bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on mu and delta opioid receptor selectivity utilizing chimeric and site-mutagenized receptors.
Authors: Wang W W , Shahrestanifar M , Jin J , Howells R D
Published: May 2006

Opioid receptors are members of the guanine nucleotide binding protein (G protein)-coupled receptor family. Three types of opioid receptors have been cloned and characterized and are referred to as the delta, kappa and mu types. Analysis of receptor chimeras and site-directed mutant receptors has provided a great deal of information about functionally important amino acid side chains that constitute the ligand-binding domains and G-protein-coupling domains of G-protein-coupled receptors. We have constructed delta/mu opioid receptor chimeras that were express in human embryonic kidney 293 cells in order to define receptor domains that are responsible for receptor type selectivity. All chimeric receptors and wild-type delta and mu opioid receptors displayed high-affinity binding of etorphine (an agonist), naloxone (an antagonist), and bremazocine (a mixed agonist/antagonist). In contrast, chimeras that lacked the putative first extracellular loop of the mu receptor did not bind the mu-selective peptide [D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO). Chimeras that lacked the putative third extracellular loop of the delta receptor did not bind the delta-selective peptide, [D-Ser2,D-Leu5]enkephalin-Thr (DSLET). Point mutations in the putative third extracellular loop of the wild-type delta receptor that converted vicinal arginine residues to glutamine abolished DSLET binding while not affecting bremazocine, etorphine, and naltrindole binding. We conclude that amino acids in the putative first extracellular loop of the mu receptor are critical for high-affinity DAMGO binding and that arginine residues in the putative third extracellular loop of the delta receptor are important for high-affinity DSLET binding.
UME6 is a central component of a developmental regulatory switch controlling meiosis-specific gene expression.
Authors: Steber C M , Esposito R E
Published: May 2006

The UME6 gene of Saccharomyces cerevisiae was identified as a mitotic repressor of early meiosis-specific gene expression. It encodes a Zn2Cys6 DNA-binding protein which binds to URS1, a promoter element needed for both mitotic repression and meiotic induction of early meiotic genes. This paper demonstrates that a complete deletion of UME6 causes not only vegetative derepression of early meiotic genes during vegetative growth but also a significant reduction in induction of meiosis-specific genes, accompanied by a severe defect in meiotic progression. After initiating premeiotic DNA synthesis the vast majority of cells (approximately 85%) become arrested in prophase and fail to execute recombination; a minority of cells (approximately 15%) complete recombination and meiosis I, and half of these form asci. Quantitative analysis of the same early meiotic transcripts that are vegetatively derepressed in the ume6 mutant, SPO11, SPO13, IME2, and SPO1, indicates a low level of induction in meiosis above their vegetative derepressed levels. In addition, the expression of later meiotic transcripts, SPS2 and DIT1, is significantly delayed and reduced. The expression pattern of early meiotic genes in ume6-deleted cells is strikingly similar to that of early meiotic genes with promoter mutations in URS1. These results support the view that UME6 and URS1 are part of a developmental switch that controls both vegetative repression and meiotic induction of meiosis-specific genes.
Frequent somatic hypermutation of the 5' noncoding region of the BCL6 gene in B-cell lymphoma.
Authors: Migliazza A , Martinotti S , Chen W , Fusco C , Ye B H , Knowles D M , Offit K , Chaganti R S , Dalla-Favera R
Published: May 2006

The BCL6 gene encodes a zinc-finger transcription factor and is altered by chromosomal arrangements in its 5' noncoding region in approximately 30% of diffuse large-cell lymphoma (DLCL). We report here that, in 22/30 (73%) DLCL and 7/15 (47%) follicular lymphoma (FL), but not in other tumor types, the BCL6 gene is also altered by multiple (1.4 x 10(-3) -1.6 x 10(-2) per bp), often biallelic, mutations clustering in its 5' noncoding region. These mutations are of somatic origin and are found in cases displaying either normal or rearranged BLC6 alleles indicating their independence from chromosomal rearrangements and linkage to immunoglobulin genes. These alterations identify a mechanism of genetic instability in malignant B cells and may have been selected during lymphomagenesis for their role in altering BCL6 expression.
A tissue-specific promoter that can drive a foreign gene to express in the suprabasal urothelial cells of transgenic mice.
Authors: Lin J H , Zhao H , Sun T T
Published: May 2006

Uroplakins are a group of integral membrane proteins that are synthesized as the major differentiation products of urothelium. The luminal portions of these proteins form 12-nm protein particles arranged in a two-dimensional crystalline array. The expression of uroplakin genes is bladder specific and differentiation dependent; little is known, however, about their molecular regulation. Here we describe the cloning of mouse uroplakin II gene and demonstrate, in transgenic mouse experiments, that a 3.6-kb 5'-flanking sequence of this gene can drive a bacterial lacZ (reporter) gene to express in the suprabasal cell layers of the urothelium. The transgene was not expressed in any tested (nonurothelial) epithelial and other tissues (except hypothalamus). These results suggest that most of the cis elements that confer the bladder-specific and differentiation-dependent expression of mouse uroplakin II gene must reside in the 3.6-kb sequence. The availability of a promoter capable of delivering a foreign molecule to the differentiated cell layers of bladder epithelium opens avenues for studying normal and pathological urothelial differentiation in transgenic mice.
Immunization of low density lipoprotein (LDL) receptor-deficient rabbits with homologous malondialdehyde-modified LDL reduces atherogenesis.
Authors: Palinski W , Miller E , Witztum J L
Published: May 2006

Atherosclerotic lesions contain oxidized LDL (OxLDL), immunoglobulins, and immune-competent cells. Low levels of circulating autoantibodies against malondialdehyde (MDA)-modified lysine, an epitope of OxLDL, occur in several species, and immune complexes between such autoantibodies and OxLDL are present in lesions. To study the potential role of autoantibodies against OxLDL in the atherogenic process, we prospectively hyperimmunized LDL receptor-deficient rabbits with homologous MDA-LDL and determined the effects of this intervention on the development of atherosclerosis. Immunization with MDA-LDL generated high titers of antibodies with similar specificity as naturally occurring autoantibodies. Immunized animals showed a significant reduction in the extent of atherosclerotic lesions in the aortic tree after 6.5 months, compared with "saline"-immunized controls (48% vs. 68%, P < 0.005). Immunization with keyhole limpet hemocyanin produced no change in lesion formation. Although the mechanisms by which immunization led to a protective effect are unknown, these results suggest an important role for the immune system in modulating the atherogenic process and may indicate a novel approach for inhibiting the progression of atherosclerosis.
Specific 5'-GGGA-3'-->5'-GGA-3' mutation of the Apc gene in rat colon tumors induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.
Authors: Kakiuchi H , Watanabe M , Ushijima T , Toyota M , Imai K , Weisburger J H , Sugimura T , Nagao M
Published: May 2006

The APC gene plays a major role in human colon carcinogenesis. We determined the genomic structure of the rat Apc gene, and we analyzed mutations in colon tumors induced in F344 rats by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), potent carcinogens contained in ordinary daily human food. Of eight PhIP-induced tumors, one tumor had two Apc mutations, two tumors had a mutation with loss of the normal allele, and one had a mutation. Two of the above five mutations were at nucleotide 1903, one at 2605, and two at 4237, all being a deletion of a guanine base at the 5'-GGGA-3' site and resulting in truncation of the APC protein. Of 13 IQ-induced tumors, 2 had an Apc mutation with loss of the normal allele. The two mutations were a missense mutation (T-->C) at nucleotide 1567 and a nonsense mutation (C-->T) at 2761. Alteration of the Apc gene was shown to play a more important role in PhIP-induced than in IQ-induced rat colon carcinogenesis. PhIP-induced tumors are characterized by their specific and unique mutation, which may be useful for mutational fingerprinting of human cancers.
Functional neuroanatomy of antisaccade eye movements investigated with positron emission tomography.
Authors: O'Driscoll G A , Alpert N M , Matthysse S W , Levy D L , Rauch S L , Holzman P S
Published: May 2006

Increasing interest in the role of the frontal lobe in relation to psychiatric and neurologic disorders has popularized tests of frontal function. One of these is the antisaccade task, in which both frontal lobe patients and schizophrenics are impaired despite normal performance on (pro)saccadic tasks. We used position emission tomography to examine the cerebral blood flow changes associated with the performance of antisaccades in normal individuals. We found that the areas of the brain that were more active during antisaccades than saccades were highly consistent with the oculomotor circuit, including frontal eye fields (FEFs), supplementary motor area, thalamus, and putamen. Superior parietal lobe and primary visual cortex were also significantly more active. In contrast, prefrontal areas 46 and 9 were not more active during antisaccades than during saccades. Performance of some frontal patients on the antisaccade task has been likened to a bradykinesia, or the inability to initiate a willed movement. It is the necessity to will the movement and inhibit competing responses that intuitively linked this task to the dorsolateral prefrontal cortex in frontal patients. Our data suggest that it is the FEFs in prefrontal cortex that differentiate between conditions in which the required oculomotor response changes while the stimulus remains the same, rather than areas 46 and 9, which, in human studies, have been linked to the performance of complex cognitive tasks. Such a conclusion is consistent with single-unit studies of nonhuman primates that have found that the FEFs, the executive portion of the oculomotor circuit, can trigger, inhibit, and set the target of saccades.
Vasopressin increases water permeability of kidney collecting duct by inducing translocation of aquaporin-CD water channels to plasma membrane.
Authors: Nielsen S , Chou C L , Marples D , Christensen E I , Kishore B K , Knepper M A
Published: May 2006

Water excretion by the kidney is regulated by the peptide hormone vasopressin. Vasopressin increases the water permeability of the renal collecting duct cells, allowing more water to be reabsorbed from collecting duct urine to blood. Despite long-standing interest in this process, the mechanism of the water permeability increase has remained undetermined. Recently, a molecular water channel (AQP-CD) has been cloned whose expression appears to be limited to the collecting duct. Previously, we immunolocalized this water channel to the apical plasma membrane (APM) and to intracellular vesicles (IVs) of collecting duct cells. Here, we test the hypothesis that vasopressin increases cellular water permeability by inducing exocytosis of AQP-CD-laden vesicles, transferring water channels from IVs to APM. Rat collecting ducts were perfused in vitro to determine water permeability and subcellular distribution of AQP-CD in the same tubules. The collecting ducts were fixed for immunoelectron microscopy before, during, and after exposure to vasopressin. Vasopressin exposure induced increases in water permeability and the absolute labeling density of AQP-CD in the APM. In parallel, the APM:IV labeling ratio increased. Furthermore, in response to vasopressin withdrawal, AQP-CD labeling density in the APM and the APM:IV labeling ratio decreased in parallel to a measured decrease in osmotic water permeability. We conclude that vasopressin increases the water permeability of collecting duct cells by inducing a reversible translocation of AQP-CD water channels from IVs to the APM.
Subunit functional studies of NAD(P)H:quinone oxidoreductase with a heterodimer approach.
Authors: Cui K , Lu A Y , Yang C S
Published: May 2006

NAD(P)H:quinone oxidoreductase (NQOR; EC 1.6.99.2) is a homodimeric enzyme which catalyzes the reduction of quinones, azo dyes, and other electron acceptors by NADPH or NADH. To pursue subunit functional studies, we expressed a wild-type/mutant heterodimer of NQOR in Escherichia coli. The wild-type subunit of the heterodimer was tagged with polyhistidine and the other subunit contained a His-194-->Ala mutation (H194A), a change known to dramatically increase the Km for NADPH. This approach enabled us to efficiently purify the heterodimer (H194A/HNQOR) from the homodimers by stepwise elution with imidazole from a nickel nitrilotriacetate column under nondenaturing conditions. The composition of the purified heterodimer was confirmed by SDS and nondenaturing polyacrylamide gel electrophoresis and immunoblot analysis. The enzyme kinetics of the purified heterodimer were studied with two two-electron acceptors, 2,6-dichloroindophenol and menadione, and a four-electron acceptor, methyl red, as the substrates. With two-electron acceptors, the Km(NADPH) and Km(NADH) values of the heterodimer H194A/HNQOR were virtually identical to those of the wild-type homodimer, but the kcat-(NADPH) and kcat(NADH) values were only about 50% those of the wild-type homodimer. With the four-electron acceptor, the Km and kcat values of H194A/HNQOR for NADPH and NADH were similar to those of the low-efficiency mutant homodimer. These results suggest that the subunits of NQOR function independently with two-electron acceptors, but dependently with a four-electron acceptor. This heterodimer approach may have general applications for studying the functional and structural relationships of subunits in dimeric or oligomeric proteins.
Internal and interfacial dielectric properties of cytochrome c from molecular dynamics in aqueous solution.
Authors: Simonson T , Perahia D
Published: May 2006

The dielectric properties of proteins are central to their stability and activity. We use the Fröhlich-Kirkwood theory of dielectrics to analyze two 1-ns molecular dynamics simulations of ferro- and ferricytochrome c in spherical droplets of 1400 water molecules. Protein and solvent are idealized as a series of concentric, spherical, dielectric media. Analysis results depend strongly on the treatment of the charged protein side chains at the protein/solvent interface. If charged side chains are viewed as part of the protein medium, then the protein dipole fluctuations are dominated by large, mutually uncorrelated, anisotropic, motions of the charged side chains. It is then incorrect to view the protein region as a single, homogeneous dielectric material. If one does take this view, estimates of the protein "dielectric constant" vary from 16 to 37, depending on the exact choice of model parameters. In contrast, if the charged portions of the charged side chains are viewed as part of the solvent medium, then theory and simulation are consistent: the protein dipole fluctuations excluding charged side chains are roughly those of a homogeneous, isotropic dielectric medium, with a dielectric constant of 4.7 +/- 1.0 (ferro) or 3.4 +/- 1.0 (ferri), in agreement with powder experiments. Statistical uncertainty and sensitivity to model parameters are small. Analysis of the radial dependence of the dipole fluctuations suggests that the inner half of the protein has a somewhat lower dielectric constant of 1.5-2, consistent with its biological function in electron transfer. These results suggest that Poisson-Boltzmann models could treat the protein bulk as a low-dielectric medium and the charged surface groups as part of the solvent region.
Independent regulation of sterol regulatory element-binding proteins 1 and 2 in hamster liver.
Authors: Sheng Z , Otani H , Brown M S , Goldstein J L
Published: May 2006

Two sterol regulatory element-binding proteins (SREBPs, designated SREBP-1 and SREBP-2), each approximately 1150 amino acids in length, are attached to membranes of the endoplasmic reticulum and nuclear envelope in human and hamster tissue culture cells. In the absence of sterols, soluble fragments of approximately 470 amino acids are released from both proteins by proteolytic cleavage. The soluble fragments enter the nucleus, where they bind to sterol regulatory elements in the promoters of genes encoding the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl CoA synthase, thereby activating transcription. Proteolytic processing of both SREBPs is blocked coordinately by sterol overloading and enhanced coordinately when sterols are depleted by treatment with an inhibitor of cholesterol synthesis. In contrast to these findings in cultured cells, the current data show that SREBP-1 and -2 are not coordinately regulated in hamster liver. In untreated animals the soluble fragment of SREBP-1, but not of SREBP-2, was detected by immunoblotting of a liver nuclear extract. Depletion of sterols by treatment with a bile acid-binding resin (colestipol) and a cholesterol synthesis inhibitor (mevinolin) led to a marked increase in the nuclear form of SREBP-2 and a reciprocal decline in the nuclear form of SREBP-1. These findings suggest that SREBP-1 is responsible for basal transcription of the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl CoA synthase genes in hamster liver and that SREBP-2 is responsible for the increased transcription that follows sterol depletion with a bile acid-binding resin and a cholesterol synthesis inhibitor.
Maize branching enzyme catalyzes synthesis of glycogen-like polysaccharide in glgB-deficient Escherichia coli.
Authors: Guan H , Kuriki T , Sivak M , Preiss J
Published: May 2006

The structure of alpha-glucan, isolated from wild-type Escherichia coli B, a glycogen branching enzyme (BE)-deficient E. coli AC71 (glgB-), or from AC71 transformed with genes coding for maize BEI and BEII individually as well as with both genes, was analyzed by high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection. Transformation of the maize BE gene(s) in AC71 (glgB-) showed complementation in branching activity. Analysis by HPAEC revealed different structures between glycogen of E. coli B and alpha-glucan of AC71 transformed with a different maize BE gene(s). The individual chains of the alpha-glucan debranched with isoamylase were distributed between chain length (CL) 3 and > 30 and the chain with CL 6 was the most abundant. In comparison with the glycogen of E. coli B, the alpha-glucan of AC71 transformed with the maize BE gene(s) consisted of a lesser amount of chains with CL 7-9 and a larger amount of chains with CL > 14. It also showed a broad peak with chains of CL 9-12 as in maize amylopectin. This study provides in vivo evidence that glycogen BE and maize BE isozymes may have different specificities in the length of chain transferred. Furthermore, this study suggests that the specificity of glycogen synthase and starch synthase and their concerted action with BE play an important role in determining the structure of the polysaccharide synthesized.
Sexual selection in plants: pros and cons.
Authors: Grant V
Published: May 2006

Sexual selection has long been regarded as a special mode of selection in animals. Various authors have argued in a growing number of publications since 1979 that sexual selection is also operative in plants, and consequently, two divergent concepts of sexual selection are now being stated in the literature, the original Darwinian concept and the new plant-centered concept. An essential feature of the Darwinian concept is the distinction between primary and secondary sexual characters. Sexual selection is a process producing secondary sexual characters in males; such characters are well known in animals but are unknown in plants. Proponents of sexual selection in plants do not mention the subject of primary and secondary sexual characters, and they make no effort to establish the existence of secondary sexual characters in plants. The evidence they do present for sexual selection in plants consists of primary sexual characters and other reproductive traits that are products of selection modes other than sexual selection.
Comparison of genetically engineered herpes simplex viruses for the treatment of brain tumors in a scid mouse model of human malignant glioma.
Authors: Chambers R , Gillespie G Y , Soroceanu L , Andreansky S , Chatterjee S , Chou J , Roizman B , Whitley R J
Published: May 2006

Genetically engineered viruses and viral genes inserted into retroviral vectors are increasingly being considered for experimental therapy of brain tumors. A primary target of these viruses and vectors is human gliomas, the most frequently occurring primary human brain tumor. To investigate the potential of genetically engineered herpes simplex viruses (HSVs) in the therapy of these tumors, we compared the attributes of two viruses, a recombinant from which the gamma 1(34.5) gene had been deleted (R3616) and a recombinant in which the gamma 1(34.5) gene had been interrupted by a stop codon (R4009). Previous studies have shown that these recombinants were completely devoid of the ability to multiply in the central nervous system of rodents. To pursue these studies, we developed a scid mouse glioma model. Tumor cell response (survival) for 10(3), 10(4), and 10(5) implanted MT539MG glioma cells was 38, 23, and 15 days, respectively. The results were as follows: (i) both R3616 and R4009 replicate and cause cytolysis in diverse glioma cell lines of murine and human origin in vitro, and (ii) Winn-type assays 10(5) MT539MG cells coinoculated with R3616 or R4009 as compared to saline significantly prolonged survival in a dose-dependent fashion. Mice that received only tumor cells or the wild-type parent strain of the recombinants, HSV-1(F), died within 15 days. Survival was greatest with R4009. These experiments define both a model for screening oncolytic viruses and a genetically engineered virus of significant potential use as an oncolytic agent.
Acute hepatitis in rats expressing human hepatitis B virus transgenes.
Authors: Takahashi H , Fujimoto J , Hanada S , Isselbacher K J
Published: May 2006

The molecular mechanisms responsible for hepatocyte death and the events leading to viral clearance in hepatitis B virus (HBV) infections are not well understood. Elucidation of the mechanisms involved have been complicated by the difficulty of infecting human hepatocytes with HBV in vitro and the lack of an appropriate animal model. We report an animal model of human HBV infection by in vivo transfection. We have directly introduced a replication-competent, cloned HBV construct into rat liver by using a membrane fusion-promoting cationic lipid. HBV mRNA and 3.2-kb HBV DNA were expressed in the liver by this in vivo transfection method. In the majority of rats, HBV virions and hepatitis B e antigen were found in the blood 3-7 days after transfection, after which antibody to the e antigen appeared. Two to three weeks after the transfection, glutamic-pyruvic transaminase levels were elevated in serum, hepatocyte death and lymphocyte infiltration were observed in the vicinity of the portal vein of liver, and HBV virions were no longer detected in the serum. Thus, transfection of HBV into rats resulted in histological and serological changes comparable to HBV-induced acute hepatitis in humans. In contrast, no hepatocellular injury was observed in T-lymphocyte-deficient nude rats transfected with the same HBV construct, and viremia was substantially prolonged, providing direct evidence that T lymphocytes play an essential role in liver cell injury and in the clearance of HBV. This rat hepatitis model will be useful for studying pathogenesis of HBV infection.
Differential expression of SNAP-25 protein isoforms during divergent vesicle fusion events of neural development.
Authors: Bark I C , Hahn K M , Ryabinin A E , Wilson M C
Published: May 2006

The presynaptic plasma membrane protein SNAP-25 (synaptosome-associated protein of 25 kDa) has been implicated as one of several neural-specific components that direct constitutive fusion mechanisms to the regulated vesicle trafficking and exocytosis of neurotransmitter release. There exist two alternatively spliced isoforms of SNAP-25, a and b, which differ in a putative membrane-interacting domain. We show that these two isoforms have distinct quantitative and anatomical patterns of expression during brain development, in neurons, and in neuroendocrine cells and that the proteins localize differently in neurites of transfected PC12 pheochromocytoma cells. These findings indicate that alternative isoforms of SNAP-25 may play distinct roles in vesicular fusion events required for membrane addition during axonal outgrowth and for release of neuromodulatory peptides and neurotransmitters.
Opposing mechanisms of regulation of a G-protein-coupled inward rectifier K+ channel in rat brain neurons.
Authors: Velimirovic B M , Koyano K , Nakajima S , Nakajima Y
Published: May 2006

In locus coeruleus neurons, substance P (SP) suppresses an inwardly rectifying K+ current via a pertussis toxin-insensitive guanine nucleotide binding protein (G protein; GnonPTX), whereas somatostatin (SOM) or [Met]enkephalin (MENK) enhances it via a pertussis toxin-sensitive G protein (GPTX). The interaction of the SP and the SOM (or MENK) effects was studied in cultured locus coeruleus neurons. In neurons loaded with guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), application of SOM (or MENK) evoked a persistent increase in the inward rectifier K+ conductance. A subsequent application of SP suppressed this conductance to a level less than that before the SOM (or MENK) application; the final conductance level was independent of the magnitude of the SOM (or MENK) response. This suppression by SP was persistent, and a subsequent SOM (or MENK) application did not reverse it. When SP was applied to GTP[gamma S]-loaded cells first, subsequent SOM elicited only a small response. In GTP-loaded neurons, application of SP temporarily suppressed the subsequent SOM- (or MENK)-induced conductance increase. These results suggest that the same inward rectifier molecule that responds to an opening signal from GPTX also responds to a closing signal from GnonPTX. The closing signal is stronger than the opening signal.
A mechanical strain-induced 1-aminocyclopropane-1-carboxylic acid synthase gene.
Authors: Botella J R , Arteca R N , Frangos J A
Published: May 2006

Ethylene production is observed in all higher plants, where it is involved in numerous aspects of growth, development, and senescence. 1-Aminocyclopropane-1-carboxylic acid synthase (ACC synthase; S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14) is the key regulatory enzyme in the ethylene biosynthetic pathway. We are reporting an ACC synthase gene in Vigna radiata (mung bean) that is inducible by mechanical strain. The ACC synthase cDNA AIM-1 was induced by mechanical strain within 10 min, reaching a maximum at 30 min, showing a dramatic reduction after 60 min, and showing no detectable message by 3 hr. The kinetics of induction for AIM-1 was similar to a mechanical strain-induced calmodulin (MBCaM-1) in V. radiata, whereas the kinetics of its decline from maximum was different. When plants were subjected to calcium-deficient conditions, supplemental calcium, calcium chelators, calcium storage releasers, calcium ionophore, or calmodulin antagonists, there was no effect on AIM-1, indicating that the mechanical strain-induced AIM-1 expression is a calcium-independent process. Induction of MBCaM-1 in all cases behaved in the same way as AIM-1, suggesting that they share similar mechanically activated cis- and/or trans-acting elements in their promoter.
Autocrine regulation of toxin synthesis by Staphylococcus aureus.
Authors: Balaban N , Novick R P
Published: May 2006

Staphylococcus aureus is a major human pathogen causing diseases which range from minor skin infection to endocarditis and toxic shock syndrome. The pathogenesis of S. aureus is due primarily to the production of toxic exoproteins, whose synthesis is controlled by a global regulatory system, agr. We show here that agr is autoinduced by a proteinaceous factor produced and secreted by the bacteria and that it is inhibited by a peptide produced by an exoprotein-deficient S. aureus mutant strain. The inhibitor, RIP, competes with the activator, RAP, and may be a mutational derivative. Our results suggest two possible approaches, independent of antibiotics, to the control of S. aureus infections. RIP may prove useful as a direct inhibitor of virulence and RAP as a vaccine against the expression of agr-induced virulence factors; either could interfere with the ability of the bacteria to establish and maintain an infection.
The RNA-binding protein TIAR is translocated from the nucleus to the cytoplasm during Fas-mediated apoptotic cell death.
Authors: Taupin J L , Tian Q , Kedersha N , Robertson M , Anderson P
Published: May 2006

We have determined the structure, intracellular localization, and tissue distribution of TIAR, a TIA-1-related RNA-binding protein. Two related isoforms of TIAR, migrating at 42 and 50 kDa, are expressed in primate cells. Unlike TIA-1, which is found in the granules of cytotoxic lymphocytes, TIAR is concentrated in the nucleus of hematopoietic and nonhematopoietic cells. Because TIAR can trigger DNA fragmentation in permeabilized thymocytes, it is a candidate effector of apoptotic cell death. Consistent with this possibility, we have found that the expression and intracellular localization of TIAR change dramatically during Fas-mediated apoptosis. TIAR moves from the nucleus to the cytoplasm within 30 min of Fas ligation. Redistribution of TIAR precedes the onset of DNA fragmentation and is not a nonspecific consequence of nuclear disintegration. Cytoplasmic redistribution of TIAR is not observed during cellular activation triggered by mitogens such as concanavalin A or phytohemagglutinin. Our results suggest that cytoplasmic redistribution of TIAR may be a general feature of the apoptotic program.
Dopamine receptors in the substantia nigra are involved in the regulation of muscle tone.
Authors: Double K L , Crocker A D
Published: May 2006

The aim of the present study was to localize the dopamine receptors involved in the regulation of muscle tone. A strategy was used whereby the effects on muscle tone of injecting the irreversible dopamine receptor antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) in discrete brain regions were assessed. Increases in muscle tone were measured as changes in electromyographic activity of the gastrocnemius and tibialis muscles of conscious, unrestrained rats. No increases in muscle tone were found after injections of EEDQ into the anterior and posterior striatum, which produced marked reductions in dopamine receptor concentration. The effects of muscle tone of injecting EEDQ into the substantia nigra pars reticulata were also assessed. Large increases in muscle tone were observed associated with inactivation of either D1 or D2 dopamine receptors in the substantia nigra. The increased muscle tone was not reduced by subcutaneous administration of apomorphine, despite the presence of a normal population of striatal dopamine receptors. These findings provide evidence that dopamine receptors in the substantia nigra play an important role in the regulation of muscle tone. Further, they challenge the hypothesis that the muscle rigidity of Parkinson disease results primarily from loss of striatal dopamine receptor stimulation.
Mechanistically different catalytic antibodies obtained from immunization with a single transition-state analog.
Authors: Guo J , Huang W , Zhou G W , Fletterick R J , Scanlan T S
Published: May 2006

The variable-region peptide sequence and steady-state kinetic behavior are compared for a family of catalytic antibodies that arose from the same immune response to a transition-state analog. The crystal structure of the most catalytically active member of the family (17E8) has been solved to 2.5 A resolution and shows that the antibody active site contains a SerH99-HisH35 (H = heavy chain) catalytic dyad analogous to the Ser-His-Asp catalytic triad of serine proteases. The variable-region peptide sequence of the next most active antibody (29G11) differs from that of 17E8 by nine heavy-chain point mutations, and results from computer modeling suggest that the three-dimensional structure of 29G11 is similar to that of 17E8. In addition, 29G11 is an efficient catalytic antibody; it possesses 26% of the hydrolytic activity of 17E8. There is one active-site mutation in 29G11 compared to 17E8; position 99 of the heavy chain of 29G11 contains a glycine residue in place of the nucleophilic serine at this position in 17E8. Consistent with this mutation, results from pH-rate studies and hydroxylamine partitioning experiments indicate that in contrast to the catalytic mechanism of 17E8, the mechanism of 29G11-catalyzed esterolysis does not feature nucleophilic catalysis.
Targeted gene transfer into hepatoma cells with lipopolyamine-condensed DNA particles presenting galactose ligands: a stage toward artificial viruses.
Authors: Remy J S , Kichler A , Mordvinov V , Schuber F , Behr J P
Published: May 2006

Optimal in vitro gene delivery with cationic lipids requires an excess of cationic charges with respect to DNA phosphates. In these conditions, in vivo delivery will be hampered by interference from cationic lipid-binding macromolecules either circulating or in the extracellular matrix. To overcome this problem, we are developing a modular transfection system based on lipid-coated DNA particles reminiscent of enveloped viruses. The particle core consists of the lipopolyamine-condensed nucleic acid in an electrically neutral ratio to which other synthetic lipids with key viral properties are hydrophobically adsorbed. As a first result, we have found that a good transfection level can be achieved simply with the neutral core particle, provided a zwitterionic lipid (dioleoyl phosphatidylethanolamine) is added to completely coat the DNA. Addition of lipids bearing a fusogenic or a nuclear localization peptide head group to the particles does not significantly improve an already efficient system, in contrast to polylysine-based gene transfer methods that rely on lysosomotropic or fusogenic agents to be effective. This emphasizes the distinctive properties of the lipopolyamines, including cell membrane destabilization, endosome buffering capacity, and possibly nuclear tropism. Most importantly, addition of lipids with a triantennary galactosyl residue drives the neutral nucleolipidic particles to the asialoglycoprotein receptor of human hepatoma HepG2 cells: Transfection increases approximately 1000-fold with 25% galactolipid. This receptor-mediated process is saturable and slightly less efficient than receptor-independent transfection obtained in vitro with a large excess of cationic lipid alone. Yet, electrically silent particles may provide an attractive solution for gene transfer in vivo where their external saccharide coat should allow them to diffuse within the organism and reach their target cells.
Allele-specific increase in basal transcription of the plasminogen-activator inhibitor 1 gene is associated with myocardial infarction.
Authors: Eriksson P , Kallin B , van 't Hooft F M , Båvenholm P , Hamsten A
Published: May 2006

Increased plasminogen-activator inhibitor 1 (PAI-1) activity is a common finding in patients with coronary heart disease. Here we provide evidence for an independent, etiological role of PAI-1 in myocardial infarction. The 4G allele of a recently described common 4/5-guanine-tract (4G/5G) polymorphism in the PAI-1 promoter is associated with higher plasma PAI-1 activity. The prevalence of the 4G allele is significantly higher in patients with myocardial infarction before the age of 45 than in population-based controls (allele frequencies of 0.63 vs. 0.53). Both alleles bind a transcriptional activator, whereas the 5G allele also binds a repressor protein to an overlapping binding site. In the absence of bound repressor, the basal level of PAI-1 transcription is increased.
SopB protein-mediated silencing of genes linked to the sopC locus of Escherichia coli F plasmid.
Authors: Lynch A S , Wang J C
Published: May 2006

Expression of a high level of F-plasmid-encoded SopB protein in Escherichia coli is found to repress genes linked to sopC, a sequence element of F consisting of 12 tandemly joined imperfect repeats of a 43-bp motif. Repression of a gene can occur over a distance of at least 10 kb from the sopC element and is not affected by the relative orientation of sopC. In the repressed state, accessibility of intracellular DNA to cellular proteins is greatly reduced in the region containing sopC, as monitored by the trapping of the covalent intermediate between DNA and DNA gyrase and by Dam methylase-catalyzed DNA methylation. These results signify the formation of a nucleoprotein structure emanating from sopC and are discussed in terms of position-dependent silencing of genes in general and the IncG type of plasmid incompatibility in particular.
The thermoelastic basis of short pulsed laser ablation of biological tissue.
Authors: Itzkan I , Albagli D , Dark M L , Perelman L T , von Rosenberg C , Feld M S
Published: May 2006

Strong evidence that short-pulse laser ablation of biological tissues is a photomechanical process is presented. A full three-dimensional, time-dependent solution to the thermoelastic wave equation is compared to the results of experiments using an interferometric surface monitor to measure thermoelastic expansion. Agreement is excellent for calibrations performed on glass and on acrylic at low laser fluences. For cortical bone, the measurements agree well with the theoretical predictions once optical scattering is included. The theory predicts the presence of the tensile stresses necessary to rupture the tissue during photomechanical ablation. The technique is also used to monitor the ablation event both before and after material is ejected.
Carbon monoxide: a role in carotid body chemoreception.
Authors: Prabhakar N R , Dinerman J L , Agani F H , Snyder S H
Published: May 2006

Carbon monoxide (CO), produced endogenously by heme oxygenase, has been implicated as a neuronal messenger. Carotid bodies are sensory organs that regulate ventilation by responding to alterations of blood oxygen, CO2, and pH. Changes in blood gases are sensed by glomus cells in the carotid body that synapse on afferent terminals of the carotid sinus nerve that projects to respiratory-related neurons in the brainstem. Using immunocytochemistry, we demonstrate that heme oxygenase 2 is localized to glomus cells in the cat and rat carotid bodies. Physiological studies show that zinc protoporphyrin IX, a potent heme oxygenase inhibitor, markedly increases carotid body sensory activity, while copper protoporphyrin IX, which does not inhibit the enzyme, is inactive. Exogenous CO reverses the stimulatory effects of zinc protoporphyrin IX. These results suggest that glomus cells are capable of synthesizing CO and endogenous CO appears to be a physiologic regulator of carotid body sensory activity.
Analysis of the human guanylin gene and the processing and cellular localization of the peptide.
Authors: Hill O , Kuhn M , Zucht H D , Cetin Y , Kulaksiz H , Adermann K , Klock G , Rechkemmer G , Forssmann W G , Mägert H J
Published: May 2006

The complete cell biological analysis of human guanylin, a recently discovered regulatory peptide, is offered in this investigation: (i) the nucleotide sequence of the gene, (ii) the isolation and characterization of its circulating molecular form, and (iii) its localization in enterochromaffin cells of the gut. As determined by molecular cloning, DNA sequencing, and comparison with the known cDNA sequence, the approximately 2.6-kbp large gene consists of three exons interrupted by two introns. The putative promoter region contains a TTTAAAA sequence motif and several potential binding sites for transcription factors such as AP-1, AP-2, Sp 1, and glucocorticoid receptors. The isolated hormonal form of guanylin is a 94-amino acid peptide with a molecular mass of 10.3 kDa. Western blot analysis of RP-HPLC fractions from blood plasma confirms this molecular form. Thus, guanylin is synthesized by gut enterochromaffin cells as a prohormone of 115 amino acids and is processed to the molecular form of 94 amino acids circulating in the blood.
Stark-effect experiments on photochemical holes in chromoproteins: protoporphyrin IX-substituted myoglobin.
Authors: Gafert J , Friedrich J , Parak F
Published: May 2006

We performed comparative Stark-effect experiments on spectral holes in a protein and a glass sample. The protein was protoporphyrin IX-substituted myoglobin in a glycerol/water solvent. The glass sample was a protoporphyrin IX-doped mixture of dimethylformamide/glycerol. As expected, in both cases the spectral holes varied linearly with the electric field. Yet, whereas in the protein the holes showed a clear splitting, they showed no splitting in the glass sample, irrespective of the chosen polarization of the laser. In both samples the hole broadened in the applied field. The magnitude of the broadening was about the same in both cases. The following conclusions were drawn. The absence of a splitting in the glass signals an effective global inversion symmetry of the chromophore, despite its low symmetry group. The dipole moment changes are random. In the protein the inversion symmetry is broken through the spatial correlation of the protein building blocks, leading to a molecular frame-fixed dipole moment difference and, hence, to the observed splitting. Despite these symmetry-breaking properties, the local structural randomness is of the same magnitude in the glass and in the protein, as is obvious from the broadening. The distinct difference in the Stark pattern shows that the range of the relevant chromophore interactions is confined to typical dimensions of the protein.
Heat shock protein hsp70 accelerates the recovery of heat-shocked mammalian cells through its modulation of heat shock transcription factor HSF1.
Authors: Kim D , Ouyang H , Li G C
Published: May 2006

The role of mammalian 70-kDa heat shock protein (hsp70) in regulating cellular response to heat shock was examined by using three closely related rat cells: control Rat-1 cells, thermotolerant Rat-1 (TT Rat-1) cells, and heat-resistant M21 cells, a derivative of Rat-1 cells that constitutively overexpress human hsp70. In all these cells, after a prescribed heat shock, the level of the phosphorylated form of heat shock transcription factor HSF1 and that of HSF1 capable of binding to its cognitive DNA sequence heat shock element (HSE) exhibit similar time dependence. The amount of a constitutive HSE-binding activity (CHBA), on the other hand, inversely correlates with those of the two aforementioned forms of HSF1. The recovery kinetics from heat shock are different for the three cell lines, with the thermal-resistant TT Rat-1 and M21 cells showing faster recovery in terms of the state of phosphorylation of HSF1 and its ability to bind HSE or in terms of the reappearance of CHBA. Treatment with okadaic acid, a serine/threonine phosphatase inhibitor, delays the recovery kinetics of Rat-1 cells but not that of thermal-resistant M21 cells. These results are interpreted in terms of a role for hsp70 in the recovery of heat-shocked mammalian cells.
Encoded reaction cassette for the highly sensitive detection of the making and breaking of chemical bonds.
Authors: Fenniri H , Janda K D , Lerner R A
Published: May 2006

A reaction cassette has been designed for the highly sensitive detection of the making and breaking of chemical bonds. The system is envisioned as a companion device to be used in the search for antibody and other novel catalysts. The cassette also may have important clinical applications in the design of diagnostic reagents. In its fully encoded format, this methodology is capable of both detecting and decoding chemical events.
Verification of 50- to 100-mer DNA and RNA sequences with high-resolution mass spectrometry.
Authors: Little D P , Thannhauser T W , McLafferty F W
Published: May 2006

Electrospray ionization with Fourier-transform mass spectrometry achieves accurate (< 50-ppm) determination of molecular weights of nucleotides, verifying structures of biological RNA and synthetic single-stranded DNA. High (1o(5)) resolving power makes possible detection of subpicomole impurities and adducts that confuse lower-resolution measurements. Molecular ions in a spectrum of 76-mer tRNA(Phe) had 34-55 Na adducts; when desalted, these show a molecular mass of 24,950.5 Da (expected, 24,950.3 Da) and minor variants at approximately -15 and +15 Da. A 50-mer DNA is characterized with < 10-ppm mass error, with detection of both N + 1 and N - 1 failure sequences. Special electrospray ionization conditions are necessary for a 72-mer to minimize fragmentation in the ion source. Despite the chemical noise from this, as well as failed sequences from automated synthesis, the spectrum of a 100-mer single-stranded DNA yielded a molecular mass of 30,702.4 +/- 1 Da, in good agreement with the expected value, 30,702.1 Da.
Metaphase chromosome analysis by ligation-mediated PCR: heritable chromatin structure and a comparison of active and inactive X chromosomes.
Authors: Hershkovitz M , Riggs A D
Published: May 2006

We report that ligation-mediated PCR (LMPCR) can be used for high-resolution study of metaphase chromosomes, and we discuss the role of metaphase chromatin structure in the preservation of differentiated cell states. The X chromosome-linked human PGK1 (phosphoglycerate kinase 1) promoter region was investigated, and euchromatic active X chromosome (Xa) metaphase chromatin was compared with interphase Xa chromatin and to heterochromatic inactive X chromosome (Xi) metaphase and interphase chromatin. We find that (i) good-quality data at single-nucleotide resolution can be obtained by LMPCR analysis of dimethyl sulfate-treated intact metaphase cells; (ii) transcription factors present on the Xa promoter of interphase chromatin are not present on metaphase chromatin, establishing that the transcription complex on the PGK1 promoter must form de novo each cell generation; and (iii) the dimethyl sulfate reactivity pattern of Xa and Xi chromatin at metaphase is very similar to that of naked DNA. These results are discussed in the context of models for heritable chromatin structure and epigenetic mechanisms for cell memory, and they are also relevant to more general aspects of chromatin structure and differences between euchromatin and heterochromatin.