bluuebunny/crossref_metadata_2025 huggingface dataset:
Glutathione deficiency leads to mitochondrial damage in brain.
Authors: Jain A , Mårtensson J , Stole E , Auld P A , Meister A
Published: May 2006

Glutathione deficiency induced in newborn rats by giving buthionine sulfoximine, a selective inhibitor of gamma-glutamylcysteine synthetase, led to markedly decreased cerebral cortex glutathione levels and striking enlargement and degeneration of the mitochondria. These effects were prevented by giving glutathione monoethyl ester, which relieved the glutathione deficiency, but such effects were not prevented by giving glutathione, indicating that glutathione is not appreciably taken up by the cerebral cortex. Some of the oxygen used by mitochondria is known to be converted to hydrogen peroxide. We suggest that in glutathione deficiency, hydrogen peroxide accumulates and damages mitochondria. Glutathione, thus, has an essential function in mitochondria under normal physiological conditions. Observations on turnover and utilization of brain glutathione in newborn, preweaning, and adult rats show that (i) some glutathione turns over rapidly (t 1/2, approximately 30 min in adults, approximately 8 min in newborns), (ii) several pools of glutathione probably exist, and (iii) brain utilizes plasma glutathione, probably by gamma-glutamyl transpeptidase-initiated pathways that account for some, but not all, of the turnover; thus, there is recovery or transport of cysteine moieties. These studies provide an animal model for the human diseases involving glutathione deficiency and are relevant to oxidative phenomena that occur in the newborn.
Molecular evolution of alcohol dehydrogenase 1 in members of the grass family.
Authors: Gaut B S , Clegg M T
Published: May 2006

The molecular evolution of the alcohol dehydrogenase 1 (Adh1; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) locus in members of the grass family is analyzed. We report the complete DNA sequence of a genomic clone of Adh1 from Pennisetum glaucum cv. Tift 23DB (pearl millet). The gene is characterized by ten exons and nine introns. The 5' flanking region of the gene contains sequences corresponding to the anaerobic regulatory element as well as sequences corresponding to the TATA box and the CAAT box identified in the maize Adh1-1S flanking regions. Exon sequences from Pennisetum and maize have been subjected to relative rate tests; the maize and Pennisetum Adh1 lineages evolve at equal rates. These results are compared with similar relative rate studies by using the chloroplast DNA encoding the ribulose-1,5-bisphosphate carboxylase (rbcL) gene. Evolutionary rates of Adh1 are estimated. Nonsynonymous rates are found to be 2.50 x 10(-10) substitutions per site per year, whereas synonymous rates are approximately 7.90 x 10(-9) substitutions per site per year. Molecular phylogenies of the Poaceae based upon Adh1 data are presented.
Comparison of constitutive and inducible transcriptional enhancement mediated by kappa B-related sequences: modulation of activity in B cells by human T-cell leukemia virus type I tax gene.
Authors: Mauxion F , Jamieson C , Yoshida M , Arai K , Sen R
Published: May 2006

The kappa B sequence (GGGACTTTCC) binds a factor, NF-kappa B, that is constitutively found in its functional, DNA binding form only in B lymphocytes. A factor with apparently indistinguishable sequence specificity can be induced in many other cell types, where it is used to regulate inducible gene expression. For example, kappa B-related sequences have been shown to be important for the transcription of a few inducible genes, such as the interleukin 2 receptor alpha-chain gene and the beta-interferon gene. However, these genes are not constitutively active in B lymphocytes, suggesting that other regulatory mechanisms must play a role in determining the patterns of expression. We have investigated the constitutive and inducible transcriptional activity mediated by five kappa B-related sequence elements in two different cell types. We show that in S194 plasma cells the activity of each element correlates well with the relative affinity of B-cell-derived NF-kappa B for that element. This leads to significantly lower transcription enhancement by sites derived from the interleukin 2 receptor or T-cell receptor genes in S194 cells. However, in either EL-4 (T) cells or S194 cells, both lower-affinity sites can be significantly induced by the tax gene product of human T-cell leukemia virus type I, showing that NF-kappa B activity can be modulated even in a B-cell line that constitutively expresses this factor.
Development and mechanism of a specific supersensitivity to nitrovasodilators after inhibition of vascular nitric oxide synthesis in vivo.
Authors: Moncada S , Rees D D , Schulz R , Palmer R M
Published: May 2006

The mechanism of the increased sensitivity to nitrovasodilators after removal of endothelial nitric oxide (NO) was investigated in vitro and in vivo. The vasoconstrictor potency of phenylephrine and the force of contraction of rat isolated aortic rings were significantly enhanced after endothelium removal or treatment with inhibitors of endothelial NO synthase. Furthermore, these procedures led to a significant decrease in the basal levels of cGMP in the vascular rings. Moreover, the potency of glyceryl trinitrate (n3Gro) and sodium nitroprusside (SNP) as relaxing agents and the ability of SNP to induce increases in cGMP in aortic rings were significantly enhanced in those rings denuded of endothelium or treated with the inhibitors. These procedures did not affect the vasodilator actions of isoprenaline or 8-bromo-cGMP. In the anesthetized rat, treatment with the inhibitors enhanced significantly the hypotensive responses to n3Gro without affecting those to isoprenaline. These data indicate that the removal of the basal NO-mediated vasodilator tone in the cardiovascular system leads, at the level of the soluble guanylate cyclase, to a specific supersensitivity to nitrovasodilators in vivo. The existence of such a phenomenon has important implications for understanding the local physiological control of blood flow, its pathological disturbances, and the mechanism of action of nitrovasodilators.
Regulation of luteinizing hormone-releasing hormone receptor binding by heterologous and autologous receptor-stimulated tyrosine phosphorylation.
Authors: Liebow C , Lee M T , Kamer A R , Schally A V
Published: May 2006

Pancreatic cancers overexpress tyrosine kinase and luteinizing hormone-releasing hormone (LH-RH) receptor (LH-RHR)-mediated tyrosine phosphatase. LH-RHR is a 60-kDa protein. One of the substrates of epidermal growth factor (EGF)-stimulated tyrosine kinase activity and LH-RH- and somatostatin-stimulated tyrosine phosphatase activity is also a 60-kDa protein. This suggests the possibility that LH-RHR regulation by tyrosine phosphatase and tyrosine kinase is mediated by (de)phosphorylation of existing LH-RHR. To test this hypothesis, membranes of MIA PaCa-2 cells, a human dedifferentiated pancreatic cancer cell line, were incubated without hormone (control) or with 0.1 microM EGF or somatostatin analogue RC-160 for 1 hr at 4 degrees C to phosphorylate the 60-kDa protein. Competition binding experiments with I125-labeled [D-Trp6]LH-RH by displacement with a nonradioactive ligand showed that the LH-RH binding in 69% of the points was increased by EGF and 85% was decreased by RC-160 compared with controls (n = 61; both significant, P less than 0.001). The specific binding was altered, increasing 50-150% after preincubation with EGF and decreasing 60-70% after RC-160. No change was seen in the binding affinity constant after pretreatment with EGF or RC-160. This shows that phosphorylation regulates binding of LH-RH and may explain the up-regulation by EGF and down-regulation by RC-160 and by LH-RH of the LH-RH response.
Ultrasensitive fluorometric detection of carbohydrates as derivatives in mixtures separated by capillary electrophoresis.
Authors: Liu J P , Shirota O , Wiesler D , Novotny M
Published: May 2006

Reducing monosaccharides and oligosaccharides, after reductive amination, were separated and detected as their 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde (CBQCA) derivatives by capillary electrophoresis/laser-induced fluorescence. Under optimized conditions, the minimum detectable quantities for monosaccharide solutes were assessed at low attomole levels (0.5 amol for the CBQCA derivative of galactose). The system has shown considerable promise for high-sensitivity analysis of both neutral and amino sugars in glycoproteins. Complex oligosaccharides, isolated from bovine fetuin by hydrazinolysis, were also successfully "mapped."
Protamine transcript sharing among postmeiotic spermatids.
Authors: Caldwell K A , Handel M A
Published: May 2006

Sharing of cytoplasmic constituents through intercellular bridges connecting postmeiotic spermatids can allow for functional equivalence of genetically nonequivalent spermatids. The technique of in situ hybridization was used to study postmeiotic distribution of transcripts from the mouse protamine 1 (Prm-1) gene among spermatids of mice with chromosomally unbalanced gametes. The Prm-1 gene is located on chromosome 16 and is expressed exclusively in haploid spermatids. Mice doubly heterozygous for two Robertsonian translocations involving chromosome 16 were used for the study of postmeiotic accumulation of transcripts of the Prm-1 gene in spermatogenic cells. The meiotic segregation pattern of chromosomal homologues in these mice produces some spermatids that are chromosomally unbalanced; some spermatids lack chromosome 16 while others have two. In situ hybridization with a cDNA probe for the Prm-1 gene transcript performed on both whole testis sections and spermatogenic cell suspensions showed that there was no statistical difference in distribution of grains over step-5 to step-10 spermatids from Robertsonian-translocation heterozygous mice and from control mice of normal karyotype. These results are consistent with sharing of transcripts of the Prm-1 gene among spermatids within a syncytium.
Calciseptine, a peptide isolated from black mamba venom, is a specific blocker of the L-type calcium channel.
Authors: de Weille J R , Schweitz H , Maes P , Tartar A , Lazdunski M
Published: May 2006

The venom of the black mamba contains a 60-amino acid peptide called calciseptine. The peptide has been fully sequenced. It is a smooth muscle relaxant and an inhibitor of cardiac contractions. Its physiological action resembles that of drugs, such as the 1,4-dihydropyridines, which are important in the treatment of cardiovascular diseases. Calciseptine, like the 1,4-dihydropyridines, selectively blocks L-type Ca2+ channels and is totally inactive on other voltage-dependent Ca2+ channels such as N-type and T-type channels. To our knowledge, it is the only natural polypeptide that has been shown to be a specific inhibitor of L-type Ca2+ channels.
Regulation of rhodopsin kinase by autophosphorylation.
Authors: Buczyłko J , Gutmann C , Palczewski K
Published: May 2006

Rhodopsin kinase (RK) catalyzes the phosphorylation of rhodopsin (Rho) as one of the steps in quenching photoactivated Rho. In this work, we investigated the autophosphorylation of RK and how it affects the interaction between RK and Rho. RK undergoes intramolecular phosphorylation, resulting in the incorporation of three or four phosphates per RK molecule. Phosphorylated RK subsequently is a substrate for protein phosphatases 2A and 2B. We isolated three forms of RK based on their differential interactions with heparin-Sepharose. Fully phosphorylated RK (alpha-RK) binds tightly to Rho but has significantly lower affinity to phosphorylated Rho, whereas unphosphorylated RK (gamma-RK) binds avidly to both forms of Rho. The heterogenous intermediately phosphorylated RK (beta-RK) was not studied. Our data support the hypothesis that RK dissociates from Rho when both Rho and RK become phosphorylated, thereby allowing the binding of arrestin to phosphorylated Rho. These results suggest that autophosphorylation plays an important role in regulating the binding of RK to Rho and that the binding sites of RK and arrestin overlap at least partially.
Nerve growth factor stimulates protein tyrosine phosphorylation in PC-12 pheochromocytoma cells.
Authors: Miyasaka T , Sternberg D W , Miyasaka J , Sherline P , Saltiel A R
Published: May 2006

The cellular actions of nerve growth factor (NGF) and epidermal growth factor (EGF) may be mediated by changes in protein phosphorylation. The tyrosine phosphorylation of two predominant proteins of molecular mass 40 and 42 kDa is seen in PC-12 cells treated with NGF or EGF, correlating with activation of a previously identified serine/threonine protein kinase that phosphorylates microtubule-associated protein (MAP). Stimulation of phosphoprotein (pp) 40 and 42 phosphorylation and MAP kinase activity by NGF but not EGF is selectively attenuated by staurosporine and K-252A. Moreover, the time courses of pp40/42 phosphorylation and MAP kinase activation produced by NGF or EGF are identical. Chromatography of lysates from growth factor-treated cells on ion-exchange or hydrophobic-interaction HPLC resolves MAP kinase into two peaks, neither of which precisely coelutes with pp40 or pp42. One of these peaks (II) exhibits no detectable phosphotyrosine. The other peak (I) has some overlap with pp40. However, the activity residing in both peaks is almost completely inhibited after treatment with alkaline phosphatase, suggesting that, at least, serine/threonine phosphorylation is required for the activity of these enzymes. These data indicate that while tyrosine phosphorylation appears to be a critical early event in NGF action, the role of this modification in activation of MAP kinases remains unclear.
Cloning of the gamma-aminobutyric acid (GABA) rho 1 cDNA: a GABA receptor subunit highly expressed in the retina.
Authors: Cutting G R , Lu L , O'Hara B F , Kasch L M , Montrose-Rafizadeh C , Donovan D M , Shimada S , Antonarakis S E , Guggino W B , Uhl G R
Published: May 2006

Type A gamma-aminobutyric acid (GABAA) receptors are a family of ligand-gated chloride channels that are the major inhibitory neurotransmitter receptors in the nervous system. Molecular cloning has revealed diversity in the subunits that compose this heterooligomeric receptor, but each previously elucidated subunit displays amino acid similarity in conserved structural elements. We have used these highly conserved regions to identify additional members of this family by using the polymerase chain reaction (PCR). One PCR product was used to isolate a full-length cDNA from a human retina cDNA library. The mature protein predicted from this cDNA sequence in 458 amino acids long and displays between 30 and 38% amino acid similarity to the previously identified GABAA subunits. This gene is expressed primarily in the retina but transcripts are also detected in the brain, lung, and thymus. Injection of Xenopus oocytes with RNA transcribed in vitro produces a GABA-responsive chloride conductance and expression of the cDNA in COS cells yields GABA-displaceable muscimol binding. These features are consistent with our identification of a GABA subunit, GABA rho 1, with prominent retinal expression that increases the diversity and tissue specificity of this ligand-gated ion-channel receptor family.
Phosphorylated Mr 32,000 dopamine- and cAMP-regulated phosphoprotein inhibits Na+,K(+)-ATPase activity in renal tubule cells.
Authors: Aperia A , Fryckstedt J , Svensson L , Hemmings H C , Nairn A C , Greengard P
Published: May 2006

Dopamine inhibits Na+,K(+)-ATPase activity in several renal tubule segments and thereby regulates urinary Na+ excretion. We now show that a phosphopeptide of 31 amino acids, corresponding to residues 8-38 of the protein phosphatase inhibitor DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of Mr 32,000), mimics the inhibitory action of dopamine on Na+,K(+)-ATPase activity in renal tubule cells from the ascending limb of the loop of Henle. The dephosphorylated form of the peptide is ineffective. The results indicate that dopamine acts through a protein phosphorylation pathway to regulate the activity of an ion pump. In addition, the data suggest that inhibition of protein phosphatase 1 by phophorylated DARPP-32 is a component of the mechanism by which dopamine regulates urinary Na+ excretion.
Increased abundance of alternatively spliced forms of D2 dopamine receptor mRNA after denervation.
Authors: Neve K A , Neve R L , Fidel S , Janowsky A , Higgins G A
Published: May 2006

The existence of two molecular forms of D2 dopamine receptors suggests that differences in the distribution or regulation of the two forms could be exploited for the pharmacological treatment of disease. Using probes selective for each alternatively spliced variant of D2 receptor mRNA, we determined that both variants were widely distributed in rat brain and pituitary but that the ratio of the forms varied among regions. mRNA for the 444-amino acid-long variant, D2(444), was the most abundant form in pituitary and neostriatum. Intermediate levels of both D2(444) mRNA and the short form, D2(415), were detected in midbrain, and low levels of D2(444) and D2(415) mRNAs were detected in all other regions examined, including hippocampus, cerebellum, and cortex. The D2(444)/D2(415) ratio was generally lower in the regions of low expression than in pituitary and neostriatum. Dopamine-depleting lesions increased the density of D2 receptors in the denervated neostriatum by 29% without altering the affinity of the receptors for [3H]spiperone. The proliferation of receptors appeared to be due to a lesion-induced increase of up to 120% in the abundance of both variants of mRNA in the neostriatum.
Prediction of the thermodynamics of protein unfolding: the helix-coil transition of poly(L-alanine).
Authors: Ooi T , Oobatake M
Published: May 2006

The method given earlier for predicting the thermodynamics of protein unfolding from the x-ray structure of a protein is applied here to the poly(L-alanine) helix. First, the fitting parameters derived earlier from a data base of 10 proteins were used to predict the unfolding thermodynamics of 4 other proteins. The agreement between the observed and predicted values is comparable to that found for the 10 proteins studied initially. Next, the temperature dependences of the Gibbs energy and enthalpy changes for unfolding of bacteriophage T4 lysozyme were predicted and compared with data in the literature. The predicted and observed temperature dependences are similar and the predicted results indicate that cold denaturation should be observed at low temperatures, as observed recently for a T4 lysozyme mutant. The fitting parameters derived from thermodynamic data for protein unfolding and for hydration of model compounds were used to predict the unfolding thermodynamics of the poly(L-alanine) helix. The results predict that helix formation is enthalpy-driven, and the predicted enthalpy change for unfolding (0.86 kcal per mol per residue) is close to the value found in a recent calorimetric study of a 50-residue alanine-rich helix.
Osteoclastic inhibition: an action of nitric oxide not mediated by cyclic GMP.
Authors: MacIntyre I , Zaidi M , Alam A S , Datta H K , Moonga B S , Lidbury P S , Hecker M , Vane J R
Published: May 2006

The osteoclast is unique in its ability to resorb bone, and excessive osteoclastic activity has been implicated in osteoporosis, Paget disease of bone, rheumatoid arthritis, and the growth of metastases in bone. The activity of this cell is controlled by the main circulating inhibitor, calcitonin, in association with locally produced modulators. We show that nitric oxide (NO) may be an important member of the latter group. NO is produced by the vascular endothelium and nervous system and is involved in both neurotransmission and the regulation of blood pressure. However, our results show that the autocoid is also a potent inhibitor of osteoclast function. NO (30 microM) produced a decrease to approximately 50% of the original osteoclast spread area. Similar effects were also produced by 3-morpholinosydnonimine or sodium nitroprusside, reagents that spontaneously release NO. These shape changes were associated with a reduction of bone resorption after a 24-hr incubation of isolated osteoclasts on devitalized bone slices. NO is thought to act by stimulating guanylate cyclase, with a consequent increase in cyclic GMP, but a different mode of action is likely in the osteoclast since dibutyryl or 8-bromo cyclic GMP have no effect. It should be noted that calcitonin can produce similar changes in shape and activity but is associated with an increase in osteoclast intracellular calcium and cessation of membrane movement; neither of these is produced by NO, suggesting that its mode of action is different. The abundance of NO-producing endothelial cells in bone marrow and their proximity to osteoclasts suggests that marrow endothelial cells may play a physiological role in the regulation of osteoclastic activity.
Nonfunctional mutants of the retinoblastoma protein are characterized by defects in phosphorylation, viral oncoprotein association, and nuclear tethering.
Authors: Templeton D J , Park S H , Lanier L , Weinberg R A
Published: May 2006

We have examined the functional consequences of mutations present in defective alleles of the retinoblastoma susceptibility gene (RB1) isolated from two spontaneously arising tumors. Unlike cDNA clones expressing the wild-type protein p110Rb, those encoding the two mutant proteins failed to induce the appearance of senescent cells in transfected Saos-2 human osteosarcoma cells. The mutant proteins were also defective in binding to the E1A oncoprotein, were unable to become hyperphosphorylated, and failed to become tightly associated with nuclear structures. We conclude that mutations in two distinct regions of the protein concomitantly affect these four aspects of p110Rb function.
Many peptide fragments of alien antigens are homologous with host proteins, thus canalizing T-cell responses.
Authors: Ohno S
Published: May 2006

All proteins of this world are constructed in compliance with the same rule. Accordingly, two totally unrelated proteins, on the average, share 30 identical tripeptides, two tetrapeptides, and one pentapeptide per 500 residues. With this in mind, the 221-residue-long influenza virus hemagglutinin II (IVHA-II), as a representative of alien antigens, was compared with three diverse proteins representing the host: 533-residue-long chicken c-src protein kinase (c-src product of the cellular oncogene of Rous sarcoma virus), 595-residue-long human estrogen receptor, and 585-residue-long human serum albumin. Forty-three tripeptides, two tetrapeptides, and one pentapeptide of IVHA-II were also found in one or the other of the three host proteins. Six regions of IVHA-II (9-22 residues long) in which oligopeptides were clustered that were identical to their host oligopeptides were defined as "host-homologous" regions, and the remaining regions were called "nonself" or "pathogen-specific" regions. Because the total number of host proteins is vastly more than three, host-homologous regions were no doubt underestimated, while only one or two regions of IVHA-II must remain as truly pathogen-specific. Nevertheless, oligopeptide analysis of two known T-cell response-eliciting peptide fragments and one known inert peptide fragment of a virus and a malarial protozoan readily revealed the latter to be a host-homologous region. Of the two known T-cell response-eliciting peptide fragments, one was more nonself than the other. Not surprisingly, the more nonself fragment elicited helper T-cell response from individuals of diverse major histocompatibility complex haplotypes, whereas the less nonself fragment elicited cytotoxic T-cell response only from HLA-A2 human individuals.
Characterization of a dopamine receptor (DA2K) in the kidney inner medulla.
Authors: Huo T , Ye M Q , Healy D P
Published: May 2006

Dopamine (DA) produces a natriuretic/diuretic response in the kidney by mechanisms that are still not well understood. There is some indication that DA2 receptors may be involved in mediating the effects of DA, but little is known regarding the nature of this receptor in the kidney. Autoradiographic localization of [3H]spiperone, a DA2 antagonist, indicated that high-density binding was restricted to inner medullary collecting ducts (IMCDs). [3H]Spiperone binding was saturable, high affinity (Kd, 17.2 +/- 1.65 nM), and high density (Bmax, 935 +/- 83 fmol per mg of protein). The photosensitive spiperone analogue N-(p-azido-m-[125I]iodophenethyl)spiperone labeled similar sized proteins of Mr = 120,000 in membranes prepared from the kidney inner medulla, striatum, and pituitary. However, the rank-order competition profile for the [3H]spiperone binding in the kidney inner medulla differed from the DA2 receptor in striatum and pituitary and, furthermore, RNA (Northern) blot analyses of kidney inner medullary RNA with brain DA2 receptor oligonucleotide probes were negative. Functionally, DA stimulated prostaglandin E2 production by IMCD cells, an effect that could be blocked by the DA2 antagonist domperidone. These results indicate that the kidney inner medulla expresses a functional DA receptor that may represent a newly identified DA receptor subtype (here designated DA2K). Moreover, these results suggest that the kidney inner medulla may be a significant site at which DA, either directly or indirectly, influences water and electrolyte excretion.
Construction, arraying, and high-density screening of large insert libraries of human chromosomes X and 21: their potential use as reference libraries.
Authors: Nizetić D , Zehetner G , Monaco A P , Gellen L , Young B D , Lehrach H
Published: May 2006

We have constructed cosmid libraries from flow-sorted human chromosomes X and 21, each of which contains greater than 30 genome equivalents, and have developed systems allowing permanent storage of primary clones, easy screening of libraries in high-density filter formats, and the simultaneous generation of fingerprinting and mapping data on the same set of cosmid clones. Clones are picked into microtiter plate wells and stored at -70 degrees C. A semiautomatic robot system allows the generation of filter replicas containing up to 10,000 clones per membrane. Sets of membranes containing 15-20 chromosome equivalents of both chromosomes will be used for the construction of ordered clone libraries by hybridization fingerprinting protocols. In addition, multiple sets of two membranes containing 4 chromosome equivalents of the human X chromosome, and one membrane containing 3 chromosome equivalents of chromosome 21, have been distributed to other interested laboratories as part of a system of reference libraries. This system allows other groups easy access to the clones and offers an efficient protocol to combine results generated in different laboratories using these libraries. Here we describe the construction of the libraries and demonstrate the use of high-density screening filters in oligonucleotide probe hybridizations and the isolation of cosmids by hybridization with probes from the X chromosome.
Modification of the coding sequence enhances plant expression of insect control protein genes.
Authors: Perlak F J , Fuchs R L , Dean D A , McPherson S L , Fischhoff D A
Published: May 2006

Increased expression of the insect control protein genes of Bacillus thuringiensis in plants has been critical to the development of genetically improved plants with agronomically acceptable levels of insect resistance. The expression of the cryIA(b) gene was compared to partially modified (3% nucleotide difference) and to fully modified (21% nucleotide difference) cryIA(b) and cryIA(c) genes in tobacco and tomato. The modified genes increased the frequency of plants that produced the proteins at quantities sufficient to control insects and dramatically increased the levels of these proteins. Among the most highly expressing transformed plants for each gene, the plants with the partially modified cryIA(b) gene had a 10-fold higher level of insect control protein and plants with the fully modified cryIA(b) had a 100-fold higher level of CryIA(b) protein compared with the wild-type gene. Similar results were obtained with the fully modified cryIA(c) gene in plants. Specific sequences of the partially modified cryIA(b) gene were analyzed for their ability to affect cryIA(b) gene expression in tobacco. The DNA sequence of a single region was identified as important to the improvement of plant expression of the cryIA(b) gene. The increased levels of cryIA(b) mRNA were not directly proportional to the increased levels of CryIA(b) protein in plants transformed with the modified cryIA(b) genes, indicating that the nucleotide sequence of these genes had an effect in improving their translational efficiency in plants.
Acidic and basic fibroblast growth factors are survival factors with distinctive activity in quiescent BALB/c 3T3 murine fibroblasts.
Authors: Tamm I , Kikuchi T , Zychlinsky A
Published: May 2006

Platelet-derived growth factor (PDGF), epidermal growth factor, and insulin-like growth factor have previously been identified as survival factors with distinctive activities for the density-inhibited quiescent BALB/c 3T3 murine fibroblasts. Fibroblast growth factor (FGF), like PDGF, renders quiescent BALB/c 3T3 cells competent to respond to epidermal growth factor and insulin-like growth factor, which mediate cell-cycle traverse through G1 into S phase [Stiles, C. D., Pledger, W. J., VanWyk, J. J., Antoniades, H. N. & Scher, C. D. (1979) Proc. Natl. Acad. Sci. USA 76, 1279-1283]. We now show that FGF possess marked cell survival-enhancing activity distinctive from that of PDGF. Both acidic FGF (aFGF) and basic FGF (bFGF) markedly enhance short-term (3-hr) survival of quiescent cells. bFGF is the more active of the two factors and shows marked long-term (20-hr) survival-promoting activity alone, whereas aFGF requires heparin for long-term activity. Protection by bFGF or aFGF plus heparin is not associated with cell-cycle traverse into S phase. Both the short-term (3-hr) and long-term (20-hr) protective actions of aFGF and bFGF critically depend on protein synthesis, whereas those of PDGF do not. The accumulated evidence shows that several growth factors can contribute to maintenance of the integrity of quiescent murine fibroblasts and that their action can involve protein kinase A- and C-mediated processes as well as protein synthesis. Different growth factors display distinctive modes of action.
The first milliseconds of the pore formed by a fusogenic viral envelope protein during membrane fusion.
Authors: Spruce A E , Iwata A , Almers W
Published: May 2006

Fibroblasts expressing the influenza virus hemagglutinin on their plasma membrane were patch clamped while they fused to erythrocytes. An increase in the fibroblast's membrane capacitance indicated the opening of the "fusion pore," the first aqueous connection between the fusing cells. We show here that the capacitance increase is preceded by a brief current transient, generated as the erythrocyte discharges its membrane potential through the nascent fusion pore. This signal allows one to calculate the pore conductance during the first milliseconds of its existence. The pore conductance jumps from 0 to approximately 150 pS and then grows more gradually over the subsequent tens of milliseconds until growth is arrested. The initial conductance is similar to that of a large ion channel and suggests that the pore is initially only 1-2 nm wide. Hence, we are probably observing events caused by only a small number of hemagglutinin molecules.
A post-transcriptional mechanism contributes to circadian cycling of a per-beta-galactosidase fusion protein.
Authors: Zwiebel L J , Hardin P E , Liu X , Hall J C , Rosbash M
Published: May 2006

The period gene (per) of Drosophila melanogaster affects circadian rhythms. Circadian fluctuations in per mRNA levels are thought to contribute to circadian fluctuations in per protein levels in the heads of adult flies. To address the mechanisms underlying these oscillatory phenomena, we have analyzed RNA and protein cycling from two per-beta-galactosidase fusion genes. These studies demonstrate that 5' noncoding sequences from per are sufficient to cause the fusion mRNA levels to cycle in a wild-type (rhythmic) background. Protein cycling requires additional sequences derived from the per coding region. The data suggest that there is a per-dependent posttranscriptional mechanism that is under circadian clock control required for per protein levels to fluctuate in a rhythmic fashion.
Physiological role of epidermal growth factor on adipose tissue development in vivo.
Authors: Serrero G , Mills D
Published: May 2006

The ability of epidermal growth factor (EGF) to affect adipose tissue development in vivo was investigated. The subcutaneous administration of EGF to newborn NBR rats for 10 days resulted in decreased body weight and fat pad weight that occurred in a dose-dependent fashion. At a dose of 1 micrograms/g of body weight, injected EGF resulted in a 50% decrease of fat pad weight. Although kidney weight was also diminished, the weight of other organs such as liver and intestine remained unchanged or even slightly increased, suggesting that the effect of EGF on fat pad development was not due to a generalized inhibitory action of EGF on the development of the neonate rats. The number of adipocyte precursors in inguinal fat pads of EGF-treated animals was higher than in control animals (1.3-fold for 0.3 micrograms/g of body weight of EGF and 1.8-fold for 1.0 micrograms/g of body weight), whereas the number of mature adipocytes and the amount of triglyceride accumulated per fat pad were concomitantly lower. Adipocyte precursors isolated from EGF-treated animals displayed a reduced differentiation ability in culture and a higher sensitivity to the inhibitory effect of EGF than did cells isolated from control animals. These experiments demonstrate that EGF can retard adipose tissue development in vivo and suggest that EGF plays an important physiological role in the control of adipocyte differentiation.
Protein-protein interactions with the acidic COOH terminus of the single-stranded DNA-binding protein of the bacteriophage T4.
Authors: Krassa K B , Green L S , Gold L
Published: May 2006

The single-stranded DNA-binding protein of the bacteriophage T4 is encoded by gene 32. Monoclonal antibodies were raised against intact gene 32 protein (gp32). We mapped the epitopes recognized by 12 of these monoclonal antibodies; the epitopes are all within the COOH-terminal region of gp32. As shown by others, removal of the COOH terminus of gp32 abolishes the ability of the intact protein to bind to many T4 proteins involved in replication, recombination, repair, and late transcription. These results suggest that the COOH terminus of gp32 is a protein-binding domain. The COOH terminus is attached to a DNA-binding domain that includes a zinc finger. We propose a model in which the DNA-binding and protein-binding domains are used in T4 replication, recombination, repair, and late transcription. The COOH terminus of gp32 is very acidic and may form four negatively charged amphipathic alpha-helices, which could fold into a four-helix bundle when associated with other proteins. At least six of the monoclonal anti-gp32 antibodies bind to the COOH terminus of gp32 and to DNA. Similarities between the COOH terminus of gp32 and DNA are explored.
Construction of a map of chromosome 16 by using radiation hybrids.
Authors: Ceccherini I , Romeo G , Lawrence S , Breuning M H , Harris P C , Himmelbauer H , Frischauf A M , Sutherland G R , Germino G G , Reeders S T
Published: May 2006

A human-hamster cell hybrid carrying a single copy of chromosome 16 as the only human genetic material was irradiated with a single dose of gamma-rays (7000 rads; 1 rad = 0.01 Gy) and then fused with a thymidine kinase-deficient hamster cell line (RJKM) to generate radiation hybrids retaining unselected fragments of this human chromosome. In two experiments, 223 hybrids were isolated in hypoxanthine/aminopterine/thymidine (HAT) medium and screened with 38 DNA probes, corresponding to anonymous DNA or gene sequences localized on chromosome 16. The most likely order and location of the 38 DNA sequences were established by multiple pairwise analysis and scaled to estimate physical distance in megabases. The order and the distances thus obtained are mostly consistent with available data on genetic and physical mapping of these markers, illustrating the usefulness of radiation hybrids for mapping.
Pertussis toxin has eukaryotic-like carbohydrate recognition domains.
Authors: Saukkonen K , Burnette W N , Mar V L , Masure H R , Tuomanen E I
Published: May 2006

Bordetella pertussis is bound to glycoconjugates on human cilia and macrophages by multiple adhesins, including pertussis toxin. The cellular recognition properties of the B oligomer of pertussis toxin were characterized and the location and structural requirements of the recognition domains were identified by site-directed mutagenesis of recombinant pertussis toxin subunits. Differential recognition of cilia and macrophages, respectively, was localized to subunits S2 and S3 of the B oligomer. Despite greater than 80% sequence homology between these subunits, ciliary lactosylceramide exclusively recognized S2 and leukocytic gangliosides bound only S3. Substitution at residue 44, 45, 50, or 51 in S2 resulted in a shift of carbohydrate recognition from lactosylceramide to gangliosides. Mutational exchange of amino acid residues 37-52 between S2 and S3 interchanged their carbohydrate and target cell specificity. Comparison of these carbohydrate recognition sequences to those of plant and animal lectins revealed that regions essential for function of the prokaryotic lectins were strongly related to a subset of eukaryotic carbohydrate recognition domains of the C type.
Transcription factor AP-1 activity is required for initiation of DNA synthesis and is lost during cellular aging.
Authors: Riabowol K , Schiff J , Gilman M Z
Published: May 2006

Activation of the AP-1 complex of transcription factors is one of the earliest nuclear responses to mitogenic stimuli. We demonstrate directly that AP-1 activity is required for human cells to proliferate in response to serum. We also find that activity of the AP-1 complex is selectively reduced in old human fibroblasts prior to their entering a fully senescent state. Levels of Fos protein induced through diverse signal transduction pathways, the amount of AP-1 DNA binding activity in vitro, and the activity of an AP-1-dependent reporter gene in vivo are substantially decreased as fibroblasts age. Moreover, the composition of the AP-1 complex changes, so that old cells produce predominantly Jun-Jun homodimers instead of Fos-Jun heterodimers. Changes in AP-1 activity may be due in part to changes in posttranslational modification of Fos protein that impair its ability to form active DNA-binding heterodimers with Jun. These data suggest that changes in AP-1 activity may contribute to the inability of senescent cells to proliferate in response to mitogens.
Importance of primer selection for the detection of hepatitis C virus RNA with the polymerase chain reaction assay.
Authors: Bukh J , Purcell R H , Miller R H
Published: May 2006

We compared four primer sets from conserved regions of the hepatitis C virus (HCV) genome for their ability to detect HCV RNA in a "nested" cDNA polymerase chain reaction assay on sera from 114 anti-HCV antibody-positive individuals from around the world. The different primer sets had equivalent sensitivity, detecting less than 1 chimpanzee ID50 (dose that infects 50%) when tested against reference strain H of HCV. We tested equal amounts of RNA extracted from the serum of each individual with the four primer sets. The set derived from two highly conserved domains within the 5' noncoding (NC) region of the HCV genome, which also share significant similarity with Pestivirus 5' NC sequences, was the most effective at detecting HCV RNA. All samples positive for HCV RNA with any other primer set were also positive with the primer set from the 5' NC region, and the latter was at least 3 times more likely to detect HCV infection than a primer set from within the nonstructural protein 3-like gene region (P less than 0.001). We had no false positive results in greater than 500 negative controls interspersed among the test samples. The 5' NC region primer set detected HCV-specific RNA, verified by high-stringency Southern blot hybridization and DNA sequencing, in 100% of 15 acute and 33 chronic non-A, non-B hepatitis patients from the United States, Europe, and Asia and 10 hepatocellular carcinoma patients from Africa and Asia that tested negative for the hepatitis B virus-encoded surface antigen. In conclusion, use of an appropriate primer set is crucial for detecting HCV RNA in the serum of infected individuals.
Long-distance restriction mapping of the proximal long arm of human chromosome 21 with Not I linking clones.
Authors: Ichikawa H , Shimizu K , Saito A , Wang D N , Oliva R , Kobayashi H , Kaneko Y , Miyoshi H , Smith C L , Cantor C R
Published: May 2006

Human chromosome 21 is the smallest of the 22 autosomes and 2 sex chromosomes. Hybridization of the human repetitive sequence Alu to pulsed-field gel-fractionated Not I-digested genomic DNA from a human-mouse hybrid cell line containing chromosome 21 as the sole human component identified chromosome 21 Not I restriction fragments. A Not I restriction map of regions of the chromosome was constructed, by identifying neighboring Alu bands with Not I linking clones. This approach simplifies the task of physical mapping and avoids ambiguities in Not I fragment assignments that arise from gel-to-gel mobility variations. A contiguous map was constructed with six Not I linking clones that covers at least the proximal one-third of the long arm of chromosome 21 and spans 20 megabases. A more detailed restriction map revealed 11 likely CpG islands in this region and localized 11 additional DNA markers.
Component H of the DNA-dependent RNA polymerases of Archaea is homologous to a subunit shared by the three eucaryal nuclear RNA polymerases.
Authors: Klenk H P , Palm P , Lottspeich F , Zillig W
Published: May 2006

The gene encoding component H of the DNA-dependent RNA polymerase (RNAP, EC 2.7.7.6) of Sulfolobus acidocaldarius has been identified by comparison of the amino acid sequence with the derived amino acid sequence of an open reading frame (ORF88) in the RNAP operon. Corresponding genes were identified in Halobacterium halobium and were cloned and sequenced from Thermococcus celer and Methanococcus vannielii. All these rpoH genes are situated between the promoters of the RNAP operons and the corresponding rpoB and rpoB2 genes. The archaeal H subunits show high sequence similarity to each other and to the C-terminal portions of the largest of four subunits shared by all three specialized nuclear RNAPs. These correlations are further evidence for the striking similarity between archaeal and eucaryal RNAP structures and transcription systems.
Phosphoglucomutase 1: complete human and rabbit mRNA sequences and direct mapping of this highly polymorphic marker on human chromosome 1.
Authors: Whitehouse D B , Putt W , Lovegrove J U , Morrison K , Hollyoake M , Fox M F , Hopkinson D A , Edwards Y H
Published: May 2006

A cDNA clone encoding the mRNA for the highly polymorphic human enzyme phosphoglucomutase 1 (PGM1; EC 5.4.2.2) has been isolated and characterized. This was achieved indirectly by first isolating a rabbit cDNA from an expression library using anti-rabbit PGM antibodies. A comparison of the nucleotide sequences shows that the homologies between human and rabbit PGM1 mRNAs are 92% and 97% for the coding nucleotide sequence and the amino acid sequence, respectively. The derived rabbit amino acid sequence is in complete agreement with the published protein sequence for rabbit muscle PGM. A physical localization of the human PGM1 gene to chromosome 1p31 has been determined by in situ hybridization. Analysis of DNA from a wide variety of vertebrates indicates a high level of PGM1 sequence conservation during evolution.
Identification of a macrophage-binding determinant on lipophosphoglycan from Leishmania major promastigotes.
Authors: Kelleher M , Bacic A , Handman E
Published: May 2006

Leishmania are obligatory intracellular parasites in mammalian macrophages that gain entry by receptor-mediated phagocytosis. Their major cell surface glycoconjugate, lipophosphoglycan (LPG), has been implicated in this process. A monoclonal antibody specific for Leishmania major LPG (WIC 79.3), which has been shown to block promastigote attachment to macrophages, was used to identify a macrophage-binding determinant of LPG. WIC 79.3 bound exclusively to the phosphorylated repeats of LPG and not to the saccharide core or lipid anchor. Furthermore, the epitope recognized by WIC 79.3 mapped to the phosphorylated oligosaccharide P5b, PO4-6[Gal(beta 1-3)Gal(beta 1-3)Gal(beta 1-3)]Gal(beta 1-4)Man(alpha 1-, which is unique to the LPG of promastigotes of L.major. Phosphorylated oligosaccharides P3, PO4-6[Gal(beta 1-3)[Gal(beta 1-4) Man(alpha 1-, and P4b, PO4-6[Gal(beta 1-3)Gal(beta 1-3)] Gal(beta 1-4)Man(alpha 1-, were also recognized by WIC 79.3 but with considerably lower (approximately 100-fold) affinities. The phosphorylated oligosaccharide P5b inhibited attachment of promastigotes of L. major to the macrophage cell line J774 to the same degree as phosphoglycan (derived from LPG) and Fab fragments of WIC 79.3, suggesting that P5b is a site of L. major LPG that is recognized by macrophage receptor(s) and is an important determinant in the attachment of promastigotes to host macrophages and initiation of infection.
A mutation in the human immunodeficiency virus type 1 transmembrane glycoprotein gp41 dominantly interferes with fusion and infectivity.
Authors: Freed E O , Delwart E L , Buchschacher G L , Panganiban A T
Published: May 2006

Several domains of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein have been identified that are involved in HIV-1-mediated membrane fusion. One domain that is involved in membrane fusion is the hydrophobic amino terminus of the HIV-1 transmembrane glycoprotein gp41. Here we show that a polar substitution at gp41 amino acid 2 (the 41.2 mutation) results in an envelope glycoprotein that dominantly interferes with both syncytium formation and infection mediated by the wild-type HIV-1 envelope glycoprotein. The interference by the 41.2 mutant is not a result of aberrant envelope glycoprotein synthesis, processing, or transport. The 41.2 mutant elicits a dominant interfering effect even in the presence of excess wild-type glycoprotein, suggesting that a higher-order envelope glycoprotein complex is involved in membrane fusion. These results shed light on the process by which the HIV-1 envelope glycoproteins induce membrane fusion reactions and present a possible approach to anti-HIV therapy.