bluuebunny/crossref_metadata_2025 huggingface dataset:
Neurotrophin 3 potentiates neuronal activity and inhibits gamma-aminobutyratergic synaptic transmission in cortical neurons.
Authors: Kim H G , Wang T , Olafsson P , Lu B
Published: May 2006

Neurotrophins have traditionally been regarded as slowly acting signals essential for neuronal survival and differentiation. However, brain-derived neurotrophic factor and neurotrophin 3 (NT-3) have recently been reported to exert an acute potentiation of synaptic activity at the amphibian neuromuscular junction. Little is known about the role of neurotrophins on functional synapses in the central nervous system. Here we show that NT-3 rapidly increased the frequency of spontaneous action potentials, and it synchronized excitatory synaptic activities in developing cortical neurons. Moreover, the inhibitory synaptic transmission mediated by gamma-aminobutyric acid (GABA) subtype A receptors was found to be reduced by NT-3. Thus, the excitatory effects of NT-3 on spontaneous action potentials were attributable to a reduction of GABAergic transmission. Our findings, together with previous reports of rapid regulation of central nervous system neurotrophin expression by neuronal activity and of the role of GABAergic transmission in cortical plasticity, suggest a mechanism for modulation of synaptic transmission and activity-dependent synaptic modulation in cortical neurons.
Adenovirus-mediated transfer of the HST-1 (FGF4) gene induces increased levels of platelet count in vivo.
Authors: Sakamoto H , Ochiya T , Sato Y , Tsukamoto M , Konishi H , Saito I , Sugimura T , Terada M
Published: May 2006

The HST-1 (fibroblast growth factor 4, FGF4) protein is a potent mitogen for a variety of cell types of mesodermal and neuroectodermal origin, including fibroblasts, endothelial cells, and melanocytes in vitro. To identify the cells and tissue targets of HST-1 in vivo, adenovirus-mediated HST-1 gene transfer was performed. In nude mice, intraperitoneal injection of 3 x 10(9) plaque-forming units of adenoviruses carrying the HST-1 gene (Adex1HST-1L) caused an increase in the number of platelets in the peripheral blood. The number of platelets reached twice the pretreated level by 12 days after the virus injection and the increased level continued up to 20-30 days thereafter. Administration of recombinant HST-1 protein resulted in a transient increase in the platelet count. The number of megakaryocytes in the bone marrow and spleen of the animals with Adex1HST-1L was increased compared with the control animals. Other hematological changes attributed to HST-1 were not observed. Although the mechanisms involved in increased levels of platelet count by HST-1 protein remain to be elucidated, these findings also suggest that adenovirus with the HST-1 gene may be efficiently used for the treatment of thrombocytopenia in various diseases.
Universal biological scaling and mortality.
Authors: Azbel MYa
Published: May 2006

Universal scaling relations for basal metabolism, heart rate, and life-span are presented in a physically transparent form with no adjustable parameters. Their accuracy is a few percent for animals ranging from invertebrates to mammals. They suggest that natural death is related to irreparable molecular damage to specific cells or molecules.
Genetic selection of growth-inhibitory sequences in mammalian cells.
Authors: Pestov D G , Lau L F
Published: May 2006

To assess the role of mitogenically activated genes in the control of cell proliferation, we have taken a genetic approach based on the premise that blocking the function of an essential gene should lead to growth inhibition. Using a newly developed selection procedure, we isolated growth-inhibitory sequences from a pool of random cDNA fragments of 19 growth-related genes associated with the G0/G1 transition. These sequences encode potential dominant negative variants of c-Fos, JunB, and p44MAPK that may interfere with their growth-related functions. We anticipate that this procedure, which allows for the selection of sequences that cause a growth-inhibition phenotype, may have broad applications in the identification and analysis of genes that regulate cell growth.
Spontaneous and restriction enzyme-induced chromosomal recombination in mammalian cells.
Authors: Godwin A R , Bollag R J , Christie D M , Liskay R M
Published: May 2006

We have derived Chinese hamster ovary (CHO) cell hybrids containing herpes simplex virus thymidine kinase (tk) heteroalleles for the study of spontaneous and restriction enzyme-induced interchromosomal recombination. These lines allowed us to make a direct comparison between spontaneous intrachromosomal and interchromosomal recombination using the same tk heteroalleles at the same genomic insertion site. We find that the frequency of interchromosomal recombination is less by a factor of at least 5000 than that of intrachromosomal recombination. Our results with mammalian cells differ markedly from results with Saccharomyces cerevisiae, with which similar studies typically give only a 10-to 30-fold difference. Next, to inquire into the fate of double-strand breaks at either of the two different Xho I linker insertion mutations, we electroporated PaeR7I enzyme, an isoschizomer of Xho I, into these hybrids. A priori, these breaks can be repaired either by recombination from the homology or by end-joining. Despite a predicted bias against recovering end-joining products in our system, all cells characterized by enzyme-induced resistance to hypoxanthine/aminopterin/thymidine were, in fact, due to nonhomologous recombination or end-joining. These results are in agreement with other studies that used extrachromosomal sequences to examine the relative efficiencies of end-joining and homologous recombination in mammalian cells, but are in sharp contrast to results of analogous studies in S. cerevisiae, wherein only products of homologous events are detected.
Infection of vaginal and colonic epithelial cells by the human immunodeficiency virus type 1 is neutralized by antibodies raised against conserved epitopes in the envelope glycoprotein gp120.
Authors: Furuta Y , Eriksson K , Svennerholm B , Fredman P , Horal P , Jeansson S , Vahlne A , Holmgren J , Czerkinsky C
Published: May 2006

The rectal and genital tract mucosae are considered to be major sites of entry for the human immunodeficiency virus (HIV) during sexual contact. We now demonstrate that vaginal epithelial cells can be infected by HIV type 1 (HIV-1) via a mechanism similar to that described for neuroglial cells and, more recently, for colorectal epithelial cells, involving initial interaction of the HIV-1 envelope glycoprotein gp120 with a cell-surface glycosphingolipid (sulfated lactosylceramide). A hyperimmune serum against gp120 was able to neutralize HIV-1 infection of vaginal epithelial cells. Site-directed immunization was employed to identify sites on gp120 recognized by antibodies neutralizing HIV-1 infection of vaginal and colonic epithelial cells. Hyperimmune sera were raised in monkeys against a series of 40 overlapping synthetic peptides covering the entire sequence of HIV-1 (HTLV-IIIB) gp120. Antisera raised against five synthetic peptides, corresponding to three relatively conserved regions and to the hypervariable region (V3 loop), efficiently neutralized HIV-1 infection of human vaginal epithelial cells in vitro. Similar results were obtained with the colonic cells. Hyperimmune sera to all five peptides have been shown earlier to neutralize HIV-1 infectivity in CD4+ T cells. These results have obvious implications for the design of mucosal subunit vaccines against sexually transmitted HIV-1 infections.
A distinct class of intracellular storage vesicles, identified by expression of the glucose transporter GLUT4.
Authors: Herman G A , Bonzelius F , Cieutat A M , Kelly R B
Published: May 2006

Some cell types have cytoplasmic storage vesicles whose fusion with the cell surface is triggered by an extracellular signal. To explore the relationship between different classes of storage vesicles, we expressed, in the neuro-endocrine cell line PC12, the facilitative glucose transporter GLUT4, which is stored in small cytoplasmic vesicles in fat and muscle cells and mobilized to the cell surface when insulin is present. PC12 cells have two known types of storage vesicles, secretory granules and synaptic vesicles, but GLUT4 is targeted to neither. It is recovered, however, in a class of small vesicles that sediment approximately twice as fast as synaptic vesicles. Immunoelectron microscopy confirmed the presence of such small vesicles in transfected PC12 cells. By velocity sedimentation analysis, GLUT4 vesicles efficiently exclude the synaptic vesicle markers synaptophysin, SV2, and synaptobrevin; the transferrin receptor, a marker of conventional endocytosis; and the polymeric immunoglobulin receptor, a marker of transcytosis. The exclusion of synaptophysin and the transferrin receptor from most of the GLUT4-containing structures was confirmed by confocal immunofluorescence microscopy. Like synaptic vesicles, therefore, GLUT4 vesicles of PC12 cells appear to be a unique type of organelle. A GLUT4-containing organelle of identical sedimentation properties was found in transfected fibroblast cell lines and in rat adipocytes. On stimulation of the adipocytes with insulin, GLUT4 was translocated from the peak of small vesicles to faster sedimenting membranes. We propose that the class of vesicles described here is present in a wide range of cell types and is involved in transient modification of the cell surface.
Sodium channel mutations in paramyotonia congenita exhibit similar biophysical phenotypes in vitro.
Authors: Yang N , Ji S , Zhou M , Ptácek L J , Barchi R L , Horn R , George A L
Published: May 2006

Mutations in the skeletal muscle voltage-gated Na+ channel alpha-subunit have been found in patients with two distinct hereditary disorders of sarcolemmal excitation: hyperkalemic periodic paralysis (HYPP) and paramyotonia congenita (PC). Six of these mutations have been functionally expressed in a heterologous cell line (tsA201 cells) using the recombinant human skeletal muscle Na+ channel alpha-subunit cDNA hSkM1. PC mutants from diverse locations in this subunit (T1313M, L1433R, R1448H, R1448C, A1156T) all exhibit a similar disturbance in channel inactivation characterized by reduced macroscopic rate, accelerated recovery, and altered voltage dependence. PC mutants had no significant abnormality in activation. In contrast, one HYPP mutation studied (T704M) has a normal inactivation rate but exhibits shifts in the midpoints of steady-state activation and inactivation along the voltage axis. These findings help to explain the phenotypic differences between HYPP and PC at the molecular and biophysical level and contribute to our understanding of Na+ channel structure and function.
Fungal degradation of recalcitrant nonphenolic lignin structures without lignin peroxidase.
Authors: Srebotnik E , Jensen K A , Hammel K E
Published: May 2006

Lignin peroxidases (LiPs) are likely catalysts of ligninolysis in many white-rot fungi, because they have the unusual ability to depolymerize the major, recalcitrant, non-phenolic structures of lignin. Some white-rot fungi have been reported to lack LiP when grown on defined medium, but it is not clear whether they exhibit full ligninolytic competence under these conditions. To address this problem, we compared the abilities of a known LiP producer, Phanerochaete chrysosporium, with those of a reported nonproducer, Ceriporiopsis subvermispora, to degrade a synthetic lignin with normal phenolic content, a lignin with all phenolic units blocked, and a dimer, 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol, that represents the major nonphenolic structure in lignin. P. chrysosporium mineralized all three models rapidly in defined medium, but C. subvermispora showed appreciable activity only toward the more labile phenolic compound under these conditions. However, in wood, its natural environment, C. subvermispora mineralized all of the models as rapidly as P. chrysosporium did. Defined media therefore fail to elicit a key component of the ligninolytic system in C. subvermispora. A double-labeling experiment with the dimeric model showed that a LiP-dependent pathway was responsible for at least half of dimer mineralization in wood by P. chrysosporium but was responsible for no more than 6-7% of mineralization by C. subvermispora in wood. Therefore, C. subvermispora has mechanisms for degradation of nonphenolic lignin that are as efficient as those in P. chrysosporium but that do not depend on LiP.
Dynamic interaction between Isp45 and mitochondrial hsp70 in the protein import system of the yeast mitochondrial inner membrane.
Authors: Kronidou N G , Oppliger W , Bolliger L , Hannavy K , Glick B S , Schatz G , Horst M
Published: May 2006

The protein import system of the yeast mitochondrial inner membrane includes at least three membrane proteins that presumably form a transmembrane channel as well as several chaperone proteins that mediate the import and refolding of precursor proteins. We show that one of the membrane proteins, Isp45, spans the mitochondrial inner membrane yet is extracted from this membrane at high pH. Solubilization of mitochondria with a nonionic detergent releases Isp45 as a complex with the chaperones mitochondrial hsp70 (mhsp70) and GrpEp. Both chaperones reversibly dissociate from Isp45 upon addition of ATP or adenosine 5'-[gamma-thio]triphosphate, suggesting that dissociation requires the binding of ATP. Control experiments indicate that the interaction between mhsp70 and Isp45 occurs in the intact mitochondria. We propose that Isp45 lines the inside of a proteinaceous channel across the inner membrane and that it is the membrane anchor for an ATP-driven "import motor" composed of mhsp70 and GrpEp. This arrangement is reminiscent of the protein transport systems of the yeast endoplasmic reticulum and the bacterial plasma membrane.
Subunit stoichiometry of staphylococcal alpha-hemolysin in crystals and on membranes: a heptameric transmembrane pore.
Authors: Gouaux J E , Braha O , Hobaugh M R , Song L , Cheley S , Shustak C , Bayley H
Published: May 2006

Elucidation of the accurate subunit stoichiometry of oligomeric membrane proteins is fraught with complexities. The interpretations of chemical cross-linking, analytical ultracentrifugation, gel filtration, and low-resolution electron microscopy studies are often ambiguous. Staphylococcal alpha-hemolysin (alpha HL), a homooligomeric toxin that forms channels in cell membranes, was believed to possess six subunits arranged around a sixfold axis of symmetry. Here, we report that analysis of x-ray diffraction data and chemical modification experiments indicate that the alpha HL oligomer is a heptamer. Self-rotation functions calculated using x-ray diffraction data from single crystals of alpha HL oligomers show a sevenfold axis of rotational symmetry. The alpha HL pore formed on rabbit erythrocyte membranes was determined to be a heptamer by electrophoretic separation of alpha HL heteromers formed from subunits with the charge of wild-type alpha HL and subunits with additional negative charge generated by targeted chemical modification of a single-cysteine mutant. These data establish the heptameric oligomerization state of the alpha HL transmembrane pore both in three-dimensional crystals and on a biological membrane.
Direct vibrational structure of protein metal-binding sites from near-infrared Yb3+ vibronic side band spectroscopy.
Authors: Roselli C , Boussac A , Mattioli T A
Published: May 2006

Near-infrared Yb3+ vibronic side band (VSB) spectroscopy is used to obtain structural information of metal binding sites in metalloproteins. This technique provides a selective "IR-like" vibrational spectrum of those ligands chelated to the Yb3+ ion. VSB spectra of various model complexes of Yb3+ representing different ligand types were studied to provide references for the VSB spectra of Yb(3+)-reconstituted metalloproteins. Ca2+ in the calcium-binding protein parvalbumin and Fe3+ in the iron-transporting protein transferrin were replaced with Yb3+. The fluorescence of Yb3+ reconstituted into these two proteins exhibits weak VSBs whose energy shifts, with respect to the main 2F5/2-->2F7/2 Yb3+ electronic transition, represent the vibrational frequencies of the Yb3+ ligands. The chemical nature of the ligands of the Yb3+ in these proteins, as deduced by the observed VSB frequencies, is entirely in agreement with their known crystal structures. For transferrin, replacement of the 12CO3(2-) metal counterion with 13CO3(2-) yielded the expected isotopic shift for the VSBs corresponding to the carbonate vibrational modes. This technique demonstrates enormous potential in elucidating the localized structure of metal binding sites in proteins.
Control of autoantibody affinity by selection against amino acid replacements in the complementarity-determining regions.
Authors: Børretzen M , Randen I , Zdárský E , Førre O , Natvig J B , Thompson K M
Published: May 2006

Rheumatoid factor (RF) autoantibodies can be produced in healthy individuals after infections or immunizations and thus escape normal tolerization mechanisms. It has not been clear whether such autoantibodies can undergo somatic hypermutation and affinity maturation similar to antibodies to exogenous antigens. We have investigated how these autoantibodies are regulated in normal individuals by analyzing the sequences of monoclonal IgM RFs obtained as hybridomas from donors after immunization. The variable regions undergo extensive hypermutation, but in contrast to antibodies against exogenous antigens, there is a strong selection against mutations that result in replacement of amino acids in the hypervariable, or complementarity-determining, regions. Furthermore, we found no increase in affinity of these RFs with the accumulation of mutations. This suggests that high-affinity variants are tolerized during the hypermutation process and there is a peripheral mechanism operating on certain autoreactive B cells that, while not deleting or anergizing all autoreactive cells, prevents the generation of high-affinity autoantibodies. Comparison of RFs by using the VH1 DP-10 heavy chain variable region segment from both normal individuals and rheumatoid arthritis (RA) patients suggests that RF from RA patients may not be subject to such a controlling mechanism.
Following the assembly of RNA polymerase-DNA complexes in aqueous solutions with the scanning force microscope.
Authors: Guthold M , Bezanilla M , Erie D A , Jenkins B , Hansma H G , Bustamante C
Published: May 2006

The capability of the scanning force microscope (SFM) to image molecules in aqueous buffers has opened the exciting possibility of following processes of molecular assembly in real time and in near-physiological environments. This capability is demonstrated in this paper by following the assembly process of RNA polymerase-DNA complexes. DNA fragments deposited on mica and imaged in Hepes/MgCl2 are shown before and after Escherichia coli RNA polymerase holoenzyme is injected in the SFM liquid chamber. The protein can recognize and bind to these DNA fragments within several seconds after injection, suggesting that the protein and the DNA retain their native configuration after deposition and during SFM imaging. A time-lapse sequence depicting the process of assembly of RNA polymerase-DNA complexes is shown. These results represent the first step for acquiring the capabilities to monitor complex biomolecular processes as they take place in ionic solutions and to characterize their spatial organization.
Enzyme replacement in a canine model of Hurler syndrome.
Authors: Shull R M , Kakkis E D , McEntee M F , Kania S A , Jonas A J , Neufeld E F
Published: May 2006

The Hurler syndrome (alpha-L-iduronidase deficiency disease) is a severe lysosomal storage disorder that is potentially amenable to enzyme-replacement therapy. Availability of a canine model of the disease and a sufficient supply of corrective enzyme have permitted a therapeutic trial lasting 3 mo. Recombinant human alpha-L-iduronidase, purified to apparent homogeneity from secretions of a stably transfected Chinese hamster ovary cell line, was administered i.v. to homozygous affected animals in doses of approximately 1 mg. The enzyme rapidly disappeared from the circulation in a biphasic manner, with t1/2 of 0.9 and 19 min, respectively, and was taken up primarily by the liver. Biopsy of the liver before and after a very short trial (seven doses administered over 12 days) showed remarkable resolution of lysosomal storage in both hepatocytes and Kupffer cells. After weekly administration of enzyme to three affected animals over a period of 3 mo, the level of enzyme was about normal in liver and spleen, lower but significant in kidney and lung, and barely detectable (0-5% of normal) in brain, heart valves, myocardium, cartilage, and cornea. Light and electron microscopic examination of numerous tissues showed normalization of lysosomal storage in liver, spleen, and kidney glomeruli, but there was no improvement in brain, heart valves, or cornea. Even though the treated dogs developed complement-activating antibodies against alpha-L-iduronidase, clinical symptoms could be prevented by slow infusion of enzyme and premedication.
Two components of transmitter release at a central synapse.
Authors: Goda Y , Stevens C F
Published: May 2006

After the arrival of a presynaptic nerve impulse at an excitatory synapse in hippocampal neurons, the rate of neurotransmitter release increases rapidly and then returns to low levels with a biphasic decay. The two kinetically distinct components are differentially affected when Sr2+ is substituted for Ca2+ ions. Our findings are comparable to those of the classical studies for the frog neuromuscular junction, and thus the basic aspects of Ca(2+)-activated transmitter release machinery appear to be conserved in central synapses. The method we have used, in addition, permits us to estimate the average neurotransmitter release rate for a single bouton. The observation of differential Ca2+/Sr2+ sensitivity is consistent with a release mechanism mediated by two Ca2+ sensors with distinct Ca2+ affinities: the low-affinity Ca2+ sensor facilitates the fast synchronous phase of release, whereas the high-affinity sensor sustains the slow asynchronous phase of release.
Sleep patterns are disturbed in cats infected with feline immunodeficiency virus.
Authors: Prospéro-García O , Herold N , Phillips T R , Elder J H , Bloom F E , Henriksen S J
Published: May 2006

Human immunodeficiency virus (HIV)-related sleep disturbances have been reported early in AIDS. Likewise, the feline immunodeficiency virus (FIV), a natural lentivirus pathogen of cats, produces a similar immunodeficiency syndrome with neurological sequelae. To identify the neurophysiological substrate of FIV infection in brain, pathogen-free cats were infected with the Maryland strain of FIV. Eight weeks after inoculation, all FIV-infected cats seroconverted and virus was detected in the cerebrospinal fluid and in the mononuclear cells of peripheral blood. Ten to 12 months after the FIV inoculation, inoculated and control cats were surgically implanted with electrodes to record the sleep/wake cycle. These sleep recordings were obtained under conditions controlling for environmental variables and instrumental adaptation. FIV-infected cats spent 50% more time awake than the sham-inoculated controls and exhibited many more sleep/waking stage shifts--i.e., 40% more than controls. In addition, FIV-infected cats showed approximately 30% of rapid eye movement (REM) sleep reduction compared to controls. The latency to sleep and REM sleep onset was also significantly delayed in FIV-infected cats. In addition, a remarkable increase in cortically recorded spindle activity (8-13 Hz) was observed during slow-wave sleep in some infected subjects, similar to changes described in HIV-infected humans. Moreover, infected cats exhibited no overt signs of systemic morbidity, such as hyperpyrexia or body weight loss. These results indicate that FIV-infected cats exhibit sleep abnormalities similar to the sleep disturbances previously described in AIDS patients and further support the feline preparation as a valuable animal model of HIV infection of the central nervous system.
Specificity analysis of blood group Lewis-y (Le(y)) antibodies generatedagainst synthetic and natural Le(y) determinants.
Authors: Kitamura K , Stockert E , Garin-Chesa P , Welt S , Lloyd K O , Armour K L , Wallace T P , Harris W J , Carr F J , Old L J
Published: May 2006

Le(y)-reactive monoclonal antibodies (mAbs) were generated in mice by immunization with synthetic Le(y) neoglycoproteins or with Le(y)-expressing cells. Serological analysis indicated that mAbs raised against synthetic Le(y) (i) reacted strongly with synthetic Le(y) but poorly with natural Le(y), (ii) cross-reacted with Le(x) or H-type 2 structures, and (iii) were IgG1, IgG2a, or IgG2b. mAbs raised against Le(y)-expressing cells (i) reacted with both synthetic Le(y) and natural Le(y), (ii) were of two types: cross-reactive with Le(x) or H-type 2 structures or specific for Le(y), and (iii) were IgM or IgG3. One of the mAbs raised against natural Le(y), mAb 3S193 (IgG3), showed high specificity for Le(y) in ELISA tests with synthetic Le(y) and Le(y) containing glycoproteins and glycolipids; it also reacted strongly in rosetting assays and cytotoxic tests with Le(y)-expressing cells. mAb 3S193 did not lyse O, A, AB, and B human erythrocytes in the presence of human complement. In flow cytometry, there was weak reactivity with granulocytes, a reactivity also observed with two previously described highly specific Le(y) mouse mAbs--BR55-2 (IgG3) and B3 (IgG1). A humanized version of mAb 3S193 has been constructed, and the specificity pattern and reactivity for Le(y) remain very similar to mouse mAb 3S193.
Assessment of genetic diversity of seagrass populations using DNA fingerprinting: implications for population stability and management.
Authors: Alberte R S , Suba G K , Procaccini G , Zimmerman R C , Fain S R
Published: May 2006

Populations of the temperate seagrass, Zostera marina L. (eelgrass), often exist as discontinuous beds in estuaries, harbors, and bays where they can reproduce sexually or vegetatively through clonal propagation. We examined the genetic structure of three geographically and morphologically distinct populations from central California (Elkhorn Slough, Tomales Bay, and Del Monte Beach), using multilocus restriction fragment length polymorphisms (DNA fingerprints). Within-population genetic similarity (Sw) values for the three eelgrass populations ranged from 0.44 to 0.68. The Tomales Bay population located in an undisturbed, littoral site possessed a within-population genetic similarity (Sw = 0.44) that was significantly lower than those of the other two populations. Cluster analysis identified genetic substructure in only the undisturbed subtidal population (Del Monte Beach). Between-population similarity values (Sb) for all pairwise comparisons ranged from 0.47 to 0.51. The three eelgrass populations show significantly less between locale genetic similarity than found within populations, indicating that gene flow is restricted between locales even though two of the populations are separated by only 30 km. The study demonstrates that (i) natural populations of Z. marina from both disturbed and undisturbed habitats possess high genetic diversity and are not primarily clonal, (ii) gene flow is restricted even between populations in close proximity, (iii) an intertidal population from a highly disturbed habital shows much lower genetic diversity than an intertidal population from an undisturbed site, and (iv) DNA fingerprinting techniques can be exploited to understand gene flow and population genetic structure in Z. marina, a widespread and ecologically important species, and as such are relevant to the management of this coastal resource.
Distinct isoforms of neuregulin are expressed in mesenchymal and neuronal cells during mouse development.
Authors: Meyer D , Birchmeier C
Published: May 2006

Neuregulin, the putative ligand of the c-neu receptor tyrosine kinase, can induce differentiation or growth of epithelia and other cells. To gain insight into the biological role of this factor, we have analyzed the expression of neuregulin during mouse embryogenesis and in the perinatal animal by a combination of in situ hybridization and RNase protection experiments. We identify sites of expression that correspond to mesenchymal cells of various parenchymal organs. Our finding implies a function of neuregulin as a mesenchymal factor that acts on epithelia. The mesenchymal expression of neuregulin could thus provide a molecular basis for the biological phenomenon of mesenchymal-epithelial interactions. It also has implications on the molecular mechanism by which amplification of c-neu can affect tumor progression of carcinomas. In addition, neuregulin expression is found in neuronal cells during development. We show by RNase protection experiments that distinct isoforms of neuregulin are expressed in the brain. Therefore, our data indicate in vivo a dual role for neuregulin as mesenchymal and neuronal factor.
Intra- and extracellular calcium modulates stereocilia stiffness on chick cochlear hair cells.
Authors: Pae S S , Saunders J C
Published: May 2006

Segments of the chick basilar papilla were isolated and maintained in culture medium. The sensory hair bundle of individual hair cells was observed with light microscopy and stimulated with a water microjet at 600 Hz. Hair bundle motion was slowed by illuminating the microscope with stroboscopic light, and water jet intensity was systematically varied in decibel (dB) steps until a visual detection level (VDL) threshold of hair bundle motion was achieved. The VDL threshold of many hair cells was measured in each isolated papilla. However, only one of eight extracellular calcium concentrations (0.0, 0.0001, 0.001, 0.01, 0.1, 1.25, 6.0, and 12.0 mM) was used with each papilla. In a second series, a calcium ionophore (ionomycin) was added to the culture medium, and VDL thresholds were again measured at seven of these extracellular calcium concentrations. With extracellular calcium alone, the stimulus level needed to achieve threshold was reduced by 2.73 dB between 0.1 and 0.01 mM. This change in threshold represented a 1.37-fold decrease in hair bundle stiffness. When ionomycin was added to the culture medium, a progressively greater stimulus intensity was needed to achieve threshold as calcium concentration increased. The 11.7-dB increase in threshold, with the addition of ionomycin, between 0.0001 and 6.0 mM extracellular calcium was equivalent to a 3.85-fold increase in bundle stiffness. These large changes in hair-bundle stiffness, as a function of the extra- or intracellular calcium environment, may play an important role in the micromechanical behavior of the hair cell during sound simulation.
Light-regulated, tissue-specific immunophilins in a higher plant.
Authors: Luan S , Albers M W , Schreiber S L
Published: May 2006

In addition to their application in organ transplantation, immunosuppressive drugs are valuable tools for studying signal transduction in eukaryotic cells. Using affinity chromatography, we have purified immunosuppressive drug receptors (immunophilins) from fava bean. Proteins belonging to both major classes of the immunophilin family identified from animal sources [FK506- and rapamycin-binding proteins (FKBPs) and cyclophilins] were present in this higher plant. FKBP13, the most abundant FKBP family member in leaf tissues, was not detected in root tissues, whereas other FKBPs were present in both tissues. While the abundance of cyclophilin A in leaves was similar to that in roots, cyclophilin B/C was expressed at a much higher level in leaf tissues than in root tissues. Subcellular localization of immunophilins in mesophyll cells showed that chloroplasts contained FKBP13 and cyclophilin B/C but not other members, which explains the preferential expression of these two proteins in leaves over roots. The abundance of chloroplast-localized immunophilins, FKBP13 and cyclophilin B/C, was regulated by light. Although etiolated leaves produced detectable levels of cyclophilin B/C, they did not express FKBP13. Illumination of etiolated plants dramatically increased the expression of both FKBP13 and cyclophilin B/C. The light-induced expression of FKBP13 is closely correlated with the accumulation of chlorophyll in the leaf tissue. Our findings suggest that FKBP13 and cyclophilin B/C may play a specific role in chloroplasts.
A small element from the Mason-Pfizer monkey virusgenome makes human immunodeficiency virus type 1 expression and replicationRev-independent.
Authors: Bray M , Prasad S , Dubay J W , Hunter E , Jeang K T , Rekosh D , Hammarskjöld M L
Published: May 2006

Replication of human immunodeficiency virus type 1
(HIV-1) is dependent on the viral Rev protein. This protein acts in concert with
the cis-acting rev-responsive element present in intron-containing RNAs to
facilitate nuclear export of these RNAs. Here we show that a cis-acting
219-nucleotide sequence from an unrelated "simple" retrovirus,
Mason-Pfizer monkey virus (MPMV), enables Rev-independent HIV-1 replication.
This sequence is present in an untranslated region near the 3' end of the
MPMV genome. The MPMV element is also able to efficiently substitute for Rev in
expression of Gag/Pol and Env proteins from subgenomic constructs. We
hypothesize that the MPMV element functions by interacting with a cellular
factor that plays a role in mRNA transport analogous to that of the Rev protein.
It might be possible to exploit this element in the development of an HIV
vaccine.
Selenol binds to iron in nitrogenase iron-molybdenumcofactor: an extended x-ray absorption fine structure study.
Authors: Conradson S D , Burgess B K , Newton W E , Di Cicco A , Filipponi A , Wu Z Y , Natoli C R , Hedman B , Hodgson K O
Published: May 2006

The biological N2-fixation reaction is catalyzed
by the enzyme nitrogenase. The metal cluster active site of this enzyme, the
iron-molybdenum cofactor (FeMoco), can be studied either while bound within the
MoFe protein component of nitrogenase or after it has been extracted into
N-methylformamide. The two species are similar but not identical. For example,
the addition of thiophenol or selenophenol to isolated FeMoco causes its rather
broad S = 3/2 electron paramagnetic resonance signal to sharpen and more closely
approach the signal exhibited by protein-bound FeMoco. The nature of this
thiol/selenol binding site has been investigated by using Se-K edge extended
x-ray absorption fine structure (EXAFS) to study selenophenol ligated to FeMoco,
and the results are reported here. EXAFS data analysis at the ligand Se-K edge
was performed with a set of software, GNXAS, that provides for direct
calculation of the theoretical EXAFS signals and least-squares fits to the
experimental data. Data analysis results show definitively that the selenol (and
by inference thiol) binds to Fe at a distance of 2.4 A. In contrast,
unacceptable fits are obtained with either Mo or S as the liganded atom (instead
of Fe). These results provide quantitative details about an exchangeable
thiol/selenol binding site on FeMoco in its isolated, solution state and
establish an Fe atom as the site of this reaction. Furthermore, the utility of
ligand-based EXAFS as a probe of coordination in polynuclear metal clusters is
demonstrated.
Inhibition of PC12 cell redox activity is a specific,early indicator of the mechanism of beta-amyloid-mediated celldeath.
Authors: Shearman M S , Ragan C I , Iversen L L
Published: May 2006

An in vitro tissue culture cell model system for
investigating the biochemical mechanisms involved in the neurodegenerative
actions of beta-amyloid has been established. Using rat pheochromocytoma PC12
cells, it was found that an early, specific response of cells to the
beta-amyloid protein or the beta-amyloid fragment 25-35 was a potent inhibition
of cellular redox activity, as measured by 3-[4,5-dimethylthiazol-2-yl]-2,
5-diphenyltetrazolium bromide (MTT) reduction. This inhibitory response was
rapid and occurred at nanomolar concentrations of peptide, concentrations at
which no equivalent decreases in cell proliferation or cell survival were
observed. The inhibition of PC12 cell MTT reduction was initially reversible
upon removal of the peptide; if sustained for several days, however, by repeated
peptide application, it became associated with a dramatic reduction in cell
survival. Inhibition of MTT reduction may, therefore, be an early indicator of
the mechanism of beta-amyloid-mediated cell death.
Ultrasensitive retrovirus detection by a reversetranscriptase assay based on product enhancement.
Authors: Pyra H , Böni J , Schüpbach J
Published: May 2006

Reverse transcriptase (RT) is an indispensable
component of infectious retroviruses. We have developed an ultrasensitive RT
test in which RNA of bacteriophage MS2 serves as the template for RT-mediated
cDNA synthesis. A fragment of the cDNA is selectively amplified by polymerase
chain reaction and the amplification product is analyzed by Southern blot
hybridization or enzyme immunoassay. The procedure was 10(6) to 10(7) times more
sensitive than a conventional RT test and detected as little as 10(-9) unit of
murine leukemia virus RT, which corresponded to 2.1 x 10(2) molecules, a number
present in 3-11 virions. As a screening assay for filterable particle-associated
RT, it was positive with supernatants from cell cultures producing human
immunodeficiency virus (HIV) type 1 or human T-cell leukemia virus (HTLV) type 1
or 2, but was negative with nonproducer cultures. It was positive with plasma
samples from all tested individuals infected with HIV-1, HIV-2, or HTLV-1 and
sera from cats infected with feline leukemia virus or feline immunodeficiency
virus. Control samples from blood donors or uninfected cats were negative.
Density banding experiments with culture supernatants showed that the RT
activity was associated with virus particles. The assay should detect all
replication-competent retroviruses or similar agents. It may be used as a
screening assay for such agents, for quantitation of the viral load, drug
susceptibility testing of RT, and control of virus inactivation in biological
products.
Post-transcriptional regulation of interleukin 1 alphain various strains of young and senescent human umbilical vein endothelialcells.
Authors: Garfinkel S , Brown S , Wessendorf J H , Maciag T
Published: May 2006

Human umbilical vein endothelial cell (HUVEC)
senescence in vitro is characterized by the loss of proliferative potential and
an increase in cell size. Because HUVEC senescence in one strain (H101) has been
characterized by the increase in the steady-state mRNA level for the
signal-peptideless cytokine, interleukin (IL) 1 alpha, we have examined young
and senescent populations of five additional HUVEC strains (H3605, H103, H928,
H929, and H930) to determine whether the elevated levels of IL-1 alpha mRNA
could be observed in all HUVEC strains. Consistent with the data from strain
H101, strains H3605 and H930 also exhibited a low steady-state level of the IL-1
alpha mRNA in young populations compared to elevated levels of IL-1 alpha mRNA
in the senescent populations. However, three strains (H103, H928, and H929) did
not exhibit reduced levels of IL-1 alpha mRNA in the young populations, and
interestingly, strain H928, at times, expressed relatively high IL-1 alpha mRNA
levels in the young populations. In addition, expression of the steady-state
level of plasminogen activator inhibitor 1 and cyclooxygenase 2 was elevated in
senescent populations of all HUVEC strains examined, whereas young populations
exhibited a low level of expression for these genes regardless of the IL-1 alpha
mRNA level. Further, the level of the IL-1 alpha polypeptide was elevated in
senescent HUVEC populations relative to young populations that expressed either
a high or low level of the IL-1 alpha mRNA. We have also demonstrated that the
elevated level of IL-1 alpha mRNA in the senescent population of strain H3605
may be regulated by mRNA stability; however, this mechanism does not apply to
all the HUVEC strains examined in this study. Thus, we suggest that while mRNA
levels of the IL-1-response genes for plasminogen activator inhibitor 1 and
cyclooxygenase 2 are appropriate markers for HUVEC senescence, HUVEC
strain-specific post-transcriptional mechanisms may exist to regulate the
function of IL-1 alpha as a modifier of HUVEC senescence in
vitro.
E-cadherin gene mutations in human gastric carcinomacell lines.
Authors: Oda T , Kanai Y , Oyama T , Yoshiura K , Shimoyama Y , Birchmeier W , Sugimura T , Hirohashi S
Published: May 2006

Reduced expression of E-cadherin has been regarded
as one of the main molecular events involved in dysfunction of the cell-cell
adhesion system, triggering cancer invasion and metastasis. However, even with a
sufficient amount of E-cadherin, cell-cell adhesion is sometimes lost in
"diffusely invasive" human carcinomas. Ten human cancer cell lines,
showing growth characterized morphologically by loose cell-cell adhesion, were
analyzed for possible structural abnormalities of their expressed E-cadherin.
Four of the cell lines showed strong mRNA and protein expression with no
nucleotide sequence abnormalities, and mRNA was absent in four other cell lines.
mRNA sequence was abnormal in the remaining two gastric carcinoma cell lines. In
MKN45 (poorly differentiated adenocarcinoma), this involved a 12-bp in-frame
deletion with strong expression of mRNA and protein. In KATO-III (signet ring
cell carcinoma), there were four mRNA species with insertions of different
sizes, among which the major transcripts (with a 7-bp insertion) caused a
frameshift, and expression of both mRNA and protein was markedly reduced. In
these two cell lines, DNA mutations were detected around exon-intron junctions,
revealing that aberrant RNA splicing was the cause of the mRNA abnormalities. In
addition, the wild-type allele of the E-cadherin locus was lost, suggesting that
the E-cadherin gene had been inactivated by two hits (mutation and allele loss),
similar to the mechanism for inactivation of tumor suppressor
genes.
Cytoplasmic dynein is involved in nuclear migration in Aspergillus nidulans.
Authors: Xiang X , Beckwith S M , Morris N R
Published: May 2006

Nuclear migration plays an important role in the growth and development of many organisms including the multinuclear fungus Aspergillus nidulans. We have identified four genes, nudA, nudC, nudF, and nudG, in which temperature-sensitive mutations affect nuclear distribution. In this report, we describe the cloning of the nudA gene by complementation of the mutant phenotype by using a chromosome VIII-specific cosmid library. A genomic fragment of nudA hybridized to an mRNA of approximately 14 kb. Sequencing analysis of nudA revealed four ATP-binding sites that are characteristic of the cytoplasmic dynein heavy chain. The amino acid sequence of the nudA gene product shows 52% overall identity with the rat brain cytoplasmic dynein heavy chain. Our study provides in vivo evidence that dynein, a microtubule motor molecule, plays a role in the nuclear migration process.
Cell cycle-dependent and kinase-specific regulation of the apical Na/H exchanger and the Na,K-ATPase in the kidney cell line LLC-PK1 by calcitonin.
Authors: Chakraborty M , Chatterjee D , Gorelick F S , Baron R
Published: May 2006

Calcitonin (CT), which regulates serum calcium through its actions in bone and the kidney tubule, also has a potent natriuretic effect in vivo. Na reabsorption in the proximal kidney tubule is mostly dependent on the activity of the Na,K-ATPase and the apical Na/H exchanger. We have previously shown that CT regulates the activity of the Na,K-ATPase in the proximal kidney tubule cell line LLC-PK1 in a cell cycle-dependent manner. We report here that, in the same cells, CT also regulates the Na/H exchanger through a cell cycle-specific activation of the Ca/calmodulin-dependent protein kinase II. In G2 phase, no changes in ethylisopropyl amiloride-sensitive 22Na uptake is observed, despite an increase in cAMP. In contrast, the hormone inhibits the apical exchanger when the cells are in S phase, resulting in an 80% inhibition of 22Na uptake. These results demonstrate that CT affects the activity of the two major proximal tubule Na transport systems and may help clarify the mechanisms by which CT regulates Na+ reabsorption.
Folded proteins occur frequently in libraries of random amino acid sequences.
Authors: Davidson A R , Sauer R T
Published: May 2006

A library of synthetic genes encoding 80- to 100-residue proteins composed mainly of random combinations of glutamine (Q), leucine (L), and arginine (R) has been expressed in Escherichia coli. These genes also encode an epitope tag and six carboxyl-terminal histidines. Screening of this library by immunoblotting showed that 5% of these QLR proteins are expressed at readily detectable levels. Three well-expressed QLR proteins were purified and characterized. Each of these proteins has significant alpha-helical content, is largely resistant to degradation by Pronase, and has a distinct oligomeric structure. In addition, one protein unfolds in a highly cooperative manner. These properties of the QLR proteins demonstrate that they possess folded structures with some native-like properties. The QLR proteins differ from most natural proteins, however, in being remarkably resistant to denaturant-induced and thermal-induced unfolding and in being relatively insoluble in the absence of denaturants.
Existence of nitric oxide synthase in rat hippocampal pyramidal cells.
Authors: Wendland B , Schweizer F E , Ryan T A , Nakane M , Murad F , Scheller R H , Tsien R W
Published: May 2006

It has been proposed that nitric oxide (NO) serves as a key retrograde messenger during long-term potentiation at hippocampal synapses, linking induction of long-term potentiation in postsynaptic CA1 pyramidal cells to expression of long-term potentiation in presynaptic nerve terminals. However, nitric oxide synthase (NOS), the proposed NO-generating enzyme, has not yet been detected in the appropriate postsynaptic cells. We here demonstrate specific NOS immunoreactivity in the CA1 region of hippocampal sections by using an antibody specific for NOS type I and relatively gentle methods of fixation. NOS immunoreactivity was found in dendrites and cell bodies of CA1 pyramidal neurons. Cultured hippocampal pyramidal cells also displayed specific immunostaining. Control experiments showed no staining with preimmune serum or immune serum that was blocked with purified NOS. These results demonstrate that CA1 pyramidal cells contain NOS, as required were NO involved in retrograde signaling during hippocampal synaptic plasticity.