bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of the complement iC3b binding site in the beta 2 integrin CR3 (CD11b/CD18).
Authors: Ueda T , Rieu P , Brayer J , Arnaout M A
Published: May 2006

The divalent cation-dependent interaction of the beta 2 integrin CR3 (CD11b/CD18) with the major complement opsonic C3 fragment iC3b is an important component of the central role of CR3 in inflammation and immune clearance. In this investigation we have identified the iC3b binding site in CR3. A recombinant fragment representing the CR3 A-domain, a 200-amino acid region in the ectodomain of the CD11b subunit, bound to iC3b directly and in a divalent cation-dependent manner. The iC3b binding site was further localized to a short linear peptide that also bound iC3b directly and inhibited iC3b binding to the A-domain as well as to CR3 expressed by human neutrophils. These data establish a major recognition function for the integrin A-domain and have important implications for development of novel antiinflammatory therapeutics.
Interleukin 12 induction of interferon gamma-dependent protection against malaria.
Authors: Sedegah M , Finkelman F , Hoffman S L
Published: May 2006

Intraperitoneal injection of recombinant Interleukin 12 (rIL-12) at 30 ng/day for 5 days beginning 1 to 2 days before sporozoite challenge or administration of a single dose of 150 ng of rIL-122 days before challenge protected 100% of BALB/c mice against challenge with 10(2) Plasmodium yoelii sporozoites. rIL-12-induced protection was eliminated in all mice by administration of a monoclonal antibody against interferon gamma and in 50% of mice by administration of NG-monomethyl-L-arginine, a competitive inhibitor of nitric oxide synthase. rIL-12 protected BALB/c mice treated with cytotoxic anti-CD4 and anti-CD8 monoclonal antibodies, as well as T-cell- and B-cell-deficient severe combined immunodeficiency mice. These data suggest that rIL-12 stimulates non-B, non-T cells to produce interferon gamma that kills intrahepatic parasites by stimulating nitric oxide production. If rIL-12 proves to be well tolerated by humans, our findings support consideration of rIL-12 as an immunoprophylactic against malaria.
Virologic and genetic studies relate Amerind origins to the indigenous people of the Mongolia/Manchuria/southeastern Siberia region.
Authors: Neel J V , Biggar R J , Sukernik R I
Published: May 2006

A commonly held theory is that the first wave of migrants into the New World was derivative from the ethnic groups then inhibiting eastern Siberia. However, these ethnic groups lack a mtDNA haplogroup (B) that is well represented in Amerindian tribes. Also, the time depth of the other three mtDNA haplogroups found in Amerindians (A, C, and D) appears to be greater in the Amerindians than in the eastern Siberian ethnic groups. In this communication we demonstrate that the human T-cell lymphotrophic virus type II, present in 11 of the 38 Amerindian tribes thus far examined, is not present in any of the 10 ethnic groups of eastern Siberia that we have studied. However, the virus has just been reported in the indigenous population of Mongolia, and mtDNA haplogroup B is also represented in this region. On the basis of these facts, we propose that the ancestors of the first migrants to the New World were not derived from north and central Siberia but from populations to the south, inhabiting the regions of Mongolia, Manchuria, and/or the extreme southeastern tip of Siberia.
Activity of the Chlamydomonas chloroplast rbcL gene promoter is enhanced by a remote sequence element.
Authors: Klein U , Salvador M L , Bogorad L
Published: May 2006

The chloroplast gene rbcL encodes the large subunit of ribulose bisphosphate carboxylase. In Chlamydomonas reinhardtii, this gene is transcribed more actively than any other protein-encoding chloroplast gene studied to date. To delineate the rbcL gene promoter, chimeric reporter genes containing fragments of the 5' region of the rbcL gene fused to the coding sequence of the bacterial uidA gene, encoding beta-glucuronidase, were stably introduced into the chloroplast genome of Chlamydomonas by microprojectile bombardment. The relative transcription rates of endogenous and introduced genes were determined in transgenic cell lines in vivo. The basic rbcL promoter is located within the region of the gene extending from positions -18 to +63, taking position +1 as the site of initiation of transcription. A chimeric reporter gene containing only the basic promoter is transcribed only 1-15% as actively as the endogenous rbcL gene, depending on the conditions under which cells are grown and tested. However, a chimeric gene containing rbcL sequences extending to position +170 or beyond is transcribed at about the same rate as the endogenous gene. Deletion of the sequence between positions +170 and +126, well within the protein-encoding region, reduces the rate of transcription to that of reporter genes with the basic promoter alone.
Calcium dependence of neurotransmitter release and rate of spontaneous vesicle fusions are altered in Drosophila synaptotagmin mutants.
Authors: Littleton J T , Stern M , Perin M , Bellen H J
Published: May 2006

Since the demonstration that Ca2+ influx into the presynaptic terminal is essential for neurotransmitter release, there has been much speculation about the Ca2+ receptor responsible for initiating exocytosis. Numerous experiments have shown that the protein, or protein complex, binds multiple Ca2+ ions, resides near the site of Ca2+ influx, and has a relatively low affinity for Ca2+. Synaptotagmin is an integral membrane protein of synaptic vesicles that contains two copies of a domain known to be involved in Ca(2+)-dependent membrane interactions. Synaptotagmin has been shown to bind Ca2+ in vitro with a relatively low affinity. In addition, synaptotagmin has been shown to bind indirectly to Ca2+ channels, positioning the protein close to the site of Ca2+ influx. Recently, a negative regulatory role for synaptotagmin has been proposed, in which it functions as a clamp to prevent fusion of synaptic vesicles with the presynaptic membrane. Release of the clamp would allow exocytosis. Here we present genetic and electrophysiological evidence that synaptotagmin forms a multimeric complex that can function as a clamp in vivo. However, upon nerve stimulation and Ca2+ influx, all synaptotagmin mutations dramatically decrease the ability of Ca2+ to promote release, suggesting that synaptotagmin probably plays a key role in activation of synaptic vesicle fusion. This activity cannot simply be attributed to the removal of a barrier to secretion, as we can electrophysiologically separate the increase in rate of spontaneous vesicle fusion from the decrease in evoked response. We also find that some syt mutations, including those that lack the second Ca(2+)-binding domain, decrease the fourth-order dependence of release on Ca2+ by approximately half, consistent with the hypothesis that a synaptotagmin complex functions as a Ca2+ receptor for initiating exocytosis.
Regional variation in expression of acetylcholinesterase mRNA in adult rat brain analyzed by in situ hybridization.
Authors: Hammond P , Rao R , Koenigsberger C , Brimijoin S
Published: May 2006

To investigate the molecular basis of regional variation in expression of brain acetylcholinesterase (AChE; EC 3.1.1.7), steady-state levels of AChE activity and mRNA were examined. Relative AChE activity in Triton extracts from six areas of the rat brain varied as follows: cortex < cerebellum < medulla < pons-midbrain < thalamus < striatum. In contralateral samples from the same brains, AChE mRNA was assessed by Northern blotting with random-primed 32P-labeled cDNA. The regional abundance of the major 2.4-kb AChE transcript differed from that of the enzyme activity: cortex < striatum < cerebellum < medulla < thalamus < pons-midbrain. In situ hybridization with a 33P-labeled antisense AChE oligonucleotide provided evidence for high levels of AChE message in cells of the nucleus basalis, nucleus accumbens, neostriatum, substantia nigra, motor nucleus of the facial nerve, and spinal nucleus of the trigeminal nerve. In the caudate-putamen, large, heavily labeled neurons were not numerous, but they were approximately as frequent as the cholinergic interneurons revealed by choline acetyltransferase immunocytochemistry. The relatively low number of these AChE-expressing cells probably explains the relative dearth of AChE mRNA-like material in the neostriatum.
Isolation of the yeast histone octamer.
Authors: Lorch Y , Kornberg R D
Published: May 2006

Procedures for the extraction and purification of the yeast histone octamer are described. Either mechanical disruption, yielding chromatin fragments, or spheroplast formation with subsequent nuclear isolation was employed. A hexahistidine tag was inserted in the N-terminal region of histone H2B, permitting resolution of the histone octamer from high-salt extracts of nuclei or chromatin to near homogeneity. The histone octamer purified in this way was fully active in reconstitution of nucleosomes.
7 beta-hydroperoxycholest-5-en-3 beta-ol, a component of human atherosclerotic lesions, is the primary cytotoxin of oxidized human low density lipoprotein.
Authors: Chisolm G M , Ma G , Irwin K C , Martin L L , Gunderson K G , Linberg L F , Morel D W , DiCorleto P E
Published: May 2006

Modification of low density lipoprotein (LDL) by free radical oxidation renders this molecular complex cytotoxic. Oxidized lipoproteins exist in vivo in atherosclerotic lesions and in the plasma of diabetic animals, suggesting that lipoprotein-induced tissue damage may occur in certain diseases. We undertook purification and identification of the major cytotoxin in oxidized LDL. The lipid extract from oxidized LDL was subjected to multiple HPLC separations, and the fractions were assayed for cytotoxicity. Mass spectrometry and nuclear magnetic resonance identified the purified toxin as 7 beta-hydroperoxycholest-5-en-3 beta-ol (7 beta-OOH-Chol). This molecule accounted for approximately 90% of the cytotoxicity of the lipids of oxidized LDL. We also found 7 beta-OOH-Chol in human atherosclerotic lesions from endarterectomy specimens obtained immediately after excision. These results are consistent with the hypothesis that the oxidized LDL present in lesions has the capacity to induce cell and tissue injury, leading to progression of the disease and the generation of the necrotic core of the lesion.
Rapamycin selectively inhibits translation of mRNAs encoding elongation factors and ribosomal proteins.
Authors: Terada N , Patel H R , Takase K , Kohno K , Nairn A C , Gelfand E W
Published: May 2006

The immunosuppressant rapamycin (RAP) has been demonstrated to specifically inhibit the activity of p70 S6 kinase (p70s6k) and subsequent phosphorylation of ribosomal S6 protein in mammalian cells. Addition of RAP to proliferating lymphoid cells resulted in inhibition of protein synthesis before any changes in the rate of cell proliferation. When the cellular composition of proteins was examined by gel electrophoresis, RAP dramatically inhibited synthesis of selective proteins, particularly elongation factor 2 (eEF-2). The inhibition of eEF-2 synthesis by RAP was at the translational level. Further, RAP inhibited the polysomal association of mRNAs encoding not only eEF-2 but also elongation factor 1-alpha and ribosomal proteins without affecting mRNA translation of any of a number of nonribosomal proteins. Since levels of activity of p70s6k are correlated with the rate of biosynthesis of eEF-2, p70s6k might be involved in coordinate translational regulation of ribosomal protein mRNAs in higher eukaryotes, which have a conserved sequence at their 5' end. Specific inhibition of ribosomal protein synthesis likely explains the differential antiproliferative effect of RAP on proliferating and mitogen-activated quiescent cells.
Attenuation of bovine leukemia virus by deletion of R3 and G4 open reading frames.
Authors: Willems L , Kerkhofs P , Dequiedt F , Portetelle D , Mammerickx M , Burny A , Kettmann R
Published: May 2006

Complex oncoviruses contain, in addition to the classical retroviral genes (gag, pol, and env), a region (X) located between the envelope sequences and the 3' long terminal repeat. The X region contains two genes, tax and rex, whose protein products are involved in transcriptional and posttranscriptional regulation of viral expression. In addition to these activators, the bovine leukemia virus (BLV) and the human T-cell leukemia virus (HTLV) contain alternative open reading frames (R3 and G4 for BLV; p30, p13, and p12 for HTLV). As a virus/animal model for HTLV-induced leukemogenesis, BLV provirus can be injected intradermally into sheep, where it induced B-lymphocyte transformation. Deletion of the R3 and G4 sequences from an infectious and tumorigenic BLV provirus greatly impaired the in vivo propagation of the viruses as demonstrated by DNA polymerase chain reaction, RNA blots, structural-protein ELISA, and immunofluorescence analysis. Our results show that the alternative open reading frames are required for maintaining high virus loads during the course of persistent infection in vivo. Thus, R3 and G4 are candidates for antiviral drug development. Furthermore, viruses with a deletion in these sequences should be tested as live attenuated vaccines.
Restoring allosterism with compensatory mutations in hemoglobin.
Authors: Kim H W , Shen T J , Sun D P , Ho N T , Madrid M , Tam M F , Zou M , Cottam P F , Ho C
Published: May 2006

Abnormal human hemoglobins (HBs) with amino acid substitutions in the alpha 1 beta 2 interface have very high oxygen affinity and greatly reduced cooperativity in O2 binding compared to normal human Hb. In such abnormal Hbs with mutations at position beta 99, the intersubunit hydrogen bonds between Asp-beta 99 and Tyr-alpha 42 and between Asp-beta 99 and Asn-alpha 97 are broken, thus destabilizing the deoxyquaternary structure of these Hbs. A molecular dynamics method has been used to design compensatory amino acid substitutions in these Hbs that can restore their allosteric properties. We have designed a compensatory mutation in a naturally occurring mutant Hb, Hb Kempsey (Asp-beta 99-->Asn), and have produced it using our Escherichia coli expression plasmid pHE2. We have determined the O2 binding properties of this recombinant double mutant Hb, Hb(Asp-beta 99-->Asn and Tyr-alpha 42-->Asp) and have used 1H NMR spectroscopy to investigate the tertiary structures around the heme groups and the quaternary structure in the alpha 1 beta 2 subunit interface. Our results clearly show that the Tyr-alpha 42-->Asp replacement can substantially compensate for the functional defect of Hb Kempsey caused by the Asp-beta 99-->Asn substitution. The structural and functional information derived from this recombinant Hb provides insights into the structural basis of allosterism and the design of compensatory amino acid substitutions to restore the functional properties of other abnormal HBs associated with hemoglobinopathies.
An ADP-ribosyltransferase as a potential target for nitric oxide action in hippocampal long-term potentiation.
Authors: Schuman E M , Meffert M K , Schulman H , Madison D V
Published: May 2006

Recent studies of long-term potentiation (LTP) in the CA1 region of the hippocampus have demonstrated that nitric oxide (NO) may be involved in some forms of LTP and have suggested that postsynaptically generated NO is a candidate to act as a retrograde messenger. However, the molecular target(s) of NO in LTP remain to be elucidated. The present study examined whether either of two potential NO targets, a soluble guanylyl cyclase or an ADP-ribosyltransferase (ADPRT; EC 2.4.2.31) plays a role in LTP. The application of membrane-permeant analogs of cGMP did not produce any long-lasting alterations in synaptic strength. In addition, application of a cGMP-dependent protein kinase inhibitor did not prevent LTP. We found that the CA1 tissue from hippocampus possesses an ADPRT activity that is dramatically stimulated by NO and attenuated by two different inhibitors of mono-ADPRT activity, phylloquinone and nicotinamide. The extracellular application of these same inhibitors prevented LTP. Postsynaptic injection of nicotinamide failed to attenuate LTP, suggesting that the critical site of ADPRT activity resides at a nonpostsynaptic locus. These results suggest that ADP-ribosylation plays a role in LTP and are consistent with the idea that an ADPRT may be a target of NO action.
Identification of a melanosomal membrane protein encoded by the pink-eyed dilution (type II oculocutaneous albinism) gene.
Authors: Rosemblat S , Durham-Pierre D , Gardner J M , Nakatsu Y , Brilliant M H , Orlow S J
Published: May 2006

The pink-eyed dilution (p) locus in the mouse is critical to melanogenesis; mutations in the homologous locus in humans, P, are a cause of type II oculocutaneous albinism. Although a cDNA encoded by the p gene has recently been identified, nothing is known about the protein product of this gene. To characterize the protein encoded by the p gene, we performed immunoblot analysis of extracts of melanocytes cultured from wild-type mice with an antiserum from rabbits immunized with a peptide corresponding to amino acids 285-298 of the predicted protein product of the murine p gene. This antiserum recognized a 110-kDa protein. The protein was absent from extracts of melanocytes cultured from mice with two mutations (pcp and p) in which transcripts of the p gene are absent or greatly reduced. Introduction of the cDNA for the p gene into pcp melanocytes by electroporation resulted in expression of the 3.3-kb mRNA and the 110-kDa protein. Upon subcellular fractionation of cultured melanocytes, the 110-kDa protein was found to be present in melanosomes but absent from the vesicular fraction; phase separation performed with the nonionic detergent Triton X-114 confirmed the predicted hydrophobic nature of the protein. These results demonstrate that the p gene encodes a 110-kDa integral melanosomal membrane protein and establish a framework by which mutations at this locus, which diminish pigmentation, can be analyzed at the cellular and biochemical levels.
Two-state transition between molten globule and unfolded states of acetylcholinesterase as monitored by electron paramagnetic resonance spectroscopy.
Authors: Kreimer D I , Szosenfogel R , Goldfarb D , Silman I , Weiner L
Published: May 2006

Cys-231 of Torpedo californica acetylcholinesterase (EC 3.1.1.7) was selectively labeled with the mercury derivative of a stable nitroxyl radical. In 1.5 M guanidinium chloride, this conjugate exists in a molten globule state (MG), whereas in 5 M denaturant, it is in an unfolded state (U). The transition between the two states is reversible. In the MG, the label is highly immobilized, whereas in the U, it is almost freely rotating. The clearly distinct electron paramagnetic resonance (EPR) spectra of the two states permits the study of this transition. Upon elevating the guanidinium chloride concentration, a decrease in the EPR signal of the MG occurs concomitantly with an increase in the U signal, the total intensity of the EPR spectra remaining constant. This behavior is characteristic of a two-state transition. The thermodynamic characteristics of this transition (delta G0 and m), whether estimated directly from the EPR data or from both CD and fluorescence data analyzed by assuming a two-state scheme, are in good agreement.
Heparin-dependent binding and autophosphorylation of epidermal growth factor (EGF) receptor by heparin-binding EGF-like growth factor but not by EGF.
Authors: Aviezer D , Yayon A
Published: May 2006

Heparin-binding EGF-like growth factor (HB-EGF) is a recently identified member of the EGF family of growth factors and a potent mitogen for smooth muscle cells and fibroblasts. Chinese hamster ovary (CHO) cells genetically engineered to express the human EGF receptor bind with high affinity both EGF and HB-EGF. CHO mutant cells lacking heparan sulfate proteoglycans (HSPG) bind EGF equally well to wild-type cells and EGF binding is not affected by exogenous heparin. However, HSPG-deficient EGF receptor-expressing cells do not bind significant levels of HB-EGF unless heparin is present in the binding medium. Moreover, binding of radiolabeled EGF to HSPG-deficient EGF receptor-expressing cells is efficiently displaced by nonlabeled HB-EGF only in the presence of heparin. Signal transduction by the EGF receptor tyrosine kinase as evidenced by receptor autophosphorylation is induced by HB-EGF only in the presence of heparin, in contrast to EGF-induced receptor autophosphorylation, which is independent of heparin. These results directly demonstrate that HB-EGF but not EGF requires heparin or cell surface HSPG for binding and activation of the EGF receptor and that HB-EGF receptor interactions can be tightly regulated by the available local concentration of heparin-like molecules.
Modulator factor-binding sequence of the sea urchin early histone H2A promoter acts as an enhancer element.
Authors: Palla F , Bonura C , Anello L , Di Gaetano L , Spinelli G
Published: May 2006

The sea urchin early H2A histone gene, like the other four members of the repeating units, is transiently expressed during very early development. To investigate the mechanisms underlying the faithful expression of the early H2A gene, we focused our attention on the modulator element. We showed by DNase I cleavage protection patterns that the modulator includes the upstream sequence element 1 (USE1) and mapped at nucleotides -137 to -108 in the early H2A gene promoter. Functional tests conducted by microinjection into sea urchin embryos then showed that the modulator element binds the transcriptional factor called modulator-binding factor 1 (MBF-1). We found in fact that coinjection of an excess of the MBF-1-binding site, either as the modulator or as the USE1, efficiently impaired the activity of the H2A promoter. An unexpected finding was the expression of the reporter gene from the early H2A promoter at the gastrula stage of embryonic development, when the early histone genes are transcriptionally silent. In addition, we also found that the modulator element was active at the gastrula stage. The potential enhancer activity of the modulator was tested by microinjecting several constructs containing single or multiple copies of the modulator element placed 5' or 3' to a thymidine kinase gene (tk) promoter in both sea urchin embryos and Xenopus laevis oocytes and determining the expression of a reporter chloramphenicol acetyltransferase gene under the control of the linked tk promoter. We found that an oligonucleotide bearing the MBF-1-binding site activates the expression of the reporter gene independently of the position and orientation. We conclude that the modulator binds the MBF-1 activator and that it is a transcriptional enhancer of the early H2A histone gene.
Singlet molecular oxygen evolution upon simple acidification of aqueous hypochlorite: application to studies on the deleterious health effects of chlorinated drinking water.
Authors: Khan A U , Kasha M
Published: May 2006

A study of the pH profile of the decomposition of aqueous hypochlorite has revealed the evolution (onset at pH 8) of single (1 delta g) molecular oxygen (singlet spin state dioxygen) detected spectroscopically (1268 nm), prior to the appearance of chlorine (onset at pH 5.5). The possible mechanism of the singlet state dioxygen evolution is presented, and the origin of its chloride ion dependence is discussed, especially in reference to chloride ion dependence of singlet molecular oxygen evolution in biological systems. Recent epidemiological analyses of the correlation of human cancer with chlorinated water supplies focus attention on the singlet oxygen mechanisms of DNA lesion formation.
Selective clustering of glutamate and gamma-aminobutyric acid receptors opposite terminals releasing the corresponding neurotransmitters.
Authors: Craig A M , Blackstone C D , Huganir R L , Banker G
Published: May 2006

Several immunocytochemical and physiological studies have demonstrated a concentration of neurotransmitter receptors at postsynaptic sites on neurons, but an overall picture of receptor distribution has not emerged. In particular, it has not been clear whether receptor clusters are selectively localized opposite terminals that release the corresponding neurotransmitter. By using antibodies against the excitatory glutamate receptor subunit GluR1 and the inhibitory type A gamma-aminobutyric acid (GABA) receptor beta 2/3 subunits, we show that these different receptor types cluster at distinct postsynaptic sites on cultured rat hippocampal neurons. The GABAA receptor beta 2/3 subunits clustered on cell bodies and dendritic shafts opposite GABAergic terminals, whereas GluR1 clustered mainly on dendritic spines and was associated with glutamatergic synapses. Chronic blockade of evoked transmitter release did not block receptor clustering at postsynaptic sites. These results suggest that complex mechanisms involving nerve terminal-specific signals are required to allow different postsynaptic receptor types to cluster opposite only appropriate presynaptic terminals.
Source of nuclear calcium signals.
Authors: Allbritton N L , Oancea E , Kuhn M A , Meyer T
Published: May 2006

Transient increases of Ca2+ concentration in the nucleus regulate gene expression and other nuclear processes. We investigated whether nuclear Ca2+ signals could be regulated independently of the cytoplasm or were controlled by cytoplasmic Ca2+ signals. A fluorescent Ca2+ indicator that is targeted to the nucleus was synthesized by coupling a nuclear localization peptide to Calcium Green dextran, a 70-kDa Ca2+ indicator. Stimulation of rat basophilic leukemia cells by antigen or by photolytic uncaging of inositol 1,4,5-trisphosphate induced transient increases in nuclear and cytosolic Ca2+ concentrations. Elevations in the nuclear Ca2+ concentration followed those in the nearby perinuclear cytosol within 200 ms. Heparin-dextran, an inhibitor of the inositol 1,4,5-trisphosphate receptor that is excluded from the nucleus, was synthesized to specifically block the release of Ca2+ from cytosolic stores. Addition of this inhibitor suppressed Ca2+ transients in the nucleus and the cytosol. We conclude that the Ca2+ level in the nucleus is not independently controlled. Rather, nuclear Ca2+ increases follow cytosolic Ca2+ increases with a short delay most likely due to Ca2+ diffusion from the cytosol through the nuclear pores.
A mutation in ribosomal protein L9 affects ribosomal hopping during translation of gene 60 from bacteriophage T4.
Authors: Herbst K L , Nichols L M , Gesteland R F , Weiss R B
Published: May 2006

Ribosomes hop over a 50-nt coding gap during translation of gene 60 mRNA from bacteriophage T4. This event occurs with near-unitary efficiency when gene 60-lacZ fusions are expressed in Escherichia coli. One of the components necessary for this hop is an RNA hairpin structure containing the 5' junction of the 50-nt coding gap. A mutant E. coli was isolated and found to significantly increase hopping when carrying gene 60-lacZ constructs with altered hairpins. The mutation, hop-1, changed Ser93 to Phe in rplI, the gene coding for ribosomal large-subunit protein L9. Ribosomal hopping on a synthetic sequence in the absence of a hairpin was also increased by this mutation. These data suggest that hop-1 may substitute for the function of the hairpin during ribosomal hopping.
Partially oxygenated sickled cells: sickle-shaped red cells found in circulating blood of patients with sickle cell disease.
Authors: Asakura T , Mattiello J A , Obata K , Asakura K , Reilly M P , Tomassini N , Schwartz E , Ohene-Frempong K
Published: May 2006

A previously uncharacterized type of sickled cell was found in venous blood of patients with sickle cell disease when blood was collected without exposure to air and fixed immediately with 1% glutaraldehyde solution equilibrated with 5% oxygen. These cells were either elongated, resembling irreversibly sickled cells (ISCs), or nonelongated, with a raisin-like shape. Both types assumed a normal discoidal shape upon full oxygenation. Since these cells exist only under partially oxygenated conditions, they are described as partially oxygenated sickled cells (POSCs). POSCs are morphologically distinct from partially deoxygenated sickled cells formed during deoxygenation by having rounded edges, while the latter have sharp edges. Transmission electron microscopy of POSCs revealed various amounts of misaligned Hb S polymers. Investigations in vitro demonstrated the formation of POSC-like cells by partial oxygenation of deoxygenated cells. Since POSCs contain intracellular fibers and sickle readily upon deoxygenation, they may have clinical and pathological significance.
Regulation of neuronal Bcl2 protein expression and calcium homeostasis by transforming growth factor type beta confers wide-ranging protection on rat hippocampal neurons.
Authors: Prehn J H , Bindokas V P , Marcuccilli C J , Krajewski S , Reed J C , Miller R J
Published: May 2006

Excessive activation of glutamate receptors accompanied by Ca2+ overloading is thought to be responsible for the death of neurons in various conditions including stroke and epilepsy. Neurons also die if deprived of important growth factors and trophic influences, conditions sensitive to certain oncogene products such as the Bcl2 protein. We now demonstrate that transforming growth factor type beta (TGF-beta) prevents neuronal Ca2+ overloading of rat hippocampal neurons in response to the glutamatergic agonist N-methyl-D-aspartate or the Ca2+ ionophore 4-Br-A23187 and, in addition, leads to a substantial increase in neuronal Bcl2 protein expression. Parallel cytotoxicity experiments demonstrate that treatment with TGF-beta protects rat hippocampal neurons from death induced by excitotoxicity, trophic factor removal, and oxidative injury. Thus, TGF-beta may protect against a wide range of toxic insults by regulating two factors with great importance for neuronal viability.
Human T-cell leukemia virus type I Tax-protein-mediated activation of NF-kappa B from p100 (NF-kappa B2)-inhibited cytoplasmic reservoirs.
Authors: Kanno T , Franzoso G , Siebenlist U
Published: May 2006

The human T-cell leukemia virus type I Tax protein transforms T cells through induced expression of many cellular genes, including those encoding the growth-related proteins interleukin 2 and the alpha chain of its receptor. Induction of these genes is mediated, at least in part, through Tax-dependent posttranslational activation of NF-kappa B, typically heterodimers of p50 (NF-kappa B1) and p65 (RelA). The preexisting NF-kappa B proteins are retained in the cytoplasm of cells by association with inhibitory ankyrin-motif-containing I kappa B proteins, primarily I kappa B-alpha but also including the precursor proteins p105 (NF-kappa B1) and p100 (NF-kappa B2). Here we demonstrate the existence of a previously undescribed multimeric cytoplasmic complex in which NF-kappa B dimers are associated with the p100 inhibitor in a manner dependent on the precursor protein's ankyrin domain. We also demonstrate an antagonistic effect of the Tax protein on the cytoplasmic sequestration function of p100; this in turn leads to nuclear translocation of NF-kappa B dimers liberated from multimeric complexes. Tax may exert these effects through the physical association with p100. Tax also relieves the p100-mediated inhibition of DNA binding by p50-p65 heterodimers in vitro. The results demonstrate a mechanism by which Tax may activate NF-kappa B in T cells.
Determination of the absolute configuration of (+)-neopentyl-1-d alcohol by neutron and x-ray diffraction analysis.
Authors: Yuan H S , Stevens R C , Bau R , Mosher H S , Koetzle T F
Published: May 2006

The absolute configuration of (+)-neopentyl-1-d alcohol, prepared by the reduction of 2,2-dimethylpropanal-1-d by actively fermenting yeast, has been determined to be S by neutron diffraction. The neutron study was carried out on the phthalate half ester of neopentyl-1-d alcohol, crystallized as its strychnine salt. The absolute configuration of the (-)-strychninium cation was first determined by an x-ray anomalous dispersion study of its iodide salt. The chiral skeleton of strychnine then served as a reference from which the absolute configuration of the -O-CHD-C(CH3)3 group of neopentyl phthalate was determined. Difference Fourier maps calculated from the neutron data showed unambiguously that the -O-CHD-C(CH3)3 groups of both independent molecules in the unit cell had the S configuration. This work proves conclusively that the yeast system reduces aldehydes by delivering hydrogen to the re face of the carbonyl group. Crystallographic details: (-)-strychninium (+)-neopentyl-1-d phthalate, space group P2(1) (monoclinic), a = 18.564(6) A, b = 7.713(2) A, c = 23.361(8) A, beta = 94.18(4) degrees, V = 3336.0(5) A3, Z = 2 (T = 100 K). Final agreement factors are R(F) = 0.073 for 2768 reflections collected at room temperature (x-ray analysis) and R(F) = 0.144 for 960 reflections collected at 100 K (neutron analysis).
High alpha-tomatine content in ripe fruit of Andean Lycopersicon esculentum var. cerasiforme: developmental and genetic aspects.
Authors: Rick C M , Uhlig J W , Jones A D
Published: May 2006

A variant of Lycopersion esculentum var. cerasiforme is described that deviates from the typical form of the entire species, including cultivated tomatoes, in possessing high levels (500-5000 micrograms/g of dry weight) of the steroidal alkaloid alpha-tomatine in its ripe fruits. This biotype is restricted to a tiny enclave in the valley of Río Mayo, Department San Martín, Peru. Among 88 accessions of var. cerasiforme from its present distribution in the Andes, a 90% association was found between high tomatine and bitter flavor; within the Mayo watershed, all samples from the upper drainage had bitterness and high tomatine; the frequency of both traits decreased to low levels toward the lower end. Tomatine therefore probably is the source of bitterness. Throughout L. esculentum tomatine is present at very high concentrations in earliest stages of fruit development, thereafter decreasing rapidly to midperiod, and finally diminishing gradually to near zero at maturity as a result of catabolism to biologically inert compounds, except in the variant described here. High tomatine content does not appear to affect adversely either the natives, among whom the bitter types are popular, or individuals who sampled them in this survey. Genetic determination of high tomatine in ripe fruits is totally recessive and appears to be monogenic with interaction with genes of minor effect. The prevailing pattern of glycoalkaloid synthesis and degradation in development of solanaceous fruits suggests a mechanism to protect against predation prior to ripening but to permit it afterward as a device to promote dispersal. In consideration of the nondegradative nature of the variant, its genetic determination, and very restricted geographic distribution, mutation to this form appears to be a random event of doubtful evolutionary significance.
The methylotrophic yeast Hansenula polymorpha contains an inducible import pathway for peroxisomal matrix proteins with an N-terminal targeting signal (PTS2 proteins).
Authors: Faber K N , Haima P , Gietl C , Harder W , Ab G , Veenhuis M
Published: May 2006

Two main types of peroxisomal targeting signals have been identified that reside either at the extreme C terminus (PTS1) or the N terminus (PTS2) of the protein. In the methylotrophic yeast Hansenula polymorpha the majority of peroxisomal matrix proteins are of the PTS1 type. Thus far, for H. polymorpha only amine oxidase (AMO) has been shown to contain a PTS2 type signal. In the present study we expressed H. polymorpha AMO under control of the strong endogenous alcohol oxidase promoter. Partial import of AMO into peroxisomes was observed in cells grown in methanol/(NH4)2SO4-containing medium. However, complete import of AMO occurred if the cells were grown under conditions that induce expression of the endogenous AMO gene. Similar results were obtained when the heterologous PTS2 proteins, glyoxysomal malate dehydrogenase from watermelon and thiolase from Saccharomyces cerevisiae, were synthesized in H. polymorpha. The import of PTS1 proteins, however, was not affected by the growth conditions. These results indicate that the reduced rate of AMO import in (NH4)2SO4-grown cells is not due to competition with PTS1 proteins for the same import pathway. Apparently, AMO is imported via a separate pathway that is induced by amines and functions for PTS2 proteins in general.
Human thrombopoietin: gene structure, cDNA sequence, expression, and chromosomal localization.
Authors: Foster D C , Sprecher C A , Grant F J , Kramer J M , Kuijper J L , Holly R D , Whitmore T E , Heipel M D , Bell L A , Ching A F
Published: May 2006

Thrombopoietin (TPO), a lineage-specific cytokine affecting the proliferation and maturation of megakaryocytes from committed progenitor cells, is believed to be the major physiological regulator of circulating platelet levels. Recently we have isolated a cDNA encoding a ligand for the murine c-mpl protooncogene and shown it to be TPO. By employing a murine cDNA probe, we have isolated a gene encoding human TPO from a human genomic library. The TPO locus spans over 6 kb and has a structure similar to that of the erythropoietin gene (EPO). Southern blot analysis of human genomic DNA reveals a hybridization pattern consistent with a single gene locus. The locus was mapped by in situ hybridization of metaphase chromosome preparations to chromosome 3q26-27, a site where a number of chromosomal abnormalities associated with thrombocythemia in cases of acute myeloid leukemia have been mapped. A human TPO cDNA was isolated by PCR from kidney mRNA. The cDNA encodes a protein with 80% identity to previously described murine TPO and is capable of initiating a proliferative signal to murine interleukin 3-dependent BaF3 cells expressing the murine or human TPO receptor.
Mechanism of electron transfer in the cytochrome b/f complex of algae: evidence for a semiquinone cycle.
Authors: Joliot P , Joliot A
Published: May 2006

The most widely accepted mechanism of electron and proton transfer within the cytochrome (Cyt) b/f complex derives from the Q-cycle hypothesis originally proposed for the mitochondrial Cyt b/c1 complex by Mitchell [Mitchell, P. (1975) FEBS Lett. 57, 135-137]. In chloroplasts, the Cyt b/f complex catalyzes the oxidation of a plastoquinol at a site, Qo (the plastoquinol binding site), close to the inner aqueous phase and the reduction of a quinone at a site, Qi (the plastoquinone binding site), close to the stromal side of the membrane. In an alternative model, the semiquinone cycle [Wikström, M. & Krab, K. (1986) J. Bioenerg. Biomembr. 18, 181-193], a charged semiquinone formed at site Qo is transferred to site Qi where it is reduced into quinol. Flash-induced kinetics of the redox changes of Cyt b and of the formation of a transmembrane potential have been measured in Chlorella sorokiniana cells incubated in reducing conditions that induce a full reduction of the plastoquinone pool. The experiments were performed in the presence of an uncoupler that collapses the permanent electrochemical proton gradient and thus accelerates the rate of the electrogenic processes. The results show that the electrogenic reaction driven by the Cyt b/f complex precedes the processes of reduction or oxidation of the b-hemes. This electrogenic process is probably due to a transmembrane movement of a charged semiquinone, in agreement with the semiquinone-cycle hypothesis. This mechanism may represent an adaptation to reducing conditions when no oxidized quinone is available at the Qi site.
Keratinocyte growth factor functions in epithelial induction during seminal vesicle development.
Authors: Alarid E T , Rubin J S , Young P , Chedid M , Ron D , Aaronson S A , Cunha G R
Published: May 2006

Development of the seminal vesicle (SV) is elicited by androgens and is dependent on epithelial-mesenchymal interactions. Androgenic signal transmission from the androgen-receptor-positive mesenchyme to the epithelium has been postulated to involve paracrine factors. Keratinocyte growth factor (KGF), a member of the fibroblast growth factor family, is produced by stromal/mesenchymal cells and acts specifically on epithelial cells. The KGF transcript was detected by reverse transcription-polymerase chain reaction in newborn mouse SVs and by Northern blot analysis of RNA from cultured neonatal SV mesenchymal cells. Newborn SVs placed in organ culture undergo androgen-dependent growth and differentiation. Addition of a KGF-neutralizing monoclonal antibody to this system caused striking inhibition of both SV growth and branching morphogenesis. This inhibition was due to a decline in epithelial proliferation and differentiation, as the mesenchymal layer was not affected by anti-KGF treatment. When KGF (100 ng/ml) was substituted for testosterone in the culture medium, SV growth was approximately 50% that observed with an optimal dose of testosterone (10(-7) M). All of these findings suggest that KGF is present during a time of active SV morphogenesis and functions as an important mediator of androgen-dependent development.
Association of a cellular myosin II with anionic phospholipids and the neuronal plasma membrane.
Authors: Li D , Miller M , Chantler P D
Published: May 2006

Myosin II has been observed in close proximity to the neuronal plasma membrane, suggesting the possibility that at least one isoform of neuronal myosin II may be capable of direct association. Here, we demonstrate that a significant fraction (> 30%, saturable around 90%) of brain myosin II, but not myosins from skeletal or cardiac muscle, can bind to lipid vesicles composed of the anionic phospholipid L-alpha-phosphatidyl-L-serine but not with vesicles made from the neutral phospholipid L-alpha-phosphatidylcholine. Binding to lipid vesicles made from L-alpha-phosphatidyl-L-serine is enhanced in the presence of millimolar amounts of free calcium. ATPase activity remains unimpaired after vesicle association. Myosin II was also shown to remain in tight association with purified plasma membranes, even after depletion of actin. The above observations suggest that mechanisms involving membrane-bound myosin II are required to facilitate metazoan cell motility.
Molecular model of the specificity pocket of the hepatitis C virus protease: implications for substrate recognition.
Authors: Pizzi E , Tramontano A , Tomei L , La Monica N , Failla C , Sardana M , Wood T , De Francesco R
Published: May 2006

We have built a model of the specificity pocket of the protease of hepatitis C virus on the basis of the known structures of trypsin-like serine proteases and of the conservation pattern of the protease sequences among various hepatitis C strains. The model allowed us to predict that the substrate of this protease should have a cysteine residue in position P1. This hypothesis was subsequently proved by N-terminal sequencing of two products of the protease. The success of this "blind" test increases our confidence in the overall correctness of our proposed alignment of the enzyme sequence with those of other proteases of known structure and constitutes a first step in the construction of a complete model of the viral protease domain.
Phylogenetic analysis of RNA editing: a primitive genetic phenomenon.
Authors: Landweber L F , Gilbert W
Published: May 2006

RNA editing by extensive uridine addition and deletion creates over 90% of the amino acid codons in the cytochrome-c oxidase subunit III (COIII) transcript in Trypanosoma brucei and Herpetomonas, whereas editing of the COIII transcripts in Leishmania tarentolae and Crithidia fasciculata generates only 6% of the amino acid codons and is limited to the 5' ends. Is extensive RNA editing a primitive or derived character? We constructed a phylogenetic tree based on nuclear small-subunit and mitochondrial large- and small-subunit ribosomal RNA sequences for nine species of kinetoplastid protozoa. Our results suggest that extensive editing is a primitive genetic phenomenon that has disappeared in recent evolutionary time and also that there have been multiple losses of the digenetic lifestyle by loss of the vertebrate host in parasite evolution.
Cytoplasmic sequences of the growth hormone receptor necessary for signal transduction.
Authors: Goujon L , Allevato G , Simonin G , Paquereau L , Le Cam A , Clark J , Nielsen J H , Djiane J , Postel-Vinay M C , Edery M
Published: May 2006

To study structure-function relationships of the growth hormone (GH) receptor (GHR), two functional systems have been developed. CHO cells were transiently cotransfected with the cDNA encoding the full-length rat GHR and with a construct consisting of the 5' flanking region of one of two GH-dependent genes encoding ovine beta-lactoglobulin or serine protease inhibitor 2.1 (Spi 2.1, formerly Spi.1; the corresponding rat gene has recently been redesignated Spin2a) coupled to the bacterial reporter gene encoding chloramphenicol acetyltransferase (CAT). Transfected cells were grown in the absence and presence of human GH and dexamethasone for the Spi 2.1 gene construct. GH was able to activate each promoter (with approximately 4-fold induction of CAT activity) in a dose-dependent manner. For both tests, the maximal effect was observed at 20 nM human GH. These tests have been used to identify functional domains of the GHR. Two truncated (T) GHRs, lacking most or part of the cytoplasmic domain [called T276 (ending at residue 276) and T436 (ending at residue 436)], were unable to stimulate CAT activity. The GHR contains a proline-rich region, called "Box I," conserved in the cytokine/GH/prolactin receptor family. Alanine substitutions for the four prolines of GHR Box I were introduced. Single proline-to-alanine mutations did not affect the functional activity of the GHR. However, modification of the four prolines together or deletion of the Box I (15 amino acids between positions 279 and 293) resulted in the complete absence of GH stimulation. Thus, the proline-rich region, shown to be important for other members of this receptor superfamily, is also critical for GH signal transduction.
A developmentally regulated basic-leucine zipper-likegene and its expression in embryonic retina and lens.
Authors: Wang S Z , Adler R
Published: May 2006

Transcriptional regulators play important roles in
the control of key developmental events. We have identified sw3-3, a likely
candidate for such a function, in tissues of the eye and other neural organs. It
encodes a basic-leucine zipper-like protein, in which two leucine zipper motifs
flank a basic domain. The latter contains helix-disturbing amino acids such as
glycine and proline, at positions occupied by conserved asparagine and alanine
residues (respectively) in "conventional" basic-leucine zipper
proteins. sw3-3 is widely expressed at early embryonic stages in the lens,
retina, and other neural tissues and is down-regulated thereafter with a spatial
and temporal pattern that correlates with the cessation of mitotic activity and
the onset of cell migration and differentiation.
Correlation of song learning and territoryestablishment strategies in the song sparrow.
Authors: Beecher M D , Campbell S E , Stoddard P K
Published: May 2006

In a field study, we show that a young song
sparrow (i) selects his songs from three or four older birds who have
neighboring territories, (ii) preferentially learns song types that these tutor
neighbors share, and (iii) ultimately sets up his territory next to, or
replaces, one of these tutor neighbors. The consequence of this song learning
strategy is that the young bird's song repertoire represents the
"logical intersection" of the song repertoires of his tutor neighbors.
We argue that this repertoire is optimally designed for mimicry (sounding like
your neighbors) and for communication between neighbors (song sparrows address
or reply to a neighbor with a song they share with that
neighbor).