bluuebunny/crossref_metadata_2025 huggingface dataset:
Regeneration of a functional RNA virus genome by recombination between deletion mutants and requirement for cowpea chlorotic mottle virus 3a and coat genes for systemic infection.
Authors: Allison R , Thompson C , Ahlquist P
Published: May 2006

RNAs 1 and 2 of the tripartite cowpea chlorotic mottle virus (CCMV) genome are sufficient for RNA replication in protoplasts, whereas systemic infection of cowpea plants additionally requires RNA3, which encodes the 3a noncapsid protein and coat protein. By using biologically active CCMV cDNA clones, we find that deletions in either RNA3 gene block systemic infection. Thus, though some plant RNA viruses are able to spread systemically without encapsidation, both the coat and 3a genes are required for systemic infection of cowpeas by CCMV. When plants were coinoculated with CCMV RNAs 1 and 2 and both the 3a and coat deletion mutants of RNA3, 30-60% rapidly developed systemic infection. Progeny RNA recovered from systemically infected leaves in such infections contained neither of the starting deletion mutants but rather a single full-length RNA3 component with both genes intact. Nucleotide substitutions introduced into the coat protein deletion mutant as an artificial marker were recovered in the full-length progeny RNA, confirming its recombinant nature. Intermolecular RNA recombination in planta can, therefore, rescue a complete infectious genome from coinoculated mutants independently disabled for systemic spread. These results have implications for the repair of defective genomes produced by frequent natural replication errors, the possible emergence of newly adapted RNA viruses upon coinfection of new hosts, and further studies of RNA virus recombination.
Enhanced polymerization of recombinant human deoxyhemoglobin beta 6 Glu----Ile.
Authors: Baudin-Chich V , Pagnier J , Marden M , Bohn B , Lacaze N , Kister J , Schaad O , Edelstein S J , Poyart C
Published: May 2006

Polymerization of the deoxy form of sickle cell hemoglobin (Hb S; beta 6 Glu----Val) involves both hydrophobic and electrostatic intermolecular contacts. These interactions drive the mutated molecules into long fibrous rods composed of seven pairs of strands. X-ray crystallography of Hb S and electron microscopy image reconstruction of the fibers have revealed the remarkable complementarity between one of the beta 6 valines of each molecule (the donor site) and an acceptor site at the EF corner of a neighboring tetramer. This interaction constitutes the major lateral contact between the two strands in a pair. To estimate the relative importance of this key hydrophobic contact in polymer formation we have generated a polymerizing Hb with isoleucine at the beta 6 position (beta E6I) by site-directed mutagenesis. The mutated beta chains were produced in Escherichia coli and reassembled into functional tetramers with native alpha chains. Compared to native Hb S, the beta E6I mutant polymerizes faster and with a shortened delay time in 1.8 M phosphate buffer, indicating an increased stability of the nuclei preceding fiber growth. The solubility of the beta E6I mutant Hb is half that of native Hb S. Computer modeling of the donor-acceptor interaction shows that the presence of an isoleucine side chain at the donor site induces increased contacts with the receptor site and an increased buried surface area, in agreement with the higher hydrophobicity of the isoleucine residue. The agreement between the predicted and experimental differences in solubility suggests that the transfer of the beta 6 valine or isoleucine side chain from water to a hydrophobic environment is sufficient to explain the observations.
Schwann cells promote the survival of rat retinal ganglion cells after optic nerve section.
Authors: Maffei L , Carmignoto G , Perry V H , Candeo P , Ferrari G
Published: May 2006

Schwann cells (SCs) are known to play an important role for the regeneration of mammalian peripheral nerves. Their effect is likely due to the production of neuronotrophic and/or supportive factors. Here we study the effect of intraocular transplant of SCs on the survival of rat retinal ganglion cells (RGCs) after the intracranial section of the optic nerve. SCs were injected intraocularly in adult hooded rats. Surviving RGCs were retrogradely labeled with horseradish peroxidase applied to the proximal stump of the optic nerve. Results show that intraocular transplants of SCs promote the survival of a large number of RGCs for periods as long as 9 and 14 weeks after optic nerve section. In experimental retinae, surviving RGCs were 2- to 8-fold more numerous than in controls. This finding suggests that SCs are the source of factors that promote the survival of RGCs. Nerve growth factor is produced by SCs, and the intraocular injection of nerve growth factor has been previously shown to promote RGC survival. The rescuing effect of SCs on RGCs is greater than that obtained by intraocular injection of nerve growth factor. This greater effect may be due to the action of other neurotrophic factors produced by SCs or by transplanted SCs producing NGF in a sustained fashion.
Abnormal structure and expression of p53 gene in human hepatocellular carcinoma.
Authors: Bressac B , Galvin K M , Liang T J , Isselbacher K J , Wands J R , Ozturk M
Published: May 2006

There is little information regarding the molecular mechanisms of hepatocarcinogenesis. We studied the p53 gene at the DNA, RNA, and protein level in seven human hepatocellular carcinoma (HCC)-derived cell lines; six of seven showed p53 abnormalities. By Southern blotting, the p53 gene was found to be partially deleted in Hep 3B and rearranged in SK-HEP-1 cells. Transcripts of the p53 gene were undetectable in Hep 3B as well as in FOCUS cells that had no apparent deletion or rearrangement of the p53 gene. Immunoprecipitation after [35S]methionine labeling of HCC cells demonstrated that p53 protein was absent in Hep 3B and FOCUS and reduced in concentration in PLC/PRF/5 cells. p53 synthesized by Mahlavu cells showed a slower migration on SDS/polyacrylamide gels suggesting it was an abnormal protein. In Huh7 cells, p53 protein had a prolonged half-life leading to its accumulation in the nuclei; increased levels of p53 protein were also found by immunoblotting. The p53 gene and its expression appeared to be unaltered in the hepatoblastoma-derived Hep G2 cell line. We found that the loss of p53 expression did not occur as a late in vitro event in the FOCUS cell line because p53 protein was also nondetectable at an early passage. We conclude that the loss of p53 expression or the presence of abnormal forms of the protein are frequently associated with HCC cell lines. These observations suggest that alterations in p53 may be important events in the transformation of hepatocytes to the malignant phenotype.
Components of fitness become effectively neutral in equilibrium populations.
Authors: Reeve R , Smith E , Wallace B
Published: May 2006

If, because of trade-offs or similar considerations, components of fitness are negatively correlated in equilibrium populations, they may then exhibit no correlation with total fitness. Lack of correlation with fitness is a fundamental characteristic of selective neutrality. Hence, selectively important traits become subsumed under neutral traits when the definition of neutrality is extended to include factors (genetic or phenotypic) that "behave as if they are neutral."
Affinity purification of spliceosomes reveals that the precursor RNA processing protein PRP8, a protein in the U5 small nuclear ribonucleoprotein particle, is a component of yeast spliceosomes.
Authors: Whittaker E , Lossky M , Beggs J D
Published: May 2006

Nuclear pre-mRNA splicing in Saccharomyces cerevisiae, as in higher eukaryotes, occurs in large RNA-protein complexes called spliceosomes. The small nuclear RNA components, U1, U2, U4, U5, and U6, have been extensively studied; however, very little is known about the protein components of yeast spliceosomes. Here we use antibodies against the precursor RNA processing protein PRP8, a protein component of the U5 small nuclear ribonucleoprotein particle, to detect its association with spliceosomes throughout the splicing reaction and in a post-splicing complex containing the excised intron. In addition, an indirect immunological approach has been developed that confirms the presence of precursor RNA processing protein PRP8 in isolated spliceosomes. This method has possible general application for the analysis of ribonucleoprotein particle complexes.
Immunolocalization in three dimensions: immunogold staining of cytoskeletal and nuclear matrix proteins in resinless electron microscopy sections.
Authors: Nickerson J A , Krockmalnic G , He D C , Penman S
Published: May 2006

We describe two methods for staining resinless thin sections with antibodies and gold-conjugated second antibodies. Immunolocalization of specific proteins is a powerful tool for cell structure studies but current techniques do not develop its full potential. Immunofluorescence provides only low-resolution localization, whereas conventional thin-section electron microscopy images and immunostains only the section surface. Resinless sections of extracted cell structures offer a simple and effective means of immuno-electron microscopy. Without embedding plastic or soluble proteins, the cell cytostructure produces high-contrast, three-dimensional images. Resinless sections of detergent-extracted cells are prepared by embedding in diethylene glycol distearate, sectioning, and removing diethylene glycol distearate before microscopy. In the first method of immunostaining, extracted cells were fixed and stained with antibodies before embedment, sectioning, removal of the embedding resin, and critical point drying. In the postembedment method, the sample was embedded and sectioned, the diethylene glycol distearate was removed, and the sample was rehydrated before antibody staining. With these techniques, specific proteins were localized with high resolution throughout the entire section. Stereoscopic micrographs of resinless sections revealed the precise localization of specific cytoskeleton and nuclear matrix proteins in three dimensions with unprecedented clarity.
Cloning, expression, and purification of human cyclophilin in Escherichia coli and assessment of the catalytic role of cysteines by site-directed mutagenesis.
Authors: Liu J , Albers M W , Chen C M , Schreiber S L , Walsh C T
Published: May 2006

The cDNA encoding human cyclophilin from the Jurkat T-cell lymphoma line has been cloned by the expression cassette polymerase chain reaction and sequenced, and an expression vector has been constructed under control of the tac promoter for efficient expression in Escherichia coli. Active cyclophilin is produced at up to 40% of soluble cell protein, facilitating a one-column purification to homogeneity. Wild-type cyclophilin was characterized for binding of the potent immunosuppressant agent cyclosporin A (Kd = 46 nM) by tryptophan fluorescence enhancement and for inhibition (IC50 = 19 nM) of cyclophilin's peptidyl-prolyl cis-trans isomerase (rotamase) activity. With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as the substrate, recombinant human cyclophilin has a high catalytic efficiency; kcat/Km is 1.4 X 10(7) M-1.S-1 at 10 degrees C. To test the prior suggestion that a cysteine residue may be essential for catalysis and immunosuppressant binding, the four cysteines at positions 52, 62, 115, and 161 were mutated individually to alanine and the purified mutant proteins were shown to retain full affinity for cyclosporin A and equivalent catalytic efficiency as a rotamase. Clearly the cysteines play no essential role in catalysis or cyclosporin A binding. These results rule out the recently proposed mechanism [Fischer, G., Wittmann-Liebold, B., Lang, K., Kiefhaber, T. & Schmid, F. X. (1989) Nature (London) 337, 476-478)] involving the formation of tetrahedral hemithioorthoamide. Whereas mechanisms that embody other tetrahedral intermediates may be operative, an alternative mechanism is considered that involves distortion of bound substrate with a twisted (90 degrees) peptidyl-prolyl amide bond.
Sustained metabolic scope.
Authors: Peterson C C , Nagy K A , Diamond J
Published: May 2006

Sustained metabolic rates (SusMR) are time-averaged metabolic rates that are measured in free-ranging animals maintaining constant body mass over periods long enough that metabolism is fueled by food intake rather than by transient depletion of energy reserves. Many authors have suggested that SusMR of various wild animal species are only a few times resting (basal or standard) metabolic rates (RMR). We test this conclusion by analyzing all 37 species (humans, 31 other endothermic vertebrates, and 5 ectothermic vertebrates) for which SusMR and RMR had both been measured. For all species, the ratio of SusMR to RMR, which we term sustained metabolic scope, is less than 7; most values fall between 1.5 and 5. Some of these values, such as those for Tour de France cyclists and breeding birds, are surely close to sustainable metabolic ceilings for the species studied. That is, metabolic rates higher than 7 times RMR apparently cannot be sustained indefinitely. These observations pose several questions: whether the proximate physiological causes of metabolic ceilings reside in the digestive tract's ability to process food or in each tissue's metabolic capacity; whether ceiling values are independent of the mode of energy expenditure; whether ceilings are set by single limiting physiological capacities or by coadjusted clusters of capacities (symmorphosis); what the ultimate evolutionary causes of metabolic ceilings are; and how metabolic ceilings may limit animals' reproductive effort, foraging behavior, and geographic distribution.
Long-term transplantation of canine keratinocytes made resistant to G418 through retrovirus-mediated gene transfer.
Authors: Flowers M E , Stockschlaeder M A , Schuening F G , Niederwieser D , Hackman R , Miller A D , Storb R
Published: May 2006

We studied cultured canine keratinocytes to determine whether they could serve as targets for retrovirus-mediated gene transfer and whether infected cells could persist after transplantation into dogs, a large random-bred model for gene transfer studies. Canine keratinocytes obtained from skin biopsy samples were cultured in vitro with lethally irradiated NIH 3T3 cells used as a feeder layer. The keratinocyte colonies consisted of squamous epithelium with numerous desmosomes, tonofilaments, and keratohyalin granules. In addition, the cells were strongly reactive with monoclonal antibodies to cytokeratin intermediate filament proteins. For the infection studies, we grew the keratinocytes on a feeder layer of lethally irradiated PA317 retrovirus packaging cells, which produced a helper-free amphotropic retroviral vector containing the neomycin phosphotransferase (neo) gene. After cocultivation, 34% (range, 10-76%) of the keratinocytes were found to be resistant to the neomycin analogue G418. Infected keratinocytes were then transplanted into the dog of origin; 1% (range, less than 0.1-3%) of the keratinocytes obtained 27-130 days after transplantation from skin biopsy samples gave rise to G418-resistant colonies. We conclude that canine keratinocytes cultured in vitro can be infected efficiently with a neo gene-containing retroviral vector, and they show persistent G418 resistance for at least 130 days after transplantation into the skin donor.
Suppression of mouse mammary tumor proviral DNA and protooncogene expression: association with nutritional regulation of mammary tumor development.
Authors: Chen R F , Good R A , Engelman R W , Hamada N , Tanaka A , Nonoyama M , Day N K
Published: May 2006

Chronic energy intake restriction (CEIR) reduces mouse mammary tumor virus (MMTV)-induced mammary tumors in C3H/Ou mice. Fewer than 10% of C3H/Ou mice developed mammary tumors during 88 wk of study when subjected to CEIR regardless of calorie source (fat vs. carbohydrate). By contrast, 100% of mice fed ad libitum diets relatively high in fat or carbohydrate or a commercial diet developed tumors by 35-40 wk. MMTV proviral DNA transcription was shown to be activated in spleen, liver, lung, kidney, small intestine, and mammary gland of mice consuming these diets ad libitum. By contrast, these messages were suppressed by CEIR in all tissues analyzed except spleen. MMTV proviral messages in liver and mammary gland increased with age in full-fed mice and were suppressed by CEIR. These findings suggest that the nutritional regulation of MMTV proviral DNA expression is tissue-specific. In CEIR mice the suppressed MMTV proviral DNA transcripts in mammary gland and liver increased with time in association with the delayed onset of mammary tumors. Mammary tumorigenesis in C3H mice is associated with integration of MMTV proviral DNA, which appears to activate a putative mammary tumor protooncogene, int-1. CEIR apparently decreases the frequency of viral reintegration adjacent to the int-1 gene and thus inhibits expression of int-1 and probably an initiation step in mammary tumorigenesis. Expression of other putative protooncogenes, int-2 and ras, in liver tissue was also reduced by CEIR. These findings indicate that both initiation and promotion of mammary tumorigenesis are influenced by CEIR in C3H/Ou mice.
Discovery of Entomophaga maimaiga in North American gypsy moth, Lymantria dispar.
Authors: Andreadis T G , Weseloh R M
Published: May 2006

An entomopathogenic fungus, Entomophaga maimaiga, was found causing an extensive epizootic in outbreak populations of the gypsy moth, Lymantria dispar, throughout many forested and residential areas of the northeastern United States. This is the first recognized occurrence of this or any entomophthoralean fungus in North American gypsy moths, and its appearance was coincident with an abnormally wet spring. Most fungal-infected gypsy moth larvae were killed in mass during the fourth and fifth stadium and were characteristically found clinging to the trunks of trees with their heads pointed downward. The fungus produces thick-walled resistant resting spores within dried gypsy moth cadavers and infectious conidia when freshly killed larvae are held in a wet environment. The morphology and development of the fungus are described. The fungus appears to have had its origin in Japan, and the current epizootic may have resulted from the survival and inapparent spread of an early introduction in 1910-1911.
Two pharmacologically distinct sodium- and chloride-coupled high-affinity gamma-aminobutyric acid transporters are present in plasma membrane vesicles and reconstituted preparations from rat brain.
Authors: Kanner B I , Bendahan A
Published: May 2006

Electrogenic sodium- and chloride-dependent gamma-aminobutyric acid (GABA) transport in crude synaptosomal membrane vesicles is partly inhibited by saturating levels of either of the substrate analogues cis-3-aminocyclohexanecarboxylic acid (ACHC) or beta-alanine. However, both of them together potently and fully inhibit the process. Transport of beta-alanine, which exhibits an apparent Km of about 44 microM, is also electrogenic and sodium and chloride dependent and competitively inhibited by GABA with a Ki of about 3 microM. This value is very similar to the Km of 2-4 microM found for GABA transport. On the other hand, ACHC does not inhibit beta-alanine transport at all. Upon solubilization of the membrane proteins with cholate and fractionation with ammonium sulfate, a fraction is obtained which upon reconstitution into proteoliposomes exhibits 4- to 10-fold-increased GABA transport. This activity is fully inhibited by low concentrations of ACHC and is not sensitive at all to beta-alanine. GABA transport in this preparation exhibits an apparent Km of about 2.5 microM and it is competitively inhibited by ACHC (Ki approximately 7 microM). These data indicate the presence of two GABA transporter subtypes in the membrane vesicles: the A type, sensitive to ACHC, and the B type, sensitive to beta-alanine.
Brainstem control of response modes in neurons of the cat's lateral geniculate nucleus.
Authors: Uhlrich D J , Tamamaki N , Sherman S M
Published: May 2006

The visual pathway from retina through the lateral geniculate nucleus to visual cortex in the cat is comprised of several parallel neuronal streams that independently analyze different aspects of the visual scene. The best known of these are the X and Y pathways that relay through the geniculate A laminae. Recent receptive-field studies of retinal and geniculate neurons suggest that there is a further elaboration of cell types at the level of the lateral geniculate nucleus. That is, two types of geniculate X cells with different temporal patterns of responses to visual stimuli are recognized, one with "nonlagged" features, exhibiting shorter response latencies and another with "lagged" features; all retinal X cells are nonlagged. We asked whether nonlagged and lagged responses represent different cell classes or two response modes of the same cells, perhaps under the control of nonretinal afferents to these relay cells. Accordingly, we studied the effects on appropriate receptive-field properties of electrical activation of the midbrain parabrachial region, which is a major nonretinal input to relay cells. Such parabrachial stimulation made each of the eight lagged X cells much more like nonlagged cells, and this stimulation completely transformed the lagged response profiles of six of the eight cells to nonlagged. We thus conclude that the property of lagged responsiveness, which is an emergent property of the lateral geniculate nucleus, is a different response mode of the same cells that can also display nonlagged responses, rather than representing different cell classes; furthermore, this switching between response modes is, at least partly, under the control of afferents from the parabrachial region.
Agonistic antibodies stimulate the kinase encoded by the neu protooncogene in living cells but the oncogenic mutant is constitutively active.
Authors: Yarden Y
Published: May 2006

The neu protooncogene (also called c-erbB2 and HER-2) undergoes oncogenic activation through a single mutation. The product of the protooncogene, p185neu, probably functions as a receptor for a peptide growth factor. To circumvent the absence of a well-characterized ligand, I generated ligand-mimicking monoclonal antibodies directed to the presumed receptor. These antibodies stimulated tyrosine phosphorylation of p185neu in living cells and also accelerated the rate of endocytosis and degradation of p185neu. A monovalent Fab fragment of such an antibody was ineffective, suggesting a role for receptor dimerization in signal transduction. Unlike the product of the protooncogene, the transforming mutant was not affected by the ligand-like antibodies. However, it undergoes constitutively high phosphorylation on tyrosine residues in living cells, and its turnover rate is remarkably rapid. Nevertheless, the pattern of phosphorylation of the mutant protein is similar to the one exhibited by an antibody-stimulated p185neu, suggesting that the mutation mimics activation by the antibody. These results suggest that the kinase of p185neu is under allosteric control that may involve ligand-induced dimerization of receptors. This mechanism is deregulated in the oncogenic mutant, which is functionally equivalent to ligand-stimulated receptor.
Specific association between an endoribonucleolytic sequence from a satellite RNA and a substrate analogue containing a 2'-5' phosphodiester.
Authors: Feldstein P A , Buzayan J M , van Tol H , deBear J , Gough G R , Gilham P T , Bruening G
Published: May 2006

Both polarities of the satellite RNA of tobacco ringspot virus are sources of self-cleaving sequences. RNA of the less abundant, negative polarity, designated sTobRV-(-)RNA, has cleaving activity that was mapped previously to two noncontiguous regions of the polyribonucleotide chain. Endoribonucleolytic oligoribonucleotides (E) corresponding to the larger of the two regions cleaved smaller substrate oligoribonucleotides, at the ApG phosphodiester that is cleaved in sTobRV(-)RNA. An analogue of the substrate, which has a 2'-5' ApG phosphodiester, was not cleaved by E but acted as a competitive inhibitor of the cleavage of substrate. The analogue served as a primer, and E served as template, for reverse transcriptase-catalyzed copying of specific E sequences. The sequences transcribed suggest base pairing between the 5' region of E and a portion of the substrate that is located 3' to, but does not include, the ApG phosphodiester. Results from other experiments indicate this base pairing is a part of the functional cleavage complex. The association of the ends of E and substrate anticipates a second, 4-base-pair association between E and a portion of substrate that is 5' to, but does not include, the ApG phosphodiester. The effects of compensating mutations in E and substrate oligoribonucleotides support the existence of this second association in the active cleavage complex.
Structure and probable genetic location of a "ribosome modulation factor" associated with 100S ribosomes in stationary-phase Escherichia coli cells.
Authors: Wada A , Yamazaki Y , Fujita N , Ishihama A
Published: May 2006

The decrease in overall translation activity occurring concomitantly with the transition from the exponential growth phase to the stationary phase of Escherichia coli cells was found to be accompanied by the appearance of 100S ribosomes (dimers of 70S ribosome monomers). Analysis of ribosomal proteins by the radical-free and highly reducing method of two-dimensional gel electrophoresis indicated that a protein, designated protein E, was exclusively associated with 100S ribosomes. From the results, we propose that protein E is a "ribosome modulation factor" (RMF), which associates with 70S ribosomes and converts them to a dimeric form. A homology search of the partial amino acid sequence of RMF using the DNA sequence data bases revealed that the rmf gene, which encodes RMF, is located next to the fabA gene at 21.8 min on the E. coli chromosome.
Ribosomal protein L4 stimulates in vitro termination of transcription at a NusA-dependent terminator in the S10 operon leader.
Authors: Zengel J M , Lindahl L
Published: May 2006

The 11-gene S10 ribosomal protein operon of Escherichia coli is under the autogenous control of L4, the product of the third gene of the operon. Ribosomal protein L4 inhibits both transcription and translation of the operon. Our in vivo studies indicated that L4 regulates transcription by causing premature termination within the untranslated S10 operon leader. We have now used an in vitro transcription system to study the effect of purified L4 on expression of the S10 operon. We find that the cell-free system reproduces the in vivo observations. Namely, in the absence of L4, most of the RNA polymerases read through the termination site in the S10 attenuator; the addition of L4 results in increased termination at this site. However, RNA polymerase does not terminate at the S10 attenuator, with or without L4, unless an additional factor, protein NusA, is added to the transcription reaction. These results suggest that the attenuator in the S10 operon is a NusA-dependent terminator whose efficiency is regulated by ribosomal protein L4.
Development of B-lineage cells in the bone marrow of scid/scid mice following the introduction of functionally rearranged immunoglobulin transgenes.
Authors: Reichman-Fried M , Hardy R R , Bosma M J
Published: May 2006

Mice homozygous for the mutation scid (scid mice) are severely immunodeficient and generally lack detectable numbers of pre-B, B, and T cells. This condition is believed to result from a defect in the mechanism responsible for rearrangement of immunoglobulin and T-cell receptor genes in developing B and T lymphocytes. To test this hypothesis and evaluate whether scid affects only the process of gene recombination, we introduced functionally rearranged immunoglobulin genes into the scid mouse genome. As scid mice appear to contain early lymphoid cells committed to the B lineage (pro-B cells), we asked whether the introduction of an IgM heavy-chain gene alone (mu-transgenic scid mice) or both IgM heavy- and kappa light-chain genes (mu kappa-transgenic scid mice) would allow further differentiation of scid pro-B cells into pre-B and B cells. We found that normal numbers of pre-B cells appeared in the bone marrow of mu-transgenic scid mice and that both pre-B and B cells appeared in the bone marrow of mu kappa-transgenic scid mice. However, in the latter case, the number of pre-B and B cells was 2- to 3-fold less than in the controls (mu kappa-transgenic scid heterozygotes) and few, if any, B cells were detectable in the peripheral lymphoid tissues. The implications of these results for the above hypothesis are discussed.
Hydrophobic surfactant-associated polypeptides: SP-C is a lipopeptide with two palmitoylated cysteine residues, whereas SP-B lacks covalently linked fatty acyl groups.
Authors: Curstedt T , Johansson J , Persson P , Eklund A , Robertson B , Löwenadler B , Jörnvall H
Published: May 2006

Pulmonary surfactant contains two hydrophobic polypeptides, SP-B and SP-C, with known amino acid sequences and with truncated subforms lacking the N-terminal residues. Treatment of SP-C with KOH releases fatty acids (palmitic acid to more than 85%) in molar ratios of 1.8-2.0 relative to the polypeptide. Furthermore, plasma-desorption mass spectrometry shows native SP-C of both the intact and truncated types to be monomers with masses about 500 units higher than those expected for the polypeptide chains. After treatment with KOH, trimethylamine, or dithioerythritol, the polypeptide masses are obtained. These results prove that native SP-C is a lipopeptide with two palmitoyl groups covalently linked to the polypeptide chain. The deacylation conditions, the presence of two cysteine residues in the polypeptide, and the absence of other possible attachment sites establish that the palmitoyl groups are thioester-linked to the two adjacent cysteine residues. In contrast, the major form of porcine SP-B is a dimer without fatty acid components. That SP-C is a true lipopeptide with covalently bound palmitoyl groups suggests possibilities for functional interactions. It gives a direct physical link between SP-C and surfactant phospholipid components. Long-chain acylation may constitute a means for association of proteins with membranes and could conceivably modulate the stability and biological activity of surfactant films.
Conversion of a 3-desoxysteroid to 3-desoxyestrogen by human placental aromatase.
Authors: Cole P A , Bean J M , Robinson C H
Published: May 2006

Human placental aromatase is a cytochrome P-450 enzyme system which converts androgens to estrogens by three successive oxidative reactions. The first two steps have been shown to be hydroxylations at the androgen 19-carbon, but the third step remains unknown. A leading theory for the third step involves ferric peroxide attack on the 19-oxo group to produce a 19,19-hydroxyferric peroxide intermediate and subsequent collapse to estrogen. We had previously developed a nonenzymatic peroxide model reaction which was based on the above-mentioned theory, and we demonstrated the importance of 3-ketone enolization in facilitating aromatization. This study discusses the synthesis and nonenzymatic and enzymatic study of a 3-desoxy-2,4-diene-19-oxo androgen analogue. This compound was found to be a potent nonenzymatic model substrate and competitive inhibitor of aromatase (Ki = 73 nM). Furthermore, in an unprecedented event, this compound served as a substrate for aromatase, with conversion to the corresponding 3-desoxyestrogen.
Osteoinductive factor inhibits formation of human osteoclast-like cells.
Authors: Kukita A , Bonewald L , Rosen D , Seyedin S , Mundy G R , Roodman G D
Published: May 2006

Osteoinductive factor (OIF) is a glycoprotein in bone that induces ectopic bone formation. Implantation of OIF plus transforming growth factor beta (TGF-beta) type 1 or 2 into subcutaneous tissues of rats induces formation of bone at the implantation site. Since TGF-beta is also present in bone matrix and inhibits formation of multinucleated cells that express an osteoclast phenotype in long-term human marrow cultures, we tested the effects of OIF on formation of these osteoclast-like cells to determine the effects of OIF on cells in the osteoclast lineage. We found that OIF inhibited total multinucleated cell (MNC) formation in a dose-dependent fashion and preferentially inhibited formation of MNCs that react with monoclonal antibody 23c6 (23c6-positive MNCs), an antibody that identifies osteoclasts. In addition, low concentrations of OIF in combination with low concentrations of TGF-beta acted synergistically to inhibit 23c6-positive MNC formation. The inhibition of 23c6-positive MNC formation by OIF was not mediated by prostaglandin synthesis. These data suggest that regulatory growth factors, such as OIF or TGF-beta, that are stored within the bone matrix and released when bone is resorbed can serve as natural inhibitors of osteoclast activity by inhibiting osteoclast formation.
A mammalian protein of 220 kDa binds pre-mRNAs in the spliceosome: a potential homologue of the yeast PRP8 protein.
Authors: Garcia-Blanco M A , Anderson G J , Beggs J , Sharp P A
Published: May 2006

A mammalian protein of approximately 220 kDa (p220) was UV-crosslinked to precursor mRNAs (pre-mRNAs) under splicing conditions. The kinetics and biochemical requirements of the UV-crosslinking of p220 corresponded to the kinetics and biochemical requirements of spliceosome formation. On Western blots, antibodies against the yeast splicing factor PRP8 recognized a doublet of proteins, the faster migrating of which comigrated with p220. Furthermore, UV-crosslinked p220 was immunoprecipitated with anti-PRP8 antisera. These results suggest structural conservation of the splicing factor PRP8 from yeast to mammals and show that this protein is in close proximity to the pre-mRNA in the spliceosome.
Platelet-activating factor may act as a second messenger in the release of icosanoids and superoxide anions from leukocytes and endothelial cells.
Authors: Stewart A G , Dubbin P N , Harris T , Dusting G J
Published: May 2006

Platelet-activating factor (PAF) is generated by endothelial cells, polymorphonuclear leukocytes, and macrophages after activation by appropriate receptor agonists, but much of the PAF remains intracellular. We have investigated whether PAF formation is important for the subsequent generation of icosanoids and superoxide anions by these cells. The generation of prostacyclin and leukotriene B4 were measured by radioimmunoassay, superoxide anion was measured by reduction of cytochrome c, and PAF was measured by bioassay. In each cell type, PAF formation preceded or accompanied icosanoid generation. Bradykinin-induced prostacyclin generation in endothelial cells was markedly reduced by the PAF receptor antagonists WEB 2086 or CV 6209. In guinea pig adherent macrophages in vitro, basal prostacyclin generation and that induced by endotoxin and fMet-Leu-Phe were inhibited by either WEB 2086 (1-100 microM) or CV 6209 (0.1-10 microM). In isolated rabbit polymorphonuclear leukocytes, fMet-Leu-Phe stimulated the generation of both leukotriene B4 and superoxide anion. WEB 2086 and CV 6209 caused concentration-dependent inhibition of both these markers of leukocyte activation. These observations lead us to suggest that PAF may be a second messenger in leukocytes and endothelial cells.
Different mouse mast cell populations express various combinations of at least six distinct mast cell serine proteases.
Authors: Reynolds D S , Stevens R L , Lane W S , Carr M H , Austen K F , Serafin W E
Published: May 2006

Mouse serosal mast cells (SMCs) and Kirsten sarcoma virus-immortalized mast cells store large amounts of mast cell carboxypeptidase A and serine proteases in their secretory granules. Secretory granule proteins from 2.6 x 10(6) purified SMCs were separated by NaDodSO4/PAGE, trans-blotted to poly(vinylidine difluoride) membranes, and subjected to amino-terminal amino acid sequencing. Four distinct mast cell serine proteases were identified. With mast cell carboxypeptidase A, these serine proteases comprise the major proteins of mouse SMC secretory granules. Each of the four SMC serine proteases was distinct from the two serine proteases present in mucosal mast cells in the intestines of helminth-infected mice. The secretory granules of a Kirsten sarcoma virus-immortalized mast cell line contained three of the SMC-derived serine proteases and one of the mucosal mast cell-derived serine proteases. Thus, the family of mouse mast cell secretory granule serine proteases has at least six distinct members that can be expressed in different combinations in different mast cell populations.
Characterization of two RNAs transcribed from the cis-regulatory region of the abd-A domain within the Drosophila bithorax complex.
Authors: Cumberledge S , Zaratzian A , Sakonju S
Published: May 2006

The infra-abdominal (iab) regions of the biothorax complex are thought to cis-regulate expression of the abdominal-A (abd-A) and Abdominal-B (Abd-B) transcripts. These large cis-regulatory regions are also actively transcribed. Here we present a detailed analysis of the transcription products of the iab-4 region. During early embryogenesis a 6.8-kilobase (kb) RNA is transcribed and processed to yield 1.7- and 2.0-kb poly(A)+ RNAs. These RNAs are expressed from the time of cellular blastoderm formation to germ-band shortening and are localized in parasegments 8-14 (the posterior second through the anterior ninth abdominal segments). Sequence analysis of the two RNAs suggests that they may not be translated. We discuss some possible functions of the 1.7/2.0-kb iab RNAs and the significance of their similarity to the bithoraxoid (bxd) RNAs and other transcripts from the iab regions of the abd-A and Abd-B domains.
Human monoclonal antibody with dual GM2/GD2 specificity derived from an immunized melanoma patient.
Authors: Yamaguchi H , Furukawa K , Fortunato S R , Livingston P O , Lloyd K O , Oettgen H F , Old L J
Published: May 2006

GM2 ganglioside is a common cell surface constituent of human melanoma and other tumors of neuroectodermal origin, and vaccination with GM2 ganglioside results in high levels of anti-GM2 antibodies in patients with melanoma. Lymphocytes from a GM2-vaccinated patient (VS) were transformed by Epstein-Barr virus and tested for production of antibodies with reactivity for GM2-positive tumor cells. A high percentage of antibody-producing B cells was detected, but antibody reactivity was generally lost during culture expansion. Two cultures, however, remained stable for antibody productivity and one was used to develop a stable hybrid line with mouse myeloma. The monoclonal antibody (designated 3-207) derived from patient VS has dual specificity for GM2 and GD2, despite the fact that only GM2 antibody could be detected in the patient's serum. Monoclonal antibody 3-207 shows high-titered reactivity with a range of melanoma, astrocytoma, neuroblastoma, and leukemia cell lines, cells with prominent cell surface expression of GM2 and GD2. The cell surface reactivity of monoclonal antibody 3-207 was not abolished by treatment of target cells with neuraminidase, as the enzyme converted GD2 to GM2, which was still detected by monoclonal antibody 3-207.
Induction of interleukin 1 and tumor necrosis factor by mycobacterial proteins: the monocyte western blot.
Authors: Wallis R S , Amir-Tahmasseb M , Ellner J J
Published: May 2006

Infection with Mycobacterium tuberculosis involves mononuclear phagocytic cells as hosts to intracellular parasites, accessory cells in the induction of the immune response, effector cells for mycobacterial killing, and targets of cytotoxic lymphocytes. When stimulated by whole mycobacteria or various mycobacterial preparations, monocytes and macrophages produce the cytokines interleukin 1 and tumor necrosis factor, which possess multiple functions, including immune induction, and may be responsible for the fever and cachexia prominent in tuberculosis. To identify mycobacterial proteins that may directly activate production of these cytokines, culture filtrate of M. tuberculosis that had been subjected to gel electrophoresis and transferred to nitrocellulose paper was used to stimulate monocyte production of cytokines. Fractions representing molecular weights of 46,000 and 20,000 consistently induced both interleukin 1 and tumor necrosis factor. The magnitude of the monocyte responses to these fractions was similar to that to intact mycobacteria or optimal concentrations of lipopolysaccharide. This stimulatory effect was not due to contamination with either bacterial lipopolysaccharide or mycobacterial lipoarabinomannan, as it was abolished by digestion with Streptomyces griseus protease but was unaffected by ammonium sulfate precipitation, preincubation with polymyxin B, or depletion of lipoarabinomannan by immunoaffinity chromatography. Proteins identified by this system may have considerable potential as immunogens, as the capacity to directly stimulate mononuclear phagocyte production of cytokines is an essential property of adjuvants.
Topography of toxin-acetylcholine receptor complexes by using photoactivatable toxin derivatives.
Authors: Chatrenet B , Trémeau O , Bontems F , Goeldner M P , Hirth C G , Ménez A
Published: May 2006

We have defined the molecular environment of a snake neurotoxin interacting with the high- and low-affinity binding sites of the nicotinic acetylcholine receptor (AcChoR). This was done by photocoupling reactions using three toxin derivatives with photoactivatable moieties on Lys-15, Lys-47, and Lys-51. Competition data showed that Lys-47 belongs to the toxin-AcChoR interacting domain whereas the other two residues are excluded from it. We first tentatively determined the threshold of covalent coupling, indicative of the proximity between the photoactivatable probes and subunits, by quantifying the coupling occurring between the same derivatives and a model compound (i.e., a toxin-specific monoclonal antibody). We then (i) quantified the coupling yields occurring when both binding sites of AcChoR were occupied by the toxin derivatives, (ii) discriminately quantified the coupling yields at the high-affinity binding site, and (iii) deduced the coupling yields at the low-affinity binding site. In the high-affinity site, the probes on Lys-15 and Lys-47 predominantly reacted with the high-affinity site of the AcChoR alpha subunit whereas the probe on Lys-51 reacted with the delta subunit. In the low-affinity site, the probe on Lys-47 predominantly reacted with the low-affinity site of the alpha chain and the beta chain whereas those on Lys-15 and Lys-51 reacted with the gamma and delta chains, respectively. A three-dimensional model showing a unique organization of AcChoR bound to two toxin molecules is presented.
Plasma membrane fatty acid-binding protein and mitochondrial glutamic-oxaloacetic transaminase of rat liver are related.
Authors: Berk P D , Wada H , Horio Y , Potter B J , Sorrentino D , Zhou S L , Isola L M , Stump D , Kiang C L , Thung S
Published: May 2006

The hepatic plasma membrane fatty acid-binding protein (h-FABPPM) and the mitochondrial isoenzyme of glutamic-oxaloacetic transaminase (mGOT) of rat liver have similar amino acid compositions and identical amino acid sequences for residues 3-24. Both proteins migrate with an apparent molecular mass of 43 kDa on SDS/polyacrylamide gel electrophoresis, have a similar pattern of basic charge isomers on isoelectric focusing, are eluted similarly from four different high-performance liquid chromatographic columns, have absorption maxima at 435 nm under acid conditions and 354 nm at pH 8.3, and bind oleate with a Ka approximately 1.2-1.4 x 10(7) M-1. Sinusoidally enriched liver plasma membranes and purified h-FABPPM have GOT enzymatic activity; the relative specific activities (units/mg) of the membranes and purified protein suggest that h-FABPPM constitutes 1-2% of plasma membrane protein in the rat hepatocyte. Monospecific rabbit antiserum against h-FABPPM reacts on Western blotting with mGOT, and vice versa. Antisera against both proteins produce plasma membrane immunofluorescence in rat hepatocytes and selectively inhibit the hepatocellular uptake of [3H]oleate but not that of [35S]sulfobromophthalein or [14C]taurocholate. The inhibition of oleate uptake produced by anti-h-FABPPM can be eliminated by preincubation of the antiserum with mGOT; similarly, the plasma membrane immunofluorescence produced by either antiserum can be eliminated by preincubation with the other antigen. These data suggest that h-FABPPM and mGOT are closely related.
Growth and atrophy of neurons labeled at their birth in a song nucleus of the zebra finch.
Authors: Konishi M , Akutagawa E
Published: May 2006

The robust nucleus of the archistriatum (RA) is one of the forebrain nuclei that control song production in birds. In the zebra finch (Poephila guttata), this nucleus contains more and larger neurons in the male than in the female. A single injection of tritiated thymidine into the egg on the 6th or 7th day of incubation resulted in labeling of many RA neurons with tritium. The size of tritium-labeled neurons and the tissue volume containing them did not differ between the sexes at 15 days after hatching. In the adult brain, tritium-labeled neurons and the tissue volume containing them were much larger in the male than in the female. Also, tritium-labeled RA neurons were large in females which received an implant of estrogen immediately after hatching. The gender differences in the neuron size and nuclear volume of the zebra finch RA are, therefore, due not to the replacement of old neurons by new ones during development but to the growth and atrophy of neurons born before hatching. Similarly, the masculinizing effects of estrogen on the female RA are due not to neuronal replacement but to the prevention of atrophy and promotion of growth in preexisting neurons.
Molecular cloning of cDNA for the carcinoma-associated antigen GA733-2.
Authors: Szala S , Froehlich M , Scollon M , Kasai Y , Steplewski Z , Koprowski H , Linnenbach A J
Published: May 2006

Defined by monoclonal antibody GA733, the GA733-2 antigen is a cell surface 40-kDa glycoprotein associated with human carcinomas of various origins. Molecular clones for the GA733-2 antigen were isolated from a colorectal carcinoma cell line cDNA library using the high-efficiency COS cell expression system. A 1.4-kilobase cDNA species was enriched by immunoselection with monoclonal antibody. The authenticity of individual clones was established by immunologic and sequence criteria. At the amino acid sequence level, GA733-2 was found to be greater than 99% identical to the previously described KSA antigen defined by monoclonal antibody KS1/4. The amino acid sequence derived from the previously described GA733-related gene, GA733-1, was found to be 49% identical to GA733-2. The positions of 12 cysteine residues in the extracellular domains of the two GA733 antigens are conserved, as is the overall distribution of hydrophobic and hydrophilic residues. A 1.45-kilobase transcript of the GA733-2/KSA gene was found to be expressed in cell lines derived from colorectal and pancreatic carcinoma.