bluuebunny/crossref_metadata_2025 huggingface dataset:
Exogenous advanced glycosylation end products induce complex vascular dysfunction in normal animals: a model for diabetic and aging complications.
Authors: Vlassara H , Fuh H , Makita Z , Krungkrai S , Cerami A , Bucala R
Published: May 2006

Advanced glycosylation end products (AGEs) have been implicated in many of the complications of diabetes and normal aging. Markedly elevated vascular tissue and circulating AGEs were linked recently to the accelerated vasculopathy of end-stage diabetic renal disease. To determine the pathogenic role of AGEs in vivo, AGE-modified albumin was administered to healthy nondiabetic rats and rabbits alone or in combination with the AGE-crosslink inhibitor aminoguanidine. Within 2-4 weeks of AGE treatment, the AGE content of aortic tissue samples rose to six times the amount found in controls (P < 0.001). Cotreatment with aminoguanidine limited tissue AGE accumulation to levels two times that of control. AGE administration was associated with a significant increase in vascular permeability, as assessed by 125I label tracer methods. This alteration was absent in animals that received aminoguanidine in addition to AGE. Significant mononuclear cell migratory activity was observed in subendothelial and periarteriolar spaces in various tissues from AGE-treated rats compared to normal cellularity noted in tissues from animals treated with aminoguanidine. Blood pressure studies of AGE-treated rats and rabbits revealed markedly defective vasodilatory responses to acetylcholine and nitroglycerin compared to controls (P < 0.001), consistent with marked NO. inactivation; aminoguanidine treatment significantly prevented this defect. These in vivo data demonstrate directly that AGEs, independent of metabolic or genetic factors, can induce complex vascular alterations resembling those seen in diabetes or aging. AGE administration represents an animal model system for the study of diabetic and aging complications as well as for assessing the efficacy of newly emerging therapies aimed at inhibiting advanced glycosylation.
Repair of DNA damaged by UV light and ionizing radiation by cell-free extracts prepared from Schizosaccharomyces pombe.
Authors: Sidik K , Lieberman H B , Freyer G A
Published: May 2006

A whole cell extract prepared from Schizosaccharomyces pombe was shown to be active in an assay for repair of plasmid DNA damaged by either ultraviolet (UV) light or gamma-radiation. The assay allows for analysis of repair synthesis at single-strand nicks generated by gamma-rays and analysis of the incision step and repair synthesis in UV-light-damaged DNA. Repair synthesis of DNA damaged by either UV light or gamma-rays was shown to depend on the presence of ATP in the reaction mixture. However, incision at pyrimidine dimers did not require the addition of exogenous ATP. These studies showed that plasmid DNA containing a single pyrimidine dimer or one single-strand nick is a suitable substrate in this assay system. S. pombe is a genetically well-defined eukaryotic organism and many radiation-sensitive mutant derivatives have already been described, making this a powerful system in which to study DNA excision repair.
Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons.
Authors: Screaton G R , Bell M V , Jackson D G , Cornelis F B , Gerth U , Bell J I
Published: May 2006

The CD44 molecule is known to display extensive size heterogeneity, which has been attributed both to alternative splicing and to differential glycosylation within the extracellular domain. Although the presence of several alternative exons has been partly inferred from cDNA sequencing, the precise intron-exon organization of the CD44 gene has not been described to date to our knowledge. In the present study we describe the structure of the human CD44 gene, which contains at least 19 exons spanning some 50 kilobases of DNA. We have identified 10 alternatively spliced exons within the extracellular domain, including 1 exon that has not been previously reported. In addition to the inclusion or exclusion of whole exons, more diversity is generated through the utilization of internal splice donor and acceptor sites within 2 of the individual exons. The variation previously reported for the cytoplasmic domain is shown to result from the alternative splicing of 2 exons. The genomic structure of CD44 reveals a remarkable degree of complexity, and we confirm the role of alternative splicing as the basis of the structural and functional diversity seen in the CD44 molecule.
Approach to a retrovirus vaccine: immunization of mice against Friend virus disease with a replication-defective Friend murine leukemia virus.
Authors: Ruan K S , Lilly F
Published: May 2006

In an initial attempt to test the ability of replication-defective retroviruses to immunize against immunologically related pathogenic viruses, we have worked with the erythroleukemogenic Friend retrovirus complex (FV), which consists of a replication-competent helper component, Friend murine leukemia virus (FMuLV), and a related defective pathogenic component, spleen focus-forming virus (SFFV). An 81-base-pair deletion was introduced into the p15E-encoding region of the env gene of an otherwise replication-competent molecular clone of the FMuLV provirus. After transfection of this clone into cells that package the viral RNA in MuLV coats, infectious virus was released into the culture medium. Mouse fibroblasts infected with this virus, here called delta FMuLV, expressed the truncated viral env gene products in their cytoplasm but not on cell surfaces, and culture fluids from these cells did not transmit the infection to fresh mouse fibroblasts. In preliminary experiments, immunization of mice of H-2-congenic BALB/c strains with delta FMuLV conferred levels of immunity to FV disease ranging from weak to relatively strong. Immunized mice developed anti-FV IgM and IgG antibodies and cytotoxic T cells. Mice observed for 15 weeks after the first of two immunizations showed no detectable pathology, but delta FMuLV DNA was detectable in livers of some immunized mice for at least 3-6 weeks. These results suggest that our approach to development of retrovirus vaccines may be a useful one.
The transition to a sustainable society.
Authors: Speth J G
Published: May 2006

Transitions in dealing with the root causes of environmental problems are advocated to achieve environmental sustainability. These transformations include (i) a demographic transition, (ii) a technology transition that includes the "green" automobile, (iii) an economic transition to one in which prices reflect full environmental costs, (iv) a transition in social equity, and (v) an institutional transition to different arrangements among governments, businesses, and peoples. Businessmen and environmentalists are urged to work together in the next decade to make the environment a personal issue, to call for government action, to recognize the environmental challenges, and to commit to accountability in order to leave a legacy of hope to the twenty-first century.
Industrial ecology: reflections on a colloquium.
Authors: Ausubel J H
Published: May 2006

Industrial ecology is the network of all industrial processes as they may interact with each other and live off each other, not only in the economic sense, but also in the sense of direct use of each other's material and energy wastes and products. This paper, which reflects upon the papers and discussions at the National Academy of Sciences Colloquium on Industrial Ecology on May 20-21, 1991, is structured around 10 questions. Do sociotechnical systems have long-range environmental goals? How is the concept of industrial ecology useful and timely? What are environmental technologies? Is there a systematic way to choose among alternatives for improving the ecology of technologies? What are ways to measure performance with respect to industrial ecology? What are the sources and rates of innovation in environmental technologies? How is the market economy performing with respect to industrial ecology? What will be the effect of the ecological modernization of the developed nations of the North on the developing countries of the South? How can creative interaction on environmental issues be fostered among diverse social groups? How must research and education change?
Isolation and characterization of the epsilon subspecies of protein kinase C from rat brain.
Authors: Koide H , Ogita K , Kikkawa U , Nishizuka Y
Published: May 2006

The epsilon subspecies of protein kinase C (epsilon PKC) was purified to near homogeneity from the soluble fraction of rat brain by successive chromatographies on DEAE-cellulose, threonine-Sepharose, phenyl-5PW, Mono Q, heparin-5PW, and hydroxyapatite columns. The enzyme from COS-7 cells that were transfected with an epsilon PKC cDNA expression plasmid showed the same elution profile. The purified enzyme from the brain was a double (96 and 93 kDa) on SDS/PAGE. Both the doublet proteins were recognized by antibodies raised against several oligopeptides that were parts of the deduced amino acid sequence of the rat brain epsilon PKC. When treated with potato acid phosphatase, both doublet proteins disappeared with the concomitant appearance of a single protein at 90 kDa, suggesting that epsilon PKC exists in the tissue as phosphorylated forms. The physiological significance of this phosphorylation is unknown. The enzymes from the rat brain and COS-7 cells were indistinguishable from each other in their kinetic and catalytic properties. Unlike alpha-, beta I-, beta II-, and gamma PKC, epsilon PKC was independent of Ca2+ but absolutely required phosphatidylserine and diacylglycerol for its activation; a tumor-promoting phorbol ester could replace diacylglycerol. epsilon PKC showed enzymological properties similar to those of delta PKC, except that epsilon PKC but not delta PKC was greatly activated by free arachidonic acid. Immunoblot analysis revealed that, in marked contrast to delta PKC, epsilon PKC is expressed predominantly in the brain tissue and only in trace amounts in heart, lung, spleen, thymus, and testis.
Synthesis of receptor antagonists of neuropeptide Y.
Authors: Tatemoto K , Mann M J , Shimizu M
Published: May 2006

We report the synthesis of receptor antagonists of neuropeptide Y (NPY) by a strategy based on synthesis of mixtures of analogs and the subsequent isolation and identification of receptor antagonists from these mixtures. After screening a series of mixtures of NPY analogs by using an NPY antagonist assay, two potent receptor antagonists, designated PYX-1 and PYX-2, were isolated from an antagonist-containing mixture. Structural analysis revealed these analogs to be Ac-[3-(2,6-dichlorobenzyl)Tyr27, D-Thr32]NPY-(27-36) amide and Ac-[3-(2,6-dichlorobenzyl)Tyr27,36,D-Thr32]NPY-(27-36) amide, respectively. The receptor antagonists inhibited release of intracellular calcium elicited by NPY in human erythroleukemia cells and displaced 3H-labeled NPY from NPY receptors in rat brain membrane. The approach of screening and identifying useful analogs from synthetic mixtures may significantly reduce the time and resources previously required for development of receptor antagonists.
Photoreceptor degeneration induced by the expression of simian virus 40 large tumor antigen in the retina of transgenic mice.
Authors: al-Ubaidi M R , Hollyfield J G , Overbeek P A , Baehr W
Published: May 2006

Expression of the viral oncogene encoding the simian virus 40 (SV40) large tumor antigen (T antigen) typically promotes tumorigenesis in mammalian cells. To generate transgenic mice that express T antigen in rod photoreceptors, a chimeric construct consisting of a mouse opsin promoter fragment fused to the coding region of SV40 T antigen was generated. Expression of T antigen in the transgenic retina began at early stages of postnatal development concomitant with expression of endogenous opsin. Instead of inducing hyperplasia or tumor formation, T-antigen expression caused a rapidly progressing photoreceptor degeneration. The degeneration was accompanied by sustained DNA synthesis in photoreceptor cells, as evidenced by incorporation of [3H]thymidine and by the appearance of mitotic figures at postnatal day 10, a stage when nontransgenic photoreceptor cells are postmitotic and quiescent. Although transgenic photoreceptor cells undergo S phase and enter mitosis, the consequences of T-antigen expression are not proliferation and tumorigenesis but proliferation and cell death.
Calcium-activated potassium channels mediate prejunctional inhibition of peripheral sensory nerves.
Authors: Stretton D , Miura M , Belvisi M G , Barnes P J
Published: May 2006

Activation of several receptors, including mu-opioid, alpha 2-adrenergic, and neuropeptide Y receptors, inhibits excitatory nonadrenergic noncholinergic (NANC) neural responses in airways, which were mediated by the release of peptides from capsaicin-sensitive sensory nerves. This raises the possibility of a common inhibitory mechanism, which may be related to an increase in K+ conductance in sensory nerves. To examine this hypothesis, we have studied whether K(+)-channel blockers inhibit the effects of neuromodulators of sensory nerves in guinea pig bronchi by using selective K(+)-channel blockers. Charybdotoxin (ChTX; 10 nM), which blocks large conductance Ca(2+)-activated K(+)-channel function, completely blocked and reversed the inhibitory effects of a mu-opioid agonist, neuropeptide Y, and an alpha 2-adrenoceptor agonist on excitatory NANC responses. Neither inhibitors of ATP-sensitive K+ channels (BRL 31660 or glibenclamide, both at 10 microM) nor an inhibitor of small conductance Ca(2+)-activated K+ channels (apamin; 0.1 microM) were effective. This suggests that ChTX-sensitive K(+)-channel activation may be a common mechanism for the prejunctional modulation of sensory nerves in airways. This may have important implications for the control of neurogenic inflammation.
p185c-neu and epidermal growth factor receptor associate into a structure composed of activated kinases.
Authors: Quian X L , Decker S J , Greene M I
Published: May 2006

The protein product of the neu protooncogene, p185c-neu, is structurally similar to the epidermal growth factor receptor (EGFR). Overexpression of these two receptor tyrosine kinases, but not either separately, leads to transformation and tumorigenicity. Heterodimerization of p185c-neu and EGFR occurs in M1 cells, which express both receptors. We have individually identified the two components of the heterodimer as EGFR and p185c-neu. Analysis of this association with relatively nondenaturing detergents and in the absence of cross-linkers indicates that noncovalent interactions are primarily responsible for heterodimer formation. The rapid reversible heterodimerization was promoted by EGF binding to its receptor. Functionally, the heterodimer is a highly active protein kinase for receptor autophosphorylation and exogenous substrate phosphorylation in vitro. The isolated heterodimer was highly phosphorylated on tyrosine residues in vivo. These results indicate that the physical association between EGFR and p185c-neu is of functional significance and define enzymatic features of complex receptor formation.
Biosynthesis of phosphatidylinositol-glycan (PI-G)-anchored membrane proteins in cell-free systems: cleavage of the nascent protein and addition of the PI-G moiety depend on the size of the COOH-terminal signal peptide.
Authors: Kodukula K , Cines D , Amthauer R , Gerber L , Udenfriend S
Published: May 2006

Nascent translation products of PI-G-anchored membrane proteins contain both NH2- and COOH-terminal signal sequences of approximately 15-30 residues that are removed during processing. Removal of the latter occurs concomitant with the addition of the PI-G moiety to the newly formed COOH terminus. In human placental alkaline phosphatase (PLAP) the COOH-terminal signal peptide contains 29 residues. An engineered form of PLAP, miniPLAP 208, containing the same NH2- and COOH-terminal signal peptides as PLAP, was used as a substrate for cell-free processing. A comparison was made with mutants (delta 202, delta 197, delta 184, and delta 179) truncated at the COOH terminus. Intact preprominiPLAP 208 and truncated delta 202 were processed to yield the same mature product which, by size and distribution between Triton X-114 and water before and after treatment with inositol-specific phospholipases, indicates that it contained the PI-G moiety. Mutants that were further truncated at the COOH terminus, miniPLAPs delta 197, delta 184, and delta 179, were processed only at their NH2 termini. Those portions of the COOH-terminal sequence in miniPLAPs delta 197 and delta 1984 that extended beyond residue 179 were not removed during processing.
Mouse model of neurodegeneration: atrophy of basal forebrain cholinergic neurons in trisomy 16 transplants.
Authors: Holtzman D M , Li Y W , DeArmond S J , McKinley M P , Gage F H , Epstein C J , Mobley W C
Published: May 2006

Vulnerability of specific brain regions and neuronal populations is a characteristic feature of Alzheimer disease and Down syndrome. Cholinergic neurons of the basal forebrain degenerate in both disorders. The basis for neuronal degeneration is unknown. Mouse trisomy 16 (Ts 16) is an animal model of Down syndrome. We sought an experimental system in which the survival and development of Ts 16 basal forebrain cholinergic neurons could be examined beyond the fetal period. As Ts 16 mice do not survive birth, we transplanted fetal Ts 16 and control basal forebrain into the hippocampus of young adult mice. Transplanted neurons survived and grew neurites in all grafts. Over time, we observed selective atrophy of cholinergic neurons in Ts 16 grafts. Denervation of the hippocampus produced a significant increase in the size of Ts 16 cholinergic neurons. This suggests that hippocampal-derived neurotrophic factors acted to prevent degeneration. beta/A4-amyloid-containing plaques were not seen. Ts 16 provides a model of spontaneous, genetically determined neurodegeneration that may be used to understand better the molecular pathogenesis of neuronal dysfunction in Alzheimer disease and Down syndrome.
The human 64-kDa polyadenylylation factor contains a ribonucleoprotein-type RNA binding domain and unusual auxiliary motifs.
Authors: Takagaki Y , MacDonald C C , Shenk T , Manley J L
Published: May 2006

Cleavage stimulation factor is one of the multiple factors required for 3'-end cleavage of mammalian pre-mRNAs. We have shown previously that this factor is composed of three subunits with estimated molecular masses of 77, 64, and 50 kDa and that the 64-kDa subunit can be UV-crosslinked to RNA in a polyadenylylation signal (AAUAAA)-dependent manner. We have now isolated cDNAs encoding the 64-kDa subunit of human cleavage stimulation factor. The 64-kDa subunit contains a ribonucleoprotein-type RNA binding domain in the N-terminal region and a repeat structure in the C-terminal region in which a pentapeptide sequence (consensus MEARA/G) is repeated 12 times and the formation of a long alpha-helix stabilized by salt bridges is predicted. An approximately 270-amino acid segment surrounding this repeat structure is highly enriched in proline and glycine residues (approximately 20% for each). When cloned 64-kDa subunit was expressed in Escherichia coli, an N-terminal fragment containing the RNA binding domain bound to RNAs in a polyadenylylation-signal-independent manner, suggesting that the RNA binding domain is directly involved in the binding of the 64-kDa subunit to pre-mRNAs.
Intrinsic anion channel activity of the recombinant first nucleotide binding fold domain of the cystic fibrosis transmembrane regulator protein.
Authors: Arispe N , Rojas E , Hartman J , Sorscher E J , Pollard H B
Published: May 2006

The first nucleotide binding fold (NBF-1) from the cystic fibrosis transmembrane regulator (CFTR) has been expressed in bacteria and found to bind ATP and to express anion channel activity when reconstituted onto a planar lipid bilayer. This evidence suggests that the NBF forms the anion-selective portion of the CFTR channel. We also found that the recombinant NBF-1 anion channel is blocked by ATP (1 mM), under which condition it appears to have a minimal conductance of approximately 9 pS and an ohmic current-voltage relationship. We further found that the recombinant NBF-1 bearing the delta F508 mutation has nearly identical anion channel activity to that of the wild-type protein but can be distinguished from wild type under bianionic conditions with chloride and gluconate. We conclude from these data that the anion channel activity of the recombinant NBF-1 could represent all or part of the anion conductance mechanism of CFTR and that the role of the ATP binding by the NBF could be to modulate this anion channel activity.
Regulation of Cl- channels in normal and cystic fibrosis airway epithelial cells by extracellular ATP.
Authors: Stutts M J , Chinet T C , Mason S J , Fullton J M , Clarke L L , Boucher R C
Published: May 2006

The rate of Cl- secretion by human airway epithelium is determined, in part, by apical cell membrane Cl- conductance. In cystic fibrosis airway epithelia, defective regulation of Cl- conductance decreases the capability to secrete Cl-. Here we report that extracytosolic ATP in the luminal bath of cultured human airway epithelia increased transepithelial Cl- secretion and apical membrane Cl- permeability. Single-channel studies in excised membrane patches revealed that ATP increased the open probability of outward rectifying Cl- channels. The latter effect occurs through a receptor mechanism that requires no identified soluble second messengers and is insensitive to probes of G protein function. These results demonstrate a mode of regulation of anion channels by binding ATP at the extracellular surface. Regulation of Cl- conductance by external ATP is preserved in cystic fibrosis airway epithelia.
Changes in cytosolic pH and calcium of guard cells precede stomatal movements.
Authors: Irving H R , Gehring C A , Parish R W
Published: May 2006

Stomatal opening is induced by indoleacetic acid (IAA), cytokinins, and fusicoccin (FC), whereas stomatal closure is induced by abscisic acid (ABA). To test the effect of these growth regulators on guard cell cytosolic Ca2+ ([Ca2+]cyt) and pH (pHcyt), epidermal strips were taken from the lower side of leaves of the orchid Paphiopedilum tonsum and were loaded with acetomethoxy-esterified forms of the Ca2+ indicator fluo-3 or the pH indicator 2',7'-bis(2-carboxyethyl)-5(6)carboxyfluorescein. Basal [Ca2+]cyt ranged from 0.05 to 0.3 M and was 0.22 +/- 0.015 (n = 21). Increases in both [Ca2+]cyt and pHcyt were observed in guard cells after application of 10-100 M ABA to open stomata, and these preceded stomatal closure. The increase in [Ca2+]cyt ranged from 1.5- to 3-fold and was seen in 7 of 10 experiments. Guard cell alkalinization began within 2 min of ABA treatment and continued for the next 8 min. The increase ranged from 0.04 to 0.3 pH unit and was seen in 13 of 14 experiments. Guard cell [Ca2+]cyt increased, whereas pHcyt decreased after treatment of closed stomata with IAA, kinetin, or FC. In response to 50-100 M IAA, [Ca2+]cyt increased 1.5- to 2-fold in all cases, and pHcyt decreased 0.2-0.4 unit within 5 min in 7 experiments. Within 12 min, 10-100 M kinetin caused [Ca2+]cyt to increase in 28 of 34 experiments (1.3- to 2.5-fold) and pHcyt fell 0.1-0.4 unit in 15 of 17 treatments. The response to 10-50 M FC was similar in both time and magnitude. These results show that stomatal opening is accompanied by an increase in [Ca2+]cyt and cytosolic acidification in the guard cells, whereas stomatal closure is preceded by an increase in [Ca2+]cyt and cytosolic alkalinization in the guard cells. The order of these events is still uncertain, but changes in pHcyt are correlated with stomatal movement, and these changes may be an important factor in the regulation of guard cell movement.
Evolutionary implications of the cDNA sequence of the single lactate dehydrogenase of a lamprey.
Authors: Stock D W , Whitt G S
Published: May 2006

All vertebrates other than lampreys exhibit multiple loci encoding lactate dehydrogenase +ADL-LDH; (S)-lactate:NAD+ oxidoreductase, EC 1.1.1.27+BD. Of these loci, Ldh-A is expressed predominantly in muscle, Ldh-B is expressed predominantly in heart, and Ldh-C (where present) exhibits different tissue-restricted patterns of expression depending on the taxon. To examine the relationship of the single LDH of lampreys to other vertebrate LDHs, we have determined the cDNA sequence of the LDH of the sea lamprey Petromyzon marinus and compared it to previously published sequences from bacteria, plants, and vertebrates. The lamprey sequence exhibits a mixture of features of both LDH-A and LDH-B at the amino acid level that may account for its intermediate kinetic properties. Both distance and maximum parsimony analyses strongly reject a relationship of lamprey LDH with mammalian LDH-C but do not significantly distinguish among remaining alternative phylogenetic hypotheses. Evolutionary parsimony analyses suggest that the lamprey LDH is related to Ldh-A and that the single locus condition has arisen as a result of the loss of Ldh-B (prior to the appearance of Ldh-C). The collection of LDH sequences for further studies of the evolution of the vertebrate LDH gene family will be facilitated by the PCR approach that we have used to obtain the lamprey sequence.
Distinct hypermethylation patterns occur at altered chromosome loci in human lung and colon cancer.
Authors: Makos M , Nelkin B D , Lerman M I , Latif F , Zbar B , Baylin S B
Published: May 2006

Regional increases in DNA methylation occur in normally unmethylated cytosine-rich areas in neoplastic cells. These changes could potentially alter chromatin structure to inactivate gene transcription or generate DNA instability. We now show that, in human lung and colon cancer DNA, hypermethylation of such a region consistently occurs on chromosome 17p in an area that is frequently reduced to homozygosity in both tumor types. Over the progression stages of colon neoplasia, this methylation change increases in extent and precedes the allelic losses on 17p that are characteristic of colon carcinomas. We also show on chromosome 3p that regional hypermethylation may nonrandomly accompany chromosome changes in human neoplasia. Increased methylation is consistent in small-cell lung carcinoma DNA at two 3p loci that are constantly reduced to homozygosity in this tumor, but it is not seen in colon cancer DNA, in which these loci are infrequently structurally altered.
Fifth mutation in human immunodeficiency virus type 1 reverse transcriptase contributes to the development of high-level resistance to zidovudine.
Authors: Kellam P , Boucher C A , Larder B A
Published: May 2006

It is recognized that high-level resistance to 3'-azido-3'-deoxythymidine (AZT, zidovudine, or Retrovir) is conferred by the presence of four mutations in the human immunodeficiency virus (HIV) reverse transcriptase [RT; deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase (RNA-directed), EC 2.7.7.49] coding sequence. However, a number of clinical isolates have been observed that exhibit high-level resistance but contain only three of the four identified mutations (Asn-67, Arg-70, and Tyr-215). Construction of a molecular clone with this genotype gave rise to only a partially resistant virus, raising the possibility that an additional mutation existed in some clinical isolates. Using an HIV marker rescue system, we have mapped and identified a fifth mutation conferring resistance to zidovudine, namely, methionine to leucine at codon 41 of HIV RT. An infectious molecular clone containing this mutation together with three previously identified mutations in the RT coding sequence yielded highly resistant HIV after transfection of T cells. Direct detection of the fifth mutation in DNA samples from cocultured peripheral blood lymphocytes by the PCR revealed that it occurred relatively early in the development of zidovudine resistance. However, this mutation was only detected after the appearance of the codon 215 change in the RT coding sequence. Identification of this mutation in addition to the other known mutations conferring resistance enables rapid and direct correlation between an RT genotype and sensitivity of the virus.
Genetic regulation of human anti-malarial antibodies in twins.
Authors: Sjöberg K , Lepers J P , Raharimalala L , Larsson A , Olerup O , Marbiah N T , Troye-Blomberg M , Perlmann P
Published: May 2006

Immune responses to defined antigens may differ between individuals in a population as the reflection of differences in genetic regulation. In experimental animals, variation in responsiveness to a given epitope may be due to major histocompatibility complex (HLA, in humans) class II restrictions, implying serious limitations for the development of subunit vaccines. For human populations, knowledge of the relative importance of genetic as opposed to environmental factors affecting the immune response is scarce. We have compared antibody levels after immunization through repeated infections to a major malarial antigen (Pf155/RESA) in monozygotic twins with those in dizygotic twins, siblings, or unrelated controls. Antibody responses to the intact antigen and to some of its immunodominant epitopes were found to be more concordant within monozygotic twin pairs than in dizygotic pairs or age- and sex-matched siblings living under similar environmental conditions. The results support the conclusion that the antibody responses were genetically regulated. When the responses were assessed for possible associations with different HLA class II DRB, DQA, and DQB alleles had haplotypes, no associations were found. This suggests that the regulation of the Pf155/RESA antibody responses seen in this study reflects the impact of factors encoded by genes outside the HLA class II regions.
Granule membrane protein 140 (GMP140) binds to carcinomas and carcinoma-derived cell lines.
Authors: Aruffo A , Dietsch M T , Wan H , Hellström K E , Hellström I
Published: May 2006

The glycoproteins granule membrane protein 140 (GMP140), endothelial-leukocyte adhesion molecule 1 (ELAM-1), and Leu-8 are members of a family of glycoprotein receptors (selectins or LEC-CAMs) that play an important role in adhesive interactions between circulating leukocytes and vascular endothelium. Recently it has been reported that ELAM-1 is able to mediate the binding of the colon carcinoma cell line HT-29 to cytokine-activated vascular endothelium, suggesting that tumor cell adhesion to vascular endothelium, a prerequisite for tumor extravasation and metastasis, is in part the result of adhesive interactions between blood-borne tumor cells and cell surface proteins expressed by vascular endothelium. Here, using an approach in which soluble immunoglobulin chimeras of the GMP140 and ELAM-1 receptors were prepared and used to carry out immunohistological studies, we establish that GMP140 binds to tumor cells in a variety of human carcinoma tissue sections (colon, lung, and breast), whereas ELAM-1 binds exclusively to tumor cells in colon carcinoma tissue sections. In addition, GMP140 was found to bind to the cell surface of a number of cell lines derived from various carcinomas but not from melanomas, whereas ELAM-1 bound only colon carcinoma cell lines. We further investigated the nature of the ligands of GMP140 and ELAM-1 on the surface of the carcinoma cells and found that the GMP140 ligand on the surface of tumor cells appears to be distinct from that expressed on the myeloid cell line HL-60. Neuraminidase treatment of a breast carcinoma cell line does not affect, or in some instances increases, GMP140 binding, whereas it completely abolishes GMP140 binding to HL-60 cells. On the other hand, the ligand of ELAM-1 on both the colon carcinoma and HL-60 cells is neuraminidase sensitive in accord with its identification as sialyl-CD15. Parallel results were obtained with neuraminidase-treated frozen carcinoma tissue sections. The present findings form the basis for investigating the role of GMP140 in tumor invasiveness and metastasis.
Strong evolutionary conservation of neuropeptide Y: sequences of chicken, goldfish, and Torpedo marmorata DNA clones.
Authors: Blomqvist A G , Söderberg C , Lundell I , Milner R J , Larhammar D
Published: May 2006

Neuropeptide Y (NPY) is an abundant and widespread neuropeptide in the nervous system of mammals. NPY belongs to a family of 36-amino acid peptides that also includes pancreatic polypeptide and the endocrine gut peptide YY as well as the fish pancreatic peptide Y. To study the evolution of this peptide family, we have isolated clones encoding NPY from central nervous system cDNA libraries of chicken, goldfish, and the ray Torpedo marmorata, as well as from a chicken genomic library. The predicted chicken NPY amino acid sequence differs from that of rat at only one position. The goldfish sequence differs at five positions and shows that bony fishes have a true NPY peptide in addition to their pancreatic peptide Y. The Torpedo sequence differs from that of rat at three positions. As Torpedo NPY has no unique positions when compared with the other sequences, it seems to be identical to the NPY of the common ancestor of cartilaginous fishes, bony fishes, and tetrapods after 420 million years of evolution. The 30-amino acid carboxyl-terminal extension of the NPY precursor also displays considerable sequence conservation. These results show that NPY is one of the most highly conserved neuroendocrine peptides.
Unification of the ferritin family of proteins.
Authors: Grossman M J , Hinton S M , Minak-Bernero V , Slaughter C , Stiefel E I
Published: May 2006

Ferritin is the iron-storage protein of eukaryotic organisms. The nucleotide sequence encoding Azotobacter vinelandii bacterioferritin, a hemoprotein, was determined. The deduced amino acid sequence reveals a high degree of identity with Escherichia coli bacterioferritin and a striking similarity to eukaryotic ferritins. Moreover, derivation of a global alignment shows that virtually all key residues specifying the unique structural motifs of eukaryotic ferritin are conserved or conservatively substituted in the A. vinelandii sequence. The alignment suggests specific methionine residues as heme-binding ligands in bacterioferritins. The overall sequence similarity with conservation of key structural residues implies that all ferritins form a unified family of proteins. The results implicate ferritins as proteins potentially common to all aerobic organisms and as such useful in taxonomic classification, evolutionary analysis, and environmental monitoring.
Regulatory genes linked to the albino locus in the mouse confer competence for inducible expression on the structural gene encoding serine dehydratase.
Authors: Lia M , Bali D , Gluecksohn-Waelsch S
Published: May 2006

A cluster of unlinked genes encoding gluconeogenic enzymes in the mouse is characterized by the failure of normal hormone-inducible expression in animals homozygous for one of several overlapping deletions mapping on chromosome 7 near the albino locus. Previous investigations have shown hormones and their receptors to be normal in the mutants and therefore not responsible for the abnormalities of inducibility. Instead, these studies have implicated a possible failure of the affected structural enzyme genes themselves to attain during prenatal development the competence for inducible gene expression. The results reported here add serine dehydratase (EC 4.2.1.13) and its structural gene to the affected group of gluconeogenic enzymes and their genes. Even though, in deletion homozygotes, serine dehydratase is expressed normally on the constitutive level, hormone-inducible expression fails to develop. The abnormality appears to reside in a defect of prenatal differentiation of cis-acting regulatory elements of the structural gene essential in the pathway of inducible gene expression.
The ankyrin repeat domains of the NF-kappa B precursor p105 and the protooncogene bcl-3 act as specific inhibitors of NF-kappa B DNA binding.
Authors: Hatada E N , Nieters A , Wulczyn F G , Naumann M , Meyer R , Nucifora G , McKeithan T W , Scheidereit C
Published: May 2006

The inducible pleiotropic transcription factor NF-kappa B is composed of two subunits, p50 and p65. The p50 subunit is encoded on the N-terminal half of a 105-kDa open reading frame and contains a rel-like domain. To date, no function has been described for the C-terminal portion. We show here that the C-terminal half of p105, when expressed as a separate molecule, binds to p50 and can rapidly disrupt protein-DNA complexes of p50 or native NF-kappa B. Deletion analysis of this precursor-derived inhibitor activity indicated a domain containing ankyrin-like repeats as necessary for inhibition. The protooncogene bcl-3, which contains seven ankyrin repeats, can equally inhibit p50 DNA binding. These observations identify bcl-3 as an inhibitor of NF-kappa B and strongly suggest that the ankyrin repeats in these factors are involved in protein-protein interactions with the rel-like domain of p50. Comparison with other ankyrin repeat-containing proteins suggests that a subclass of these proteins acts as regulators of rel-like transcription factors.
Simulations of the folding pathway of triose phosphate isomerase-type alpha/beta barrel proteins.
Authors: Godzik A , Skolnick J , Kolinski A
Published: May 2006

Simulations of the folding pathways of two large alpha/beta proteins, the alpha subunit of tryptophan synthase and triose phosphate isomerase, are reported using the knight's walk lattice model of globular proteins and Monte Carlo dynamics. Starting from randomly generated unfolded states and with no assumptions regarding the nature of the folding intermediates, for the tryptophan synthase subunit these simulations predict, in agreement with experiment, the existence and location of a stable equilibrium intermediate comprised of six beta strands on the amino terminus of the molecule. For the case of triose phosphate isomerase, the simulations predict that both amino- and carboxyl-terminal intermediates should be observed. In a significant modification of previous lattice models, this model includes a full heavy atom side chain description and is capable of representing native conformations at the level of 2.5- to 3-A rms deviation for the C alpha positions, as compared to the crystal structure. With a well-balanced compromise between accuracy of the protein description and the computer requirements necessary to perform simulations spanning biologically significant amounts of time, the lattice model described here brings the possibility of studying important biological processes to present-day computers.
sar: a genetic mouse model for human sarcosinemia generated by ethylnitrosourea mutagenesis.
Authors: Harding C O , Williams P , Pflanzer D M , Colwell R E , Lyne P W , Wolff J A
Published: May 2006

A mouse mutant with sarcosinemia was found by screening the progeny of ethylnitrosourea-mutagenized mice for aminoacidurias. Paper chromatography, column chromatography, and gas chromatography-mass spectrometry identified high levels of sarcosine in the urine of the mutant mice. While sarcosine cannot be detected in the urine of plasma of normal mice, the urinary sarcosine level of 102 +/- 58 mmol per g of creatinine in the mutant mice was at the upper range of the urinary levels (1.5-4.5 mmol of sarcosine per g of creatinine) observed in humans with sarcosinemia. Similarly, the plasma sarcosine level of 785 +/- 153 mumol/liter in the sarcosinemic mice was at the upper range of the plasma sarcosine levels (53-760 mumol/liter) observed in affected humans. Sarcosine dehydrogenase [sarcosine:(acceptor) oxidoreductase (demethylating), EC 1.5.99.1] activity was deficient in sarcosinemic mice. The sarcosinuria phenotype in these mice was inherited as an autosomal recessive trait. This mouse mutant provides a useful genetic model for human sarcosinemia and for development of therapeutic approaches for genetic disease.
Corticothalamic activation modulates thalamic firing through glutamate "metabotropic" receptors.
Authors: McCormick D A , von Krosigk M
Published: May 2006

The mammalian thalamus forms an obligatory relay for nearly all sensory information that reaches the cerebral cortex. The transmission of sensory information by the thalamus varies in a state-dependent manner, such that during slow wave sleep or drowsiness thalamic responsiveness is markedly reduced, whereas during the waking, attentive state transmission is enhanced. Although activation of brainstem inputs to thalamic neurons has long been assumed to underlie this gating of sensory transfer through the thalamus, numerically the largest input to thalamic relay neurons derives from layer VI cells of the cerebral cortex. Here we report that activation of corticothalamic fibers causes a prolonged excitatory postsynaptic potential in guinea pig dorsal lateral geniculate relay neurons resulting from the reduction of a potassium conductance, consistent with the activation of glutamatergic "metabotropic" receptors. This slow depolarization can switch firing of thalamic neurons from the burst firing mode, which is prevalent during slow wave sleep, to the single spike mode, which is prevalent during waking, thereby facilitating transmission of sensory information through the thalamus. This prolonged enhancement of thalamic transfer may allow the cerebral cortex to gate or control selective fields of sensory inputs in a manner that facilitates arousal, attention, and cognition.
Both p21ras and pp60v-src are required, but neither alone is sufficient, to activate the Raf-1 kinase.
Authors: Williams N G , Roberts T M , Li P
Published: May 2006

The raf genes encode a family of cytoplasmic proteins with intrinsic protein-serine/threonine kinase activity. The c-raf gene is the cellular homolog of v-raf, the transforming gene of murine sarcoma virus 3611. The constitutive kinase activity of the v-Raf protein has been implicated in transformation and mitogenesis. The activity of Raf-1, the protein product of the c-raf gene, is normally suppressed by a regulatory N-terminal domain. Activation of various tyrosine-kinase growth factor receptors results in activation of Raf-1 and its hyperphosphorylation. Further, Raf-1 has been shown to act either downstream or independently of the p21ras protein, as indicated by experiments involving microinjection of anti-Ras antibodies. To investigate the potential role of p21ras in the activation of Raf-1 by tyrosine kinases, we have used the baculovirus/Sf9 cell system to overproduce various wild-type and mutant forms of pp60src, p21ras, and Raf-1 proteins. We show that either pp60v-src or p21c-ras can independently activate the autokinase activity of Raf-1, but only to a limited extent. Surprisingly, both pp60v-src and p21c-ras are required to fully activate Raf-1. Analysis of the Raf-1 autokinase activity in vitro shows that Raf-1 autophosphorylation sites are distributed equally on serine and threonine residues. When Raf-1 is analyzed by immunoblotting, as previously reported for mammalian cell experiments, a marked increase in the apparent molecular weight of Raf-1 is seen only when it is coexpressed with both pp60v-src and p21ras.
Cloning and expression of a cytosolic megakaryocyte protein-tyrosine-phosphatase with sequence homology to retinaldehyde-binding protein and yeast SEC14p.
Authors: Gu M , Warshawsky I , Majerus P W
Published: May 2006

Protein tyrosine phosphorylation is important in the regulation of cell growth, the cell cycle, and malignant transformation. We have cloned a cDNA that encodes a cytosolic protein-tyrosine-phosphatase (PTPase), MEG2, from MEG-01 cell and human umbilical vein endothelial cell cDNA libraries. The 4-kilobase cDNA sequence of PTPase MEG2 corresponds in length to the mRNA transcript detected by Northern blotting. The predicted open reading frame encodes a 68-kDa protein composed of 593 amino acids and has no apparent signal or transmembrane sequences, suggesting that it is a cytosolic protein. The C-terminal region has a PTPase catalytic domain that has 30-40% amino acid identity to other known PTPases. The N-terminal region has 254 amino acids that are 28% identical to cellular retinaldehyde-binding protein and 24% identical to yeast SEC14p, a protein that has phosphatidylinositol transfer activity and is required for protein secretion through the Golgi complex in yeast. Recombinant PTPase MEG2 expressed in Escherichia coli possesses PTPase activity. PTPase MEG2 mRNA was detected in 12 cell lines tested, which suggests that this phosphatase is widely expressed. The structure of PTPase MEG2 implies that a tyrosine phosphatase could participate in the transfer of hydrophobic ligands or in functions of the Golgi apparatus.
Addition of extra origins of replication to a minichromosome suppresses its mitotic loss in cdc6 and cdc14 mutants of Saccharomyces cerevisiae.
Authors: Hogan E , Koshland D
Published: May 2006

Many cell division cycle (cdc) mutants of Saccharomyces cerevisiae exhibit elevated mitotic loss of pDK243, a 14-kilobase minichromosome with a centromere and one autonomous replicating sequence (ARS). Tandem copies of different ARSs were added to pDK243. The addition of these ARS clusters to pDK243 had no effect on its mitotic loss in cdc7 (protein kinase), cdc9 (DNA ligase), or cdc16 or cdc17 (DNA polymerase) mutants. However, in cdc6 and cdc14 mutants, the mitotic loss of pDK243 with an ARS cluster was suppressed by a factor of 6-8 compared to pDK243 without the cluster. This suppression was dependent upon the number of ARSs in the cluster and the integrity of the ARS consensus sequence in each ARS of the cluster. ARSs are known to be DNA replication origins. Therefore, the suppression of mini-chromosome loss by ARSs in cdc6 and cdc14 mutants suggests that these mutants are defective in the initiation of DNA replication. Since the CDC6 protein appears to act at the G1/S phase transition, the CDC6 protein may be a factor required at the beginning of S phase to initiate DNA replication at origins. In contrast, the CDC14 protein acts after mitosis. We suggest that the CDC14 protein performs a function late in the cell cycle that may be required for efficient initiation of DNA replication during S phase of the next cell cycle.
The effects of dietary restriction on immune function and development of autoimmune disease in BXSB mice.
Authors: Kubo C , Gajar A , Johnson B C , Good R A
Published: May 2006

Chronic energy intake restriction (CEIR) prolonged the median life span and inhibited autoimmunity and development of autoimmune disease in BXSB mice, as has been established for mice of several other autoimmune-prone, short-lived strains. Whether imposed just after weaning or delayed until manifestations of disease had appeared, CEIR inhibited or reversed development of autoimmunity and immune complex-based renal disease in male BXSB mice. CEIR also prevented the formation of anti-DNA antibodies and prevented the increase in circulating immune complex levels that is typically observed in male mice of this strain. Moreover, CEIR inhibited development of splenomegaly and prevented the normal age-associated decline of a number of immunological functions, including interleukin 2 production, cell-mediated cytotoxic responses, and mixed lymphocyte reactivity. The observed improvement in cell-mediated immune responses was attributed largely to the capacity of CEIR to inhibit development of the splenomegaly that occurs concomitant with expansion of a non-T, non-B lymphoid cell population. These findings emphasize that CEIR, even when imposed relatively late in life in BXSB mice, can influence expression of autoimmunities and autoimmune diseases of different genetic origins and presumed pathogenetic bases.