺ (Ca_V) 2.1 channels by Ca²⁺ sensor (CaS) proteins contributes to the facilitation and rapid depression of synaptic transmission in cultured neurons that transiently express Ca_V2.1 channels. To examine the modulation of endogenous Ca_V2.1 channels by CaS proteins in native synapses, we introduced a mutation (IM-AA) into the CaS protein-binding site in the C-terminal domain of Ca_V2.1 channels in mice, and tested synaptic facilitation and depression in neuromuscular junction synapses that use exclusively Ca_V2.1 channels for Ca²⁺ entry that triggers synaptic transmission. Even though basal synaptic transmission was unaltered in the neuromuscular synapses in IM-AA mice, we found reduced short-term facilitation in response to paired stimuli at short interstimulus intervals in IM-AA synapses. In response to trains of action potentials, we found increased facilitation at lower frequencies (10–30 Hz) in IM-AA synapses accompanied by slowed synaptic depression, whereas synaptic facilitation was reduced at high stimulus frequencies (50–100 Hz) that would induce strong muscle contraction. As a consequence of altered regulation of Ca_V2.1 channels, the hindlimb tibialis anterior muscle in IM-AA mice exhibited reduced peak force in response to 50 Hz stimulation and increased muscle fatigue. The IM-AA mice also had impaired motor control, exercise capacity, and grip strength. Taken together, our results indicate that regulation of Ca_V2.1 channels by CaS proteins is essential for normal synaptic plasticity at the neuromuscular junction and for muscle strength, endurance, and motor coordination in mice in vivo.Classic work on the frog neuromuscular junction (NMJ) first described facilitation and depression of synaptic transmission during trains of action potentials (1). These forms of short-term plasticity are widespread among different types of synapses, and they transmit information encoded in the frequency and pattern of action potential generation to postsynaptic cells (2). Calcium (Ca²⁺)-dependent synaptic transmission is mediated by multiple types of voltage-gated Ca²⁺ (Ca_V) channels. Mature mammalian NMJ synapses use exclusively Ca_V2.1 channels to initiate synaptic transmission (3), in contrast to central nervous system synapses that use combinations of Ca_V2.1, Ca_V2.2, and Ca_V2.3 channels (4–6). Disruption of Ca_V2.1 channels by elimination of their pore-forming α1 subunit greatly reduces facilitation at the calyx of Held synapse (7, 8) and the NMJ (9) in mice, suggesting a key role for Ca_V2.1 channels in short-term synaptic plasticity.Ca_V2.1 channels in transfected nonneuronal cells are regulated in a biphasic manner by calmodulin and other related Ca²⁺ sensor (CaS) proteins through interaction with a bipartite regulatory site in their C-terminal domain composed of an IQ-like motif (IM) and a calmodulin-binding domain (CBD) (10–13). CaS proteins interact with the IM motif to initiate Ca²⁺-dependent facilitation in response to local increases in Ca²⁺, and then interact with the CBD to induce Ca²⁺-dependent inactivation in response to longer, more global increases in Ca²⁺ (10–13).The facilitation and inactivation of Ca_V2.1 channels during trains of repetitive stimuli induces synaptic facilitation, followed by a rapid phase of synaptic depression in cultured superior cervical ganglion neurons transiently expressing Ca_V2.1 channels (14). The Ile-Met→Ala-Ala (IM-AA) mutation prevents this synaptic plasticity by altering the interaction of Ca_V2.1 channels with CaS proteins (14). Other CaS proteins can displace calmodulin from their common regulatory site, enhance either facilitation or inactivation of the Ca²⁺ current, and thereby control the direction and amplitude of synaptic plasticity in cultured superior cervical ganglion neurons (15–18). Although previous studies revealed that regulation of Ca_V2.1 channels by CaS proteins can induce and regulate short-term synaptic plasticity in transfected neurons in cell culture, whether this mechanism makes an important contribution to short-term synaptic plasticity in native synapses has remained unknown.To define the functional role of regulation of endogenous Ca_V2.1 channels by CaS proteins in short-term synaptic plasticity in vivo, we introduced the IM-AA mutation into the CaS protein-binding site in the C-terminal domain of Ca_V2.1 channels in mice, and investigated the effects of this mutation on synaptic transmission and short-term synaptic plasticity of NMJ synapses. The IM-AA mutation did not affect basal neuromuscular transmission; however, this mutation blocked short-term synaptic facilitation in response to paired stimuli with short interstimulus intervals (ISIs). Similarly, during high-frequency trains in which the intervals between stimuli are short, the IM-AA mutation reduced synaptic facilitation. In contrast, during trains of stimuli at low frequency with long ISIs, the IM-AA mutation slowed synaptic depression and thereby allowed increased synaptic facilitation. Hindlimb tibialis anterior (TA) muscles of IM-AA mice exhibited reduced peak specific force at 50-Hz stimulation, along with increased muscle fatigue. These defects in muscle function were accompanied by impaired motor control, reduced exercise capacity, and loss of grip strength in IM-AA mice in vivo.Forceful muscle contractions require high-frequency stimulation by the presynaptic motor nerve. Therefore, our results with IM-AA mice link reduced paired-pulse facilitation (PPF) at short ISIs and reduced facilitation during high-frequency trains of stimuli at the cellular level with impaired strength, coordination, and exercise capacity in vivo. These findings demonstrate a critical role for the modulation of Ca_V2.1 channels by CaS proteins in regulating short-term synaptic plasticity, with important consequences for muscle strength and motor control.     

Neuregulins stimulate the functional expression of Ca2+-activated K+ channels in developing chicken parasympathetic neurons

The developmental expression of macroscopic Ca²⁺-activated K⁺ currents (I_K[Ca]) in chicken ciliary ganglion (CG) neurons is dependent in part on trophic factors released from preganglionic nerve terminals. Neuregulins are expressed in the preganglionic neurons that innervate the chicken CG and are therefore plausible candidates for this activity. Application of 1 nM β1-neuregulin peptide for 12 hr evokes a large (7- to 10-fold) increase in I_K[Ca] in embryonic day 9 CG neurons, even in the presence of a translational inhibitor. A similar posttranslational effect is produced by high concentrations (10 nM) of epidermal growth factor and type α transforming growth factor but not by 10 nM α2-neuregulin peptide or by neurotrophins at 40 ngml⁻¹. β1-neuregulin treatment for 12 hr also confers Ca²⁺ sensitivity onto large-conductance (285 pS) K⁺ channels observed in inside–out patches. β-Neuregulins have no effect on voltage-activated Ca²⁺ currents of CG neurons. These data support the hypothesis that β-neuregulins mediate the trophic effects of preganglionic nerve terminals on the electrophysiological differentiation of developing CG neurons.     

Neto2 is a KCC2 interacting protein required for neuronal Cl? regulation in hippocampal neurons

KCC2 is a neuron-specific K⁺–Cl⁻ cotransporter that is essential for Cl⁻ homeostasis and fast inhibitory synaptic transmission in the mature CNS. Despite the critical role of KCC2 in neurons, the mechanisms regulating its function are not understood. Here, we show that KCC2 is critically regulated by the single-pass transmembrane protein neuropilin and tolloid like-2 (Neto2). Neto2 is required to maintain the normal abundance of KCC2 and specifically associates with the active oligomeric form of the transporter. Loss of the Neto2:KCC2 interaction reduced KCC2-mediated Cl⁻ extrusion, resulting in decreased synaptic inhibition in hippocampal neurons.     

From the Cover: Presenilins regulate calcium homeostasis and presynaptic function via ryanodine receptors in hippocampal neurons

Presenilin (PS) plays a central role in the pathogenesis of Alzheimer’s disease, and loss of PS causes progressive memory impairment and age-related neurodegeneration in the mouse cerebral cortex. In hippocampal neurons, PS is essential for neurotransmitter release, NMDA receptor-mediated responses, and long-term potentiation. PS is also involved in the regulation of calcium homeostasis, although the precise site of its action is less clear. Here we investigate the mechanism by which PS regulates synaptic function and calcium homeostasis using acute hippocampal slices from PS conditional knockout mice and primary cultured postnatal hippocampal neurons, in which PS is inducibly inactivated. Using two different calcium probes, Fura-2 and Mag–Fura-2, we found that inactivation of PS in primary hippocampal neurons does not affect calcium concentration in the endoplasmic reticulum. Rather, in the absence of PS, levels of ryanodine receptor (RyR) are reduced in the hippocampus, measured by Western analysis and radioligand binding assay, although the mRNA expression is unaffected. RyR-mediated function is also impaired, as indicated by reduced RyR agonist-induced calcium release from the ER and RyR-mediated synaptic responses in the absence of PS. Furthermore, knockdown of RyR expression in wild-type hippocampal neurons by two independent shRNAs to levels comparable with the RyR protein reduction in PS-deficient hippocampal neurons mimics the defects exhibited in calcium homeostasis and presynaptic function. Collectively, our findings show that PS regulates calcium homeostasis and synaptic function via RyR and suggest that disruption of intracellular calcium homeostasis may be an early pathogenic event leading to presynaptic dysfunction in Alzheimer’s disease.     

Dysbindin regulates hippocampal LTP by controlling NMDA receptor surface expression

Abnormalities in NMDA receptor (NMDAR) function have been implicated in schizophrenia. Here, we show that dysbindin, a schizophrenia-susceptibility gene widely expressed in the forebrain, controls the surface expression of NMDARs in a subunit-specific manner. Imaging analyses revealed a marked increase in surface NR2A, but not NR2B, in hippocampal neurons derived from dysbindin-null mutant mice (Dys−/−). Exogenous expression of dysbindin reduced NR2A surface expression in both wild-type and Dys−/− neurons. Biotinylation experiments also revealed an increase in surface expression of endogenous NR2A in Dys−/− neurons. Disruption of the dysbindin gene dramatically increased NR2A-mediated synaptic currents, without affecting AMPA receptor currents, in hippocampal CA1 neurons. The Dys−/− hippocampal slices exhibited an enhanced LTP, whereas basal synaptic transmission, presynaptic properties, and LTD were normal. Thus, dysbindin controls hippocampal LTP by selective regulation of the surface expression of NR2A. These results reveal subunit-specific regulation of NMDARs by dysbindin, providing an unexpected link between these two proteins implicated in schizophrenia.     

Ca2+-induced rebound potentiation of γ-aminobutyric acid-mediated currents requires activation of Ca2+/calmodulin-dependent kinase II

In cerebellar Purkinje neurons, γ-aminobutyric acid (GABA)-mediated inhibitory synaptic transmission undergoes a long-lasting “rebound potentiation” after the activation of excitatory climbing fiber inputs. Rebound potentiation is triggered by the climbing-fiber-induced transient elevation of intracellular Ca²⁺ concentration and is expressed as a long-lasting increase of postsynaptic GABA_A receptor sensitivity. Herein we show that inhibitors of the Ca²⁺/calmodulin-dependent protein kinase II (CaM-KII) signal transduction pathway effectively block the induction of rebound potentiation. These inhibitors have no effect on the once established rebound potentiation, on voltage-gated Ca²⁺ channel currents, or on the basal inhibitory transmission itself. Futhermore, a protein phosphatase inhibitor and the intracellularly applied CaM-KII markedly enhanced GABA-mediated currents in Purkinje neurons. Our results demonstrate that CaM-KII activation and the following phosphorylation are key steps for rebound potentiation.     

Endocrine modulation of the neurotoxicity of gp120: Implications for AIDS-related dementia complex

HIV infection often involves the development of AIDS-related dementia complex, a variety of neurologic, neuropsychologic, and neuropathologic impairments. A possible contributor to AIDS-related dementia complex is the HIV envelope glycoprotein gp120, which damages neurons via a complex glutamate receptor- and calcium-dependent cascade. We demonstrate an endocrine modulation of the deleterious effects of gp120 in primary hippocampal and cortical cultures. Specifically, we observe that gp120-induced calcium mobilization and neurotoxicity are exacerbated by glucocorticoids, the adrenal steroids secreted during stress. Importantly, this deleterious synergy can occur between gp120 and synthetic glucocorticoids (such as prednisone or dexamethasone) that are used clinically in high concentrations to treat severe cases of the Pneumocystis carinii pneumonia typical of HIV infection. Conversely, we also observe that estradiol protects neurons from the deleterious actions of gp120, reducing toxicity and calcium mobilization.     

Conformation-dependent phosphorylation of p53

Phosphorylation of the p53 tumor suppressor protein is known to modulate its functions. Using bacterially produced glutathione S-transferase (GST)-p53 fusion protein and baculovirus-expressed histidine-tagged p53 (_Hisp53), we have determined human p53 phosphorylation by purified forms of jun-N-kinase (JNK), protein kinase A (PKA), and β subunit of casein kinase II (CKIIβ) as well as by kinases present in whole cell extracts (WCEs). We demonstrate that PKA is potent p53 kinase, albeit, in a conformation- and concentration-dependent manner, as concluded by comparing full-length with truncated forms of p53. We further demonstrate JNK interaction with GST-p53 and the ability of JNK to phosphorylate truncated forms of GST-p53 or full-length _Hisp53. Dependence of phosphorylation on conformation of p53 is further supported by the finding that the wild-type form of p53 (p53^wt) undergoes better phosphorylation by CKIIβ and by WCE kinases than mutant forms of p53 at amino acid 249 (p53²⁴⁹) or 273 (p53²⁷³). Moreover, shifting the kinase reaction’s temperature from 37°C to 18°C reduces the phosphorylation of mutant p53 to a greater extent than of p53^wt. Comparing truncated forms of p53 revealed that the ability of CKIIβ, PKA, or WCE kinases to phosphorylate p53 requires amino acids 97–155 within the DNA-binding domain region. Among three 20-aa peptides spanning this region we have identified residues 97–117 that increase p53 phosphorylation by CKIIβ while inhibiting p53 phosphorylation by PKA or WCE kinases. The importance of this region is further supported by computer modeling studies, which demonstrated that mutant p53²⁴⁹ exhibits significant changes to the conformation of p53 within amino acids 97–117. In summary, phosphorylation-related analysis of different p53 forms in vitro indicates that conformation of p53 is a key determinant in its availability as a substrate for different kinases, as for the phosphorylation pattern generated by the same kinase.     

Cytoplasmic sequestration of an O6-methylguanine-DNA methyltransferase enhancer binding protein in DNA repair-deficient human cells

O⁶-Methylguanine-DNA methyltransferase (MGMT), an enzyme that repairs adducts at O⁶ of guanine in DNA, is a major determinant of susceptibility to simple methylating carcinogens or of tumor response to anticancer chloroethylating drugs. To investigate the mechanisms underlying cellular expression of this DNA repair enzyme, we focused on the role of a 59-bp enhancer of the human MGMT gene in the regulation of its expression. By using chloramphenicol acetyltransferase reporter assays, we found that the enhancer activity, which was present in both MGMT-expressing (Mer⁺) and -deficient (Mer⁻) cells, correlated with the endogenous MGMT activity in Mer⁺ cell lines. Band-shift assays and deletion analysis of the 59-bp sequence defined a minimal 9-mer cis element (5′-CTGGGTCGC-3′) for specific trans factor binding. The MGMT enhancer binding protein (MEBP), 45 kDa by Southwestern blot analysis, was present in the nuclei of all Mer⁺ cells tested but was apparently restricted to the cytoplasm of Mer⁻ cells. We conclude that the MEBP–enhancer interaction plays an important role in regulating constitutive MGMT expression in Mer⁺ cells and that MEBP exclusion from the nucleus may account for the down-regulation of MGMT in Mer⁻ cells.     

Genetically determined chloride-sensitive hypertension and stroke

The stroke-prone spontaneously hypertensive rat (SHRSP) is a genetically determined model of “salt-sensitive” stroke and hypertension whose full phenotypic expression is said to require a diet high in Na⁺ and low in K⁺. We tested the hypothesis that dietary Cl⁻ determines the phenotypic expression of the SHRSP. In the SHRSP fed a normal NaCl diet, supplementing dietary K⁺ with KCl exacerbated hypertension, whereas supplementing either KHCO₃ or potassium citrate (KB/C) attenuated hypertension, when blood pressure (BP) was measured radiotelemetrically, directly and continually. Supplemental KCl, but not KB/C, induced strokes, which occurred in all and only those rats in the highest quartiles of both BP and plasma renin activity (PRA). PRA was higher with KCl than with KB/C. These observations demonstrate that with respect to both severity of hypertension and frequency of stroke the phenotypic expression of the SHRSP is (i) either increased or decreased, depending on whether the anionic component of the potassium salt supplemented is, or is not, Cl⁻; (ii) increased by supplementing Cl⁻ without supplementing Na⁺, and despite supplementing K⁺; and hence (iii) both selectively Cl⁻-sensitive and Cl⁻-determined. The observations suggest that in the SHRSP selectively supplemented with Cl⁻ the likelihood of stroke depends on the extent to which both BP and PRA increase.     

Defective placental vasculogenesis causes embryonic lethality in VHL-deficient mice

Inheritance of an inactivated form of the VHL tumor suppressor gene predisposes patients to develop von Hippel–Lindau disease, and somatic VHL inactivation is an early genetic event leading to the development of sporadic renal cell carcinoma. The VHL gene was disrupted by targeted homologous recombination in murine embryonic stem cells, and a mouse line containing an inactivated VHL allele was generated. While heterozygous VHL (+/−) mice appeared phenotypically normal, VHL −/− mice died in utero at 10.5 to 12.5 days of gestation (E10.5 to E12.5). Homozygous VHL −/− embryos appeared to develop normally until E9.5 to E10.5, when placental dysgenesis developed. Embryonic vasculogenesis of the placenta failed to occur in VHL −/− mice, and hemorrhagic lesions developed in the placenta. Subsequent hemorrhage in VHL −/− embryos caused necrosis and death. These results indicate that VHL expression is critical for normal extraembryonic vascular development.