A novel seizure-induced synaptotagmin gene identified by differential display

Systemic administration of kainic acid, a cyclic analogue of glutamate, produces many of the clinical features of human temporal lobe epilepsy and status epilepticus in rats, including the induction of motor convulsions and the degeneration of neurons in the hippocampus and piriform cortex. Differential display PCR was used to identify mRNAs that are differentially expressed between degenerating and nondegenerating tissues in the brain after kainic acid-induced seizure activity. A novel cDNA fragment expressed in the degenerating hippocampus and piriform cortex, but not in the nondegenerating parietal cortex, was identified, cloned, and sequenced. This novel cDNA fragment identified a new member of the synaptotagmin gene family that is rapidly and transiently induced in response to seizure activity. Differential expression of this synaptotagmin gene, syt X, was confirmed by Northern blot analysis and in situ hybridization. This novel, inducible synaptotagmin gene may provide a direct link between seizure-induced neuronal gene expression and subsequent modulation of synaptic structure and function.     

Studies using double mutants of the conformational transitions in influenza hemagglutinin required for its membrane fusion activity

Amino acid substitutions widely distributed throughout the influenza hemagglutinin (HA) influence the pH of its membrane fusion activity. We have combined a number of these substitutions in double mutants and determined the effects on the pH of fusion and on the pH at which the refolding of HA required for fusion occurs. By analyzing combinations of mutations in three regions of the metastable neutral-pH HA that are rearranged at fusion pH we obtain evidence for both additive and nonadditive effects and for an apparent order of dominance in the effects of amino acid substitutions in particular regions on the pH of fusion. We conclude that there are at least three components in the structural transition required for membrane fusion activity and consider possible pathways for the transition in relation to the known differences between neutral and fusion pH HA structures.     

Role of leptin in hypothalamic–pituitary function

A defect in the structure of the obese gene is responsible for development of obesity in the ob/ob mouse. The product of expression of the gene is the protein hormone leptin. Leptin causes weight loss in ob/ob and normal mice, it is secreted by adipocytes, and it is an important controller of the size of fat stores by inhibiting appetite. The ob/ob mouse is infertile and has a pattern of gonadotropin secretion similar to that of prepubertal animals. Consequently, we hypothesized that leptin might play a role in the control of gonadotropin secretion and initiated studies on its possible acute effects on hypothalamic–pituitary function. After a preincubation period, hemi-anterior pituitaries of adult male rats were incubated with leptin for 3 hr. Leptin produced a dose-related increase in follicle-stimulating hormone (FSH) and luteinizing hormone (LH) release, which reached peaks with 10⁻⁹ and 10⁻¹¹ M leptin, respectively. Gonadotropin release decreased at higher concentrations of leptin to values indistinguishable from that of control pituitaries. On the other hand, prolactin secretion was greatly increased in a dose-related manner but only with leptin concentrations (10⁻⁷–10⁻⁵ M). Incubation with leptin of median eminence–arcuate nuclear explants from the same animals produced significant increases in LH-releasing hormone (LHRH) release only at the lowest concentrations tested (10⁻¹²–10⁻¹⁰ M). As the leptin concentration was increased, LHRH release decreased and was significantly less than control release at the highest concentration tested (10⁻⁶ M). To determine if leptin can also release gonadotropins in vivo, ovariectomized females bearing implanted third ventricle cannulae were injected with 10 μg of estradiol benzoate s.c., followed 72 hr later by microinjection into the third ventricle of leptin (0.6 nmol in 5 μl) or an equal volume of diluent. There was a highly significant increase in plasma LH, which peaked 10–50 min after injection of leptin. Leptin had no effect on plasma FSH concentrations, and the diluent had no effect on either plasma FSH or LH. Thus, leptin at very low concentrations stimulated LHRH release from hypothalamic explants and FSH and LH release from anterior pituitaries of adult male rats in vitro and released LH, but not FSH, in vivo. The results indicate that leptin plays an important role in controlling gonadotropin secretion by stimulatory hypothalamic and pituitary actions.     

Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein

Human P-glycoprotein (Pgp) confers multidrug resistance to cancer cells by ATP-dependent extrusion of a great many structurally dissimilar hydrophobic compounds. The manner in which Pgp recognizes these different substrates is unknown. The protein shows internal homology between its N- and C-terminal halves, each comprised of six putative transmembrane helices and a consensus ATP binding/utilization site. Photoactive derivatives of certain Pgp substrates specifically label two regions, one on each half of the protein. In this study, using [¹²⁵I]iodoarylazidoprazosin ([¹²⁵I]IAAP), a photoactive analog of prazosin, we have demonstrated the presence of two nonidentical drug-interaction sites within Pgp. Taking advantage of a highly susceptible trypsin cleavage site in the linker region of Pgp, we characterized the [¹²⁵I]IAAP binding to the N- and C-terminal halves. cis(Z)-Flupentixol, a modulator of Pgp function, preferentially increased the affinity of [¹²⁵I]IAAP for the C-terminal half of the protein (C-site) by reducing the K_d from 20 to 6 nM without changing the labeling or affinity (K_d = 42–46 nM) of the N-terminal half (N-site). Also, the concentration of vinblastine (Pgp substrate) and cyclosporin A (Pgp modulator) required for 50% inhibition of [¹²⁵I]IAAP binding to the C-site was increased 5- to 6-fold by cis(Z)-flupentixol without any effect on the N-site. In addition, [¹²⁵I]IAAP binding to the N-site was less susceptible than to C-site to inhibition by vanadate which blocks ATP hydrolysis and drug transport. These data demonstrate the presence of at least two nonidentical substrate interaction sites in Pgp.     

Modulation of potassium channel function by methionine oxidation and reduction

Oxidation of amino acid residues in proteins can be caused by a variety of oxidizing agents normally produced by cells. The oxidation of methionine in proteins to methionine sulfoxide is implicated in aging as well as in pathological conditions, and it is a reversible reaction mediated by a ubiquitous enzyme, peptide methionine sulfoxide reductase. The reversibility of methionine oxidation suggests that it could act as a cellular regulatory mechanism although no such in vivo activity has been demonstrated. We show here that oxidation of a methionine residue in a voltage-dependent potassium channel modulates its inactivation. When this methionine residue is oxidized to methionine sulfoxide, the inactivation is disrupted, and it is reversed by coexpression with peptide methionine sulfoxide reductase. The results suggest that oxidation and reduction of methionine could play a dynamic role in the cellular signal transduction process in a variety of systems.     

Reconstitution of hyperacetylated, DNase I-sensitive chromatin characterized by high conformational flexibility of nucleosomal DNA

Increased acetylation at specific N-terminal lysines of core histones is a hallmark of active chromatin in vivo, yet the structural consequences of acetylation leading to increased gene activity are only poorly defined. We employed a new approach to characterize the effects of histone acetylation: A Drosophila embryo-derived cell-free system for chromatin reconstitution under physiological conditions was programmed with exogenous histones to assemble hyperacetylated or matching control chromatin of high complexity. Hyperacetylated chromatin resembled unmodified chromatin at similar nucleosome density with respect to its sensitivity toward microccal nuclease, its nucleosomal repeat length, and the incorporation of the linker histone H1. In contrast, DNA in acetylated chromatin showed an increased sensitivity toward DNase I and a surprisingly high degree of conformational flexibility upon temperature shift pointing to profound alterations of DNA/histone interactions. This successful reconstitution of accessible and flexible chromatin outside of a nucleus paves the way for a thorough analysis of the causal relationship between histone acetylation and gene function.     

Glucocorticoid enhancement of memory storage involves noradrenergic activation in the basolateral amygdala

Evidence indicates that the modulatory effects of the adrenergic stress hormone epinephrine as well as several other neuromodulatory systems on memory storage are mediated by activation of β-adrenergic mechanisms in the amygdala. In view of our recent findings indicating that the amygdala is involved in mediating the effects of glucocorticoids on memory storage, the present study examined whether the glucocorticoid-induced effects on memory storage depend on β-adrenergic activation within the amygdala. Microinfusions (0.5 μg in 0.2 μl) of either propranolol (a nonspecific β-adrenergic antagonist), atenolol (a β₁-adrenergic antagonist), or zinterol (a β₂-adrenergic antagonist) administered bilaterally into the basolateral nucleus of the amygdala (BLA) of male Sprague–Dawley rats 10 min before training blocked the enhancing effect of posttraining systemic injections of dexamethasone (0.3 mg/kg) on 48-h memory for inhibitory avoidance training. Infusions of these β-adrenergic antagonists into the central nucleus of the amygdala did not block the dexamethasone-induced memory enhancement. Furthermore, atenolol (0.5 μg) blocked the memory-enhancing effects of the specific glucocorticoid receptor (GR or type II) agonist RU 28362 infused concurrently into the BLA immediately posttraining. These results strongly suggest that β-adrenergic activation is an essential step in mediating glucocorticoid effects on memory storage and that the BLA is a locus of interaction for these two systems.     

Loss of heterozygosity induced by a chromosomal double-strand break

The repair of chromosomal double-strand breaks (DSBs) is necessary for genomic integrity in all organisms. Genetic consequences of misrepair include chromosomal loss, deletion, and duplication resulting in loss of heterozygosity (LOH), a common finding in human solid tumors. Although work with radiation-sensitive cell lines suggests that mammalian cells primarily rejoin DSBs by nonhomologous mechanisms, alternative mechanisms that are implicated in chromosomal LOH, such as allelic recombination, may also occur. We have examined chromosomal DSB repair between homologs in a gene targeted mammalian cell line at the retinoblastoma (Rb) locus. We have found that allelic recombinational repair occurs in mammalian cells and is increased at least two orders of magnitude by the induction of a chromosomal DSB. One consequence of allelic recombination is LOH at the Rb locus. Some of the repair events also resulted in other types of genetic instability, including deletions and duplications. We speculate that mammalian cells may have developed efficient nonhomologous DSB repair processes to bypass allelic recombination and the potential for reduction to homozygosity.     

Activation of the cell death program by inhibition of proteasome function

Activation of proteolytic enzymes, including cysteine proteases of the ced-3/ICE family, is a characteristic feature of the apoptotic program. In contrast, the role of the proteasome as the major nonlysosomal machinery to degrade or process proteins by ATP/ubiquitin-dependent proteolysis in this process is less clear. In human leukemic HL60 cells, inhibition of proteasome-mediated proteolysis by specific proteasomal inhibitors leads to the rapid induction of apoptosis as judged by morphological changes as well as by nuclear condensation and DNA fragmentation. HL60 apoptosis is due to activation of CPP32, a member of the ced-3/ICE family of cysteine proteases, and appears to occur independently from ICE activity. HL60 apoptosis is accompanied by an increase in the concentration of the cyclin-dependent kinase inhibitor p27^Kip1. Labeling of the cells by the TUNEL technique demonstrates that HL60 cells undergoing apoptosis are primarily in the G₁ phase of the cell cycle. Proteasomal activity therefore appears to be required in proliferating, but not in quiescent, HL60 cells for cell survival as well as normal progression through the cell cycle.     

Dominant negative inhibition by fragments of a monomeric enzyme

Dominant negative inhibition is most commonly seen when a mutant subunit of a multisubunit protein is coexpressed with the wild-type protein so that assembly of a functional oligomer is impaired. By analogy, it should be possible to interfere with the functional assembly of a monomeric enzyme by interfering with the folding pathway. Experiments in vitro by others suggested that fragments of a monomeric enzyme might be exploited for this purpose. We report here dominant negative inhibition of bacterial cell growth by expression of fragments of a tRNA synthetase. Inhibition is fragment-specific, as not all fragments cause inhibition. An inhibitory fragment characterized in more detail forms a specific complex with the intact enzyme in vivo, leading to enzyme inactivation. This fragment also associated stoichiometrically with the full-length enzyme in vitro after denaturation and refolding, and the resulting complex was catalytically inactive. Inhibition therefore appears to arise from an interruption in the folding pathway of the wild-type enzyme, thus suggesting a new strategy to design dominant negative inhibitors of monomeric enzymes.     

Limited oral bioavailability and active epithelial excretion of paclitaxel (Taxol) caused by P-glycoprotein in the intestine

In mice, the mdr1a and mdr1b genes encode drug-transporting proteins that can cause multidrug resistance in tumor cells by lowering intracellular drug levels. These P-glycoproteins are also found in various normal tissues such as the intestine. Because mdr1b P-glycoprotein is not detectable in the intestine, mice with a homozygously disrupted mdr1a gene [mdr1a(−/−) mice] do not contain functional P-glycoprotein in this organ. We have used these mdr1a(−/−) mice to study the effect of gut P-glycoprotein on the pharmacokinetics of paclitaxel. The area under the plasma concentration-time curves was 2- and 6-fold higher in mdr1a(−/−) mice than in wild-type (wt) mice after i.v. and oral drug administration, respectively. Consequently, the oral bioavailability in mice receiving 10 mg paclitaxel per kg body weight increased from only 11% in wt mice to 35% in mdr1a(−/−) mice. The cumulative fecal excretion (0–96 hr) was markedly reduced from 40% (after i.v. administration) and 87% (after oral administration) of the administered dose in wt mice to below 3% in mdr1a(−/−) mice. Biliary excretion was not significantly different in wt and mdr1a(−/−) mice. Interestingly, after i.v. drug administration of paclitaxel (10 mg/kg) to mice with a cannulated gall bladder, 11% of the dose was recovered within 90 min in the intestinal contents of wt mice vs. <3% in mdr1a(−/−) mice. We conclude that P-glycoprotein limits the oral uptake of paclitaxel and mediates direct excretion of the drug from the systemic circulation into the intestinal lumen.     

Structural basis for methylesterase CheB regulation by a phosphorylation-activated domain

We report the x-ray crystal structure of the methylesterase CheB, a phosphorylation-activated response regulator involved in reversible modification of bacterial chemotaxis receptors. Methylesterase CheB and methyltransferase CheR modulate signaling output of the chemotaxis receptors by controlling the level of receptor methylation. The structure of CheB, which consists of an N-terminal regulatory domain and a C-terminal catalytic domain joined by a linker, was solved by molecular replacement methods using independent search models for the two domains. In unphosphorylated CheB, the N-terminal domain packs against the active site of the C-terminal domain and thus inhibits methylesterase activity by directly restricting access to the active site. We propose that phosphorylation of CheB induces a conformational change in the regulatory domain that disrupts the domain interface, resulting in a repositioning of the domains and allowing access to the active site. Structural similarity between the two companion receptor modification enzymes, CheB and CheR, suggests an evolutionary and/or functional relationship. Specifically, the phosphorylated N-terminal domain of CheB may facilitate interaction with the receptors, similar to the postulated role of the N-terminal domain of CheR. Examination of surfaces in the N-terminal regulatory domain of CheB suggests that despite a common fold throughout the response regulator family, surfaces used for protein–protein interactions differ significantly. Comparison between CheB and other response regulators indicates that analogous surfaces are used for different functions and conversely, similar functions are mediated by different molecular surfaces.     

GIRK1 immunoreactivity is present predominantly in dendrites, dendritic spines, and somata in the CA1 region of the hippocampus

Electron microscopic analysis of the CA1 region of the rat hippocampus revealed that specific immunoreactivity (IR) for a G protein-gated, inwardly rectifying potassium channel (GIRK1) was present exclusively in neurons and predominantly located in spiny dendrites of pyramidal cells. Within stratum lacunosum-moleculare and the superficial stratum radiatum, GIRK1-IR was often present immediately adjacent to asymmetric (excitatory-type) postsynaptic densities in dendritic spines. The subcellular localization of GIRK1-IR in the Golgi apparatus of pyramidal cell somata and in the plasma membrane of dendrites and dendritic spines confirms the hypothesis that GIRK1 is synthesized by pyramidal cells and transported to the more distal dendritic processes. G protein-coupled receptor activation of a dendritic potassium conductance would attenuate the propagation of excitatory synaptic inputs and thereby produce postsynaptic inhibition. Thus, these results show that the GIRK family of channels joins the list of voltage-sensitive channels now known to be expressed in dendritic spines.     

Regulation of microtubule dynamics by the neuronal growth-associated protein SCG10

Dynamic assembly and disassembly of microtubules is essential for cell division, cell movements, and intracellular transport. In the developing nervous system, microtubule dynamics play a fundamental role during neurite outgrowth, elongation, and branching, but the molecular mechanisms involved are unknown. SCG10 is a neuron-specific protein that is membrane-associated and highly enriched in growth cones. Here we show that SCG10 binds to microtubules, inhibits their assembly, and can induce microtubule disassembly. We also show that SCG10 overexpression enhances neurite outgrowth in a stably transfected neuronal cell line. These data identify SCG10 as a key regulator of neurite extension through regulation of microtubule instability.     

Strained DNA is kinked by low concentrations of Zn2+

A novel atomic force microscope with a magnetically oscillated tip has provided unprecedented resolution of small DNA fragments spontaneously adsorbed to mica and imaged in situ in the presence of divalent ions. Kinks (localized bends of average angle 78°) were observed in axially strained minicircles consisting of tandemly repeated d(A)₅ and d(GGGCC[C]) sequences. The frequency of kinks in identical minicircles increased 4-fold in the presence of 1 mM Zn²⁺ compared with 1 mM Mg²⁺. Kinking persisted in mixed Mg²⁺/Zn²⁺ electrolytes until the Zn²⁺ concentration dropped below 100 μM, indicating that this type of kinking may occur under physiological conditions. Kinking appears to replace intrinsic bending, and statistical analysis shows that kinks are not localized within any single sequence element. A surprisingly small free energy is associated with kink formation.