bluuebunny/crossref_metadata_2025 huggingface dataset:
Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells.
Authors: Broxmeyer H E , Douglas G W , Hangoc G , Cooper S , Bard J , English D , Arny M , Thomas L , Boyse E A
Published: May 2006

The purpose of this study was to evaluate human umbilical cord blood as an alternative to bone marrow in the provision of transplantable stem/progenitor cells for hematopoietic reconstitution. Although no direct quantitative assay for human hematopoietic repopulating cells is at present available, the granulocyte-macrophage progenitor cell (CFU-GM) assay has been used with success as a valid indicator of engrafting capability. We examined greater than 100 collections of human umbilical cord blood for their content of nucleated cells and granulocyte-macrophage, erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells, in many cases both before and after cryopreservation. First it was determined that granulocyte-macrophage, erythroid, and multipotential progenitor cells remained functionally viable in cord blood untreated except for addition of anticoagulant for at least 3 days at 4 degrees C or 25 degrees C (room temperature), though not at 37 degrees C, implying that these cells could be satisfactorily studied and used or cryopreserved for therapy after transport of cord blood by overnight air freight carriage from a remote obstetrical service. Granulocyte-macrophage progenitor cells from cord blood so received responded normally to stimulation by purified recombinant preparations of granulocyte-macrophage, granulocyte, and macrophage colony-stimulating factors and interleukin 3. The salient finding, based on analysis of 101 cord blood collections, is that the numbers of progenitor cells present in the low-density (less than 1.077 gm/ml) fraction after Ficoll/Hypaque separation typically fell within the range that has been reported for successful engraftment by bone marrow cells. Another observation of practical importance is that procedures to remove erythrocytes or granulocytes prior to freezing, and washing of thawed cells before plating, entailed large losses of progenitor cells, the yield of unwashed progenitor cells from unfractionated cord blood being many times greater. The provisional inference is that human umbilical cord blood from a single individual is typically a sufficient source of cells for autologous (syngeneic) and for major histocompatibility complex-matched allogeneic hematopoietic reconstitution.
Cloning immunoglobulin variable domains for expression by the polymerase chain reaction.
Authors: Orlandi R , Güssow D H , Jones P T , Winter G
Published: May 2006

We have designed a set of oligonucleotide primers to amplify the cDNA of mouse immunoglobulin heavy and light chain variable domains by the polymerase chain reaction. The primers incorporate restriction sites that allow the cDNA of the variable domains to be force-cloned for sequencing and expression. Here we have applied the technique to clone and sequence the variable domains of five hybridoma antibodies and to express a mouse-human chimeric antibody that binds to the human mammary carcinoma line MCF-7. The technique should also lead to the cloning of antigen-binding specificities directly from immunoglobulin genes.
Mechanism of inhibition of growth of 3T3-L1 fibroblasts and their differentiation to adipocytes by dehydroepiandrosterone and related steroids: role of glucose-6-phosphate dehydrogenase.
Authors: Shantz L M , Talalay P , Gordon G B
Published: May 2006

Dehydroepiandrosterone (DHEA) and certain structural analogues block the differentiation of 3T3-L1 mouse embryo fibroblasts to adipocytes. These steroids also are potent uncompetitive inhibitors of mammalian glucose-6-phosphate dehydrogenases (G6PDs). We provide direct evidence that treatment of the 3T3-L1 cells with DHEA and its analogues results in intracellular inhibition of G6PD, which is associated with the block of differentiation: (i) Levels of 6-phosphogluconate and other products of the pentose phosphate pathway are decreased; (ii) the magnitude of these decreases depends on the potency of steroids as inhibitors of G6PD and on concentration and duration of exposure, and it is accompanied by a proportionate block of differentiation; (iii) in cells exposed to 16 alpha-bromoepiandrosterone (a more potent inhibitor of G6PD than DHEA) at concentrations that block differentiation, introduction of exogenous 6-phosphogluconate in liposomes raises the levels of 6-phosphogluconate and other products of the pentose phosphate pathway and partially relieves the steroid block of cell growth and differentiation.
Role of coupling entropy in establishing the nature and magnitude of allosteric response.
Authors: Reinhart G D , Hartleip S B , Symcox M M
Published: May 2006

The coupling free energy between an allosteric ligand and a substrate, delta Gax, is an explicit measure of the nature as well as the magnitude of impact that an allosteric ligand has on the binding of the substrate ligand to the enzyme, with positive values indicating inhibition and negative values indicating activation. By measuring the variation with temperature of the coupling free energy between the allosteric ligand and the substrate, it is possible to determine the enthalpic and entropic components that give rise to the coupling free energy. We have performed this analysis on two different K-type allosteric systems: the allosteric inhibition of rat liver phosphofructokinase by MgATP, and the allosteric activation of beef heart NAD+-dependent isocitrate dehydrogenase by ADP. In both cases the coupling free energy arises as the net result of opposing enthalpic and entropic components, with the coupling enthalpy (delta Hax) favoring activation and the coupling entropy (delta Sax) favoring inhibition. For phosphofructokinase at 25 degrees C, the absolute value of T delta Sax is greater than the absolute value of delta Hax, and net inhibition of rat liver phosphofructokinase by MgATP is realized. For isocitrate dehydrogenase, delta Hax dominates; however, the net activation is substantially mitigated by the magnitude of T delta Sax. Hence, the coupling entropy plays an important role in establishing both the nature and magnitude of the allosteric response. We hypothesize that the negative coupling entropy arises from the particular constraint placed upon the internal dynamical properties of the enzyme by the simultaneous binding of both allosteric and substrate ligands.
Cell-specific transcriptional control of the mouse DNA-binding protein mC/EBP.
Authors: Xanthopoulos K G , Mirkovitch J , Decker T , Kuo C F , Darnell J E
Published: May 2006

The mRNA encoding the mouse homolog of C/EBP, a rat DNA-binding protein that participates in activating a number of genes in hepatocytes, is present in liver cells at a far higher concentration than in most other cells, including spleen, kidney, muscle, and the majority of the brain. However, fat cells and intestinal cells contain 25-50% as much mRNA as liver cells. "Run-on" experiments show that the basis for the restricted cellular distribution of the mouse C/EBP mRNA is transcriptional regulation of the gene. We also show that disruption of cell-cell contacts incident to liver cell dispersion results in a prompt and extensive reduction in mouse C/EBP transcription as we had earlier shown to be the case for a group of 10 genes transcribed in a hepatocyte-specific fashion. In contrast, breaking cell contacts and plating the hepatocytes in culture leads to a prolonged increase in transcription of the Jun-B gene that encodes a widely distributed transcription factor. These results illustrate that the regulation of expression of a mammalian regulatory protein with limited tissue distribution is controlled at the level of transcription and depends on cell contacts.
Intercellular adhesion molecule 1 is induced on isolated endocrine islet cells by cytokines but not by reovirus infection.
Authors: Campbell I L , Cutri A , Wilkinson D , Boyd A W , Harrison L C
Published: May 2006

The selective destruction of the pancreatic islet beta cells in type 1 diabetes mellitus is thought to be mediated by a cellular autoimmune process, possibly triggered by virus infection in genetically susceptible individuals. Because of the potentially important role of cell-cell adhesion in the immune response, we investigated whether cytokine products of mononuclear cells, or virus infection, induced the expression of intercellular adhesion molecule 1 (ICAM-1) on human endocrine islet cells. By flow cytofluorimetry, control islet cells did not express detectable ICAM-1. However, after a 72-hr exposure of islets to interferon gamma (IFN-gamma) and/or tumor necrosis factor alpha (TNF-alpha) (each at 250 units/ml), ICAM-1 was induced on greater than 85% of islet cells. IFN-gamma was 50% more potent than TNF-alpha; together, their effects were additive. Class I major histocompatibility complex (MHC) protein expression, detected on control islet cells, was also stimulated by IFN-gamma and/or TNF-alpha. In contrast, infection with reovirus type 3 did not induce ICAM-1 on islet cells, although it stimulated the expression of class I MHC proteins. By double-label indirect immunofluorescence microscopy, ICAM-1 expression was identified on both beta (insulin-secreting) and delta (somatostatin-secreting) islet cells. Monoclonal antibody to ICAM-1 precipitated protein of Mr 97,000 from [35S]methionine-labeled islets exposed to IFN-gamma and TNF-alpha, but not from control islets. RNA blot analysis revealed a major species of 3.3 kilobases and a minor species of 2.2 kilobases induced in islets exposed to the cytokines. These findings have implications for the molecular mechanisms of beta-cell destruction in type 1 diabetes, in that expression of ICAM-1 by beta cells may facilitate adhesion of antigen-targeted immune cells.
Rapid increase in enzyme and peptide mRNA in sympathetic ganglia after electrical stimulation in humans.
Authors: Schalling M , Stieg P E , Lindquist C , Goldstein M , Hökfelt T
Published: May 2006

Thoracic ganglia in humans were studied after electrical, preganglionic stimulation using in situ hybridization with synthetic oligonucleotide probes against the catecholamine-synthesizing enzymes tyrosine hydroxylase (EC 1.14.16.2) and dopamine beta-hydroxylase (EC 1.14.17.1) and neuropeptide tyrosine. Immunohistochemical analysis was also performed. Following short peroperative stimulation a severalfold increase in all three mRNAs was found in principal ganglion cells, whereas no definite changes could be detected in enzyme or peptide levels with immunohistochemistry. The results suggest a very rapid and sensitive regulation of genes involved in signal transmission in the sympathetic nervous system of humans. Moreover, they indicate that electrical stimulation of neurons and/or pathways combined with in situ hybridization may be used as a method to define neuronal projections by visualizing increases in mRNAs for transmitter enzymes and/or peptide in target cells.
Identification of genes from pattern formation, tyrosine kinase, and potassium channel families by DNA amplification.
Authors: Kamb A , Weir M , Rudy B , Varmus H , Kenyon C
Published: May 2006

The study of gene family members has been aided by the isolation of related genes on the basis of DNA homology. We have adapted the polymerase chain reaction to screen animal genomes very rapidly and reliably for likely gene family members. Using conserved amino acid sequences to design degenerate oligonucleotide primers, we have shown that the genome of the nematode Caenorhabditis elegans contains sequences homologous to many Drosophila genes involved in pattern formation, including the segment polarity gene wingless (vertebrate int-1), and homeobox sequences characteristic of the Antennapedia, engrailed, and paired families. In addition, we have used this method to show that C. elegans contains at least five different sequences homologous to genes in the tyrosine kinase family. Lastly, we have isolated six potassium channel sequences from humans, a result that validates the utility of the method with large genomes and suggests that human potassium channel gene diversity may be extensive.
Molecular cloning of the alpha-subunit of human prolyl 4-hydroxylase: the complete cDNA-derived amino acid sequence and evidence for alternative splicing of RNA transcripts.
Authors: Helaakoski T , Vuori K , Myllylä R , Kivirikko K I , Pihlajaniemi T
Published: May 2006

Prolyl 4-hydroxylase [procollagen-proline, 2-oxyglutarate 4-dioxygenase; procollagen-L-proline, 2-oxoglutarate:oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2], an alpha 2 beta 2 tetramer, catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages. We report here on the isolation of cDNA clones encoding the alpha-subunit of the enzyme from human tumor HT-1080, placenta, and fibroblast cDNA libraries. Eight overlapping clones covering almost all of the corresponding 3000-nucleotide mRNA, including all the coding sequences, were characterized. These clones encode a polypeptide of 517 amino acid residues and a signal peptide of 17 amino acids. Previous characterization of cDNA clones for the beta-subunit of prolyl 4-hydroxylase has indicated that its C terminus has the amino acid sequence Lys-Asp-Glu-Leu, which, it has been suggested, is necessary for the retention of a polypeptide within the lumen of the endoplasmic reticulum. The alpha-subunit does not have this C-terminal sequence, and thus one function of the beta-subunit in the prolyl 4-hydroxylase tetramer appears to be to retain the enzyme within this cell organelle. Interestingly, three of the cDNA clones for the alpha-subunit contained a 64-nucleotide sequence homologous but not identical to the corresponding 64-nucleotide sequence found in four other cDNA clones. Nuclease S1 mapping experiments demonstrated that this difference was due to the existence of two types of mRNA present in approximately equal amounts. Southern blot analyses of human genomic DNA with a cDNA probe for the alpha-subunit suggested the presence of only one gene encoding the two types of mRNA, which appear to result from mutually exclusive alternative splicing of primary transcripts of one gene.
Metabolite specific proton magnetic resonance imaging.
Authors: Hurd R E , Freeman D M
Published: May 2006

An imaging method is described that makes use of proton double quantum nuclear magnetic resonance (NMR) to construct images based on selected metabolites such as lactic acid. The optimization of the method is illustrated in vitro, followed by in vivo determination of lactic acid distribution in a solid tumor model. Water suppression and editing of lipid signals are such that two-dimensional spectra of lactic acid may be obtained from a radiation-induced fibrosarcoma (RIF-1) tumor in under 1 min and lactic acid images from the same tumor in under 1 hr at 2.0 T. This technique provides a fast and reproducible method at moderate magnetic field strength for mapping biologically relevant metabolites.
Escherichia coli DNA helicase II (uvrD gene product) catalyzes the unwinding of DNA.RNA hybrids in vitro.
Authors: Matson S W
Published: May 2006

DNA helicase II is a well-characterized Escherichia coli enzyme capable of unwinding duplex DNA and known to be involved in both methyl-directed mismatch repair and excision repair of pyrimidine dimers. Here it is shown that this enzyme also catalyzes the ATP-dependent unwinding of a DNA.RNA hybrid consisting of a radioactively labeled RNA molecule annealed on M13 single-stranded DNA. The DNA.RNA unwinding reaction required less protein to unwind more base pairs than the corresponding unwinding of duplex DNA. In addition, the rate of unwinding of the DNA.RNA hybrid was more than an order of magnitude faster than unwinding of a DNA partial duplex of similar length. The unwinding of the DNA.RNA hybrid is a property unique to helicase II since helicase I, Rep protein, and helicase IV failed to catalyze the reaction. In light of these results it seems likely that helicase II is involved in some previously unrecognized aspect of nucleic acid metabolism, in addition to its known roles in DNA repair reactions.
Use of a laser-induced optical force trap to study chromosome movement on the mitotic spindle.
Authors: Berns M W , Wright W H , Tromberg B J , Profeta G A , Andrews J J , Walter R J
Published: May 2006

A laser-induced optical force trap was used to alter the movement of chromosomes in mitotic cells in vitro. The trap was produced by using a 1.06-microns neodymium YAG (yttrium/aluminum garnet) laser focused through a phase-contrast microscope. The trap was applied to one side of centrophilic chromosomes off the mitotic spindle and to late-moving chromosomes on the mitotic spindle. In both situations, chromosome movement was initiated in the direction opposite to that of the applied force. When the force was applied, chromosomes moved at velocities 10-20 times normal. These studies verify and extend the feasibility of using this new technique to study factors that influence organelle motility.
An activated form of transforming growth factor beta is produced by cocultures of endothelial cells and pericytes.
Authors: Antonelli-Orlidge A , Saunders K B , Smith S R , D'Amore P A
Published: May 2006

Using an in vitro coculture system to mimic the interactions between the cells of the vessel wall, we have previously shown that pericytes and smooth muscle cells (SMC) inhibit the growth of capillary endothelial cells (EC). We have undertaken studies to determine the mechanism of this inhibition. Using conditioned media and affinity-purified antibodies to transforming growth factor beta (TGF-beta), we now demonstrate that activated TGF-beta produced in these cocultures mediates EC growth inhibition. No inhibitory activity was detected when media conditioned by individual cultures of EC, SMC, or pericytes were examined for their effect on EC growth. In contrast, media conditioned by cocultures of EC-SMC and EC-pericytes inhibited EC proliferation to the same degree as the coculture itself. Immunoadsorption of coculture-derived conditioned media with antibodies to TGF-beta eliminated the inhibitory activity. Acid activation of serum-free media conditioned by any of the cells cultured alone yielded inhibitory activity, whereas activation of coculture conditioned media did not increase its inhibitory activity. Addition of anti-TGF-beta neutralizing antibodies to cocultures blocked the pericyte-mediated EC growth inhibition. These results indicate that latent TGF-beta is produced by these cells and it is activated by a mechanism that requires contact between the two cell types.
Vgr-1, a mammalian gene related to Xenopus Vg-1, is a member of the transforming growth factor beta gene superfamily.
Authors: Lyons K , Graycar J L , Lee A , Hashmi S , Lindquist P B , Chen E Y , Hogan B L , Derynck R
Published: May 2006

The transforming growth factor beta (TGF-beta)-related products of the Xenopus Vg-1 and Drosophila decapentaplegic (DPP) genes have been implicated in the control of growth and differentiation during embryogenesis. We have isolated a mouse cDNA, Vgr-1, that encodes a polypeptide structurally related to Xenopus Vg-1. Sequence comparisons indicate that the Vgr-1 protein belongs to a family of DPP-like gene products within the TGF-beta superfamily. The levels of Vgr-1 RNA were determined in embryos and tissues isolated at various stages of development. A 3.5-kilobase mRNA increases throughout development and into adulthood in many tissues and in F9 teratocarcinoma cells differentiating into endoderm in response to retinoic acid and cAMP. The amino acid homologies and patterns of expression suggest that, like the DPP gene product, Vgr-1 plays a role at various stages of development.
Physical linkage of a guanine nucleotide-binding protein-related gene to the chicken major histocompatibility complex.
Authors: Guillemot F , Billault A , Auffray C
Published: May 2006

Several genes were found closely associated with major histocompatibility class I and class II beta-chain genes in chicken genomic DNA clusters by hybridizing tissue-specific cDNA probes to cosmid clones. A cDNA probe for one of these genes, probe C12.3 isolated from a chicken liver cDNA library, was used to clone the homologous sequence H12.3 from a human B-lymphoblastoid cell line cDNA library. C12.3 and H12.3 encode exactly the same 317-residue-long protein. The sequence of 12.3 shows significant homology with the two known guanine nucleotide-binding protein beta subunits (GP beta 1 and GP beta 2) and other proteins that all share the same segmented structure with seven internal homologous repeats about 45 residues in length. Unlike the chicken gene, the human H12.3 gene and its mouse counterpart are not located on the same chromosome as the major histocompatibility complex. A possible involvement of the C12.3 gene product in major histocompatibility complex-linked control of lymphocyte proliferation in chickens is discussed.
Loss of alleles from the distal short arm of chromosome 1 occurs late in melanoma tumor progression.
Authors: Dracopoli N C , Harnett P , Bale S J , Stanger B Z , Tucker M A , Housman D E , Kefford R F
Published: May 2006

The gene for familial malignant melanoma and its precursor lesion, the dysplastic nevus, has been assigned to a region of the distal short arm of chromosome 1, which is frequently involved in karyotypic abnormalities in melanoma cells. We have examined loci on chromosome 1p for loss-of-constitutional heterozygosity in 35 melanomas and 21 melanoma cell lines to analyze the role of these abnormalities in melanocyte transformation. Loss-of-heterozygosity at loci on chromosome 1p was identified in 15/35 (43%) melanomas and 11/21 (52%) melanoma cell lines. Analysis of multiple metastases derived from the same patient and of melanoma and lymphoblastoid samples from a family with hereditary melanoma showed that the loss-of-heterozygosity at loci on distal 1p is a late event in tumor progression, rather than the second mutation that would occur if melanoma were due to a cellular recessive mechanism. Comparisons with neuroblastoma and multiple endocrine neoplasia (MEN2) suggest that the frequent 1p loss-of-heterozygosity in these malignancies is a common late event of neuroectodermal tumor progression.
Deficiencies of polyunsaturated fatty acids and replacement by nonessential fatty acids in plasma lipids in multiple sclerosis.
Authors: Holman R T , Johnson S B , Kokmen E
Published: May 2006

Fatty acid compositions of plasma phospholipids, cholesteryl esters, triacylglycerols, and nonesterified fatty acids of 14 clinically proven and graded cases of multiple sclerosis were determined by capillary gas chromatography and compared with the values obtained for 100 normal, healthy subjects. In phospholipids, linoleic acid (18:2 omega 6; 18 carbon atoms, 2 double bonds, 6 carbon atoms beyond last double bond) was normal and 18:3 omega 6 was increased, but all subsequent omega 6 acids were subnormal (P less than 0.001), indicating impairment of chain elongation. All omega 3 acids were subnormal. The paucity of polyunsaturated fatty acids was compensated mass-wise by an increase in saturated acids. Disproportionate increases in short-chain, saturated, and monounsaturated acids, decreases in long-chain homologs, and increases of branched and odd-chain acids were observed. Loss of polyunsaturated fatty acids and replacement by nonessential acids lowered mean chain length and raised mean melting point significantly, suggesting that lowered membrane fluidity was only partially compensated by endogenous synthesis of lower-melting, nonessential acids. This phenomenon was not observed in cholesteryl esters or triacylglycerols. Nonesterified fatty acids showed significant changes in pattern of possible autacoid precursors. The abnormal profile of fatty acids in multiple sclerosis has features in common with profiles of other syndromes involving viral infections.
Expression of immunogenic epitopes of hepatitis B surface antigen with hybrid flagellin proteins by a vaccine strain of Salmonella.
Authors: Wu J Y , Newton S , Judd A , Stocker B , Robinson W S
Published: May 2006

A nonvirulent Salmonella dublin flagellin-negative, aromatic-dependent live vaccine strain has been used to express hepatitis B virus surface antigen epitopes in an immunogenic form. The envelope proteins of the virion are encoded by the S gene, which contains the pre-S1, pre-S2, and S coding regions. Synthetic oligonucleotides corresponding to amino acid residues S-(122-137) and pre-S2-(120-145) were inserted in-frame into the hypervariable region of a cloned Salmonella flagellin gene, and the recombinant plasmids were introduced into a flagellin-negative aroA mutant live vaccine strain of S. dublin, SL5928. The flagellin gene was expressed in bacteria carrying the plasmids as detected by immunoblotting with anti-flagellin (H1-d) serum. Both the S and pre-S2 epitopes were detected in bacteria carrying the relevant plasmid by immunoblotting with anti-HBs (antibody to hepatitis B virus surface antigen) and anti-peptide antisera. Animals immunized intramuscularly or orally with the live recombinant bacteria developed antibodies specific to these hepatitis B virus epitopes as detected by ELISA.
Thymidine kinase-negative herpes simplex virus mutants establish latency in mouse trigeminal ganglia but do not reactivate.
Authors: Coen D M , Kosz-Vnenchak M , Jacobson J G , Leib D A , Bogard C L , Schaffer P A , Tyler K L , Knipe D M
Published: May 2006

Herpes simplex virus infection of mammalian hosts involves lytic replication at a primary site, such as the cornea, translocation by axonal transport to sensory ganglia and replication, and latent infection at a secondary site, ganglionic neurons. The virus-encoded thymidine kinase, which is a target for antiviral drugs such as acyclovir, is not essential for lytic replication yet evidently is required at the secondary site for replication and some phase of latent infection. To determine the specific stage in viral pathogenesis at which this enzyme is required, we constructed virus deletion mutants that were acyclovir resistant and exhibited no detectable thymidine kinase activity. After corneal inoculation of mice, the mutants replicated to high titers in the eye but were severely impaired for acute replication in trigeminal ganglia and failed to reactivate from ganglia upon cocultivation with permissive cells. Nevertheless, latency-associated transcripts were expressed in neuronal nuclei of ganglia from mutant-infected mice and superinfection of the ganglia with a second virus rescued the latent mutant virus. Thus, contrary to a widely accepted hypothesis, the thymidine kinase-negative mutants established latent infections, implying that neither thymidine kinase activity nor ganglionic replication is necessary for establishment of latency. Rather, thymidine kinase appears to be necessary for reactivation from latency. These results suggest that acyclovir-resistant viruses could establish latent infections in clinical settings and have implications for the use of genetically engineered herpesviruses to deliver foreign genes to neurons.
Fc-receptor and M-protein genes of group A streptococci are products of gene duplication.
Authors: Heath D G , Cleary P P
Published: May 2006

The partial nucleotide sequence for an Fc-receptor gene from an M-type 76 group A streptococcus was determined. DNA sequence analysis revealed considerable sequence similarity between the Fc-receptor and M-protein genes in their proposed promoter regions, signal sequences, and 3' termini. Additional analysis indicated that the deduced Fc-receptor protein contains a proline-rich region and membrane anchor region highly similar to that of M protein. In view of these results, we postulated that Fc-receptor and M-protein genes of group A streptococci are the products of gene duplication from a common ancestral gene. It is proposed that DNA sequence similarity between these two genes may allow for extragenic homologous recombination as a means of generating antigenic diversity in these two surface proteins.
Visual experience regulates gene expression in the developing striate cortex.
Authors: Neve R L , Bear M F
Published: May 2006

We have examined the regulation of expression of the genes for the neuronal growth-associated protein GAP43, the type II calcium/calmodulin-dependent protein kinase, and glutamic acid decarboxylase in the kitten visual cortex during normal postnatal development and after a period of visual deprivation. We find that the mRNA transcripts of these genes display very different patterns of normal development but are all increased in the visual cortex of animals reared in the dark. Upon exposure to light, the transcript of the GAP43 gene drops to near-normal levels within 12 hr.
Characterization of a cDNA clone encoding human filaggrin and localization of the gene to chromosome region 1q21.
Authors: McKinley-Grant L J , Idler W W , Bernstein I A , Parry D A , Cannizzaro L , Croce C M , Huebner K , Lessin S R , Steinert P M
Published: May 2006

Filaggrins are an important class of intermediate filament-associated proteins that interact with keratin intermediate filaments of terminally differentiating mammalian epidermis. They show wide species variations and their aberrant expression has been implicated in a number of keratinizing disorders. We have isolated a cDNA clone encoding human filaggrin and used this to demonstrate that the human gene encodes a polyprotein precursor containing numerous tandem filaggrin repeats. This structure is similar to that of mouse; however, the human filaggrin repeat is much longer (972 base pairs; 324 amino acids) and shows little sequence homology to the mouse protein. Also, data presented here reveal that the human filaggrin repeats show considerable sequence variations; such polymorphism is not found in the mouse. Furthermore, chromosomal mapping data revealed that the human gene is located at 1q21, indicating that the polymorphism is confined to a single locus. By peptide mapping, we define a short linker sequence within the human filaggrin repeat that is excised by proteolysis to yield functional molecules. Finally, we show by in situ hybridization that human filaggrin precursor gene expression is tightly regulated at the transcriptional level in terminally differentiating epidermis and that this represents a useful system in which to study intermediate filament-intermediate filament-associated protein interactions as well as disorders of keratinization.
cAMP stimulates transcription of the beta 2-adrenergic receptor gene in response to short-term agonist exposure.
Authors: Collins S , Bouvier M , Bolanowski M A , Caron M G , Lefkowitz R J
Published: May 2006

In addition to conveying cellular responses to an effector molecule, receptors are often themselves regulated by their effectors. We have demonstrated that epinephrine modulates both the rate of transcription of the beta 2-adrenergic receptor (beta 2AR) gene and the steady-state level of beta 2AR mRNA in DDT1MF-2 cells. Short-term (30 min) exposure to epinephrine (100 nM) stimulates the rate of beta 2AR gene transcription, resulting in a 3- to 4-fold increase in steady-state beta 2AR mRNA levels. These effects are mimicked by 1 mM N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate (Bt2cAMP) or foskolin but not by phorbol esters. The half-life of the beta 2AR mRNA after addition of actinomycin D (46.7 +/- 10.2 min; mean +/- SEM; n = 5) remained unchanged after 30 min of epinephrine treatment (46.8 +/- 10.6 min; mean +/- SEM; n = 4), indicating that a change in transcription rate is the predominant factor responsible for the increase of beta 2AR mRNA. Whereas brief exposure to epinephrine or Bt2cAMP does not significantly affect the total number of cellular beta 2ARs (assessed by ligand binding), continued exposure results in a gradual decline in beta 2AR number to approximately 20% (epinephrine) or approximately 45% (Bt2cAMP) of the levels in control cells by 24 hr. Similar decreases in agonist-stimulated adenylyl cyclase activity are observed. This loss of receptors with prolonged agonist exposure is accompanied by a 50% reduction in beta 2AR mRNA. Transfection of the beta 2AR promoter region cloned onto a reporter gene (bacterial chloramphenicol acetyltransferase) allowed demonstration of a 2- to 4-fold induction of transcription by agents that elevate cAMP levels, such as forskolin or phosphodiesterase inhibitors. These results establish the presence of elements within the proximal promoter region of the beta 2AR gene responsible for the transcriptional enhancing activity of cAMP and demonstrate that beta 2AR gene expression is regulated by a type of feedback mechanism involving the second messenger cAMP.
Multiple alpha subunits of guanine nucleotide-binding proteins in Dictyostelium.
Authors: Pupillo M , Kumagai A , Pitt G S , Firtel R A , Devreotes P N
Published: May 2006

Previous results have shown that chemotaxis and the expression of several classes of genes in Dictyostelium discoideum are regulated through a cell surface cAMP receptor interacting with guanine nucleotide-binding proteins (G proteins). We now describe cloning and sequencing of cDNAs encoding two G alpha protein subunits from Dictyostelium. The derived amino acid sequences show that they are 45% identical to each other and to G alpha protein subunits from mammals and yeast. Both cDNAs are complementary to multiple mRNAs that are differentially expressed during development. This evidence and analysis of mutants presented elsewhere suggest that they have distinct physiological functions.
cDNA cloning and expression of the human A-type platelet-derived growth factor (PDGF) receptor establishes structural similarity to the B-type PDGF receptor.
Authors: Claesson-Welsh L , Eriksson A , Westermark B , Heldin C H
Published: May 2006

The primary structure of the human A-type receptor for platelet-derived growth factor (PDGF) has been determined. A 6.5-kilobase (kb) transcript was identified through low-stringency hybridization with a probe derived from the B-type PDGF receptor cDNA. The sequence of a cDNA clone corresponding to the 6.5-kb transcript contains an open reading frame that predicts a 1089-amino acid growth factor receptor-like molecule, which displays 44% overall amino acid similarity with the PDGF B-type receptor. The two receptors have a similar domain organization, with five immunoglobulin-like domains extracellularly and an intracellular split protein tyrosine kinase domain. Transfection of the new cDNA into COS cells led to the expression of a protein specifically recognized by an antiserum previously shown to react with the PDGF A-type receptor. The expressed protein was shown to display high-affinity binding of all three 125I-labeled dimeric forms of PDGF A and B chains in a manner that is characteristic for the PDGF A-type receptor.
Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin
Authors: Hicks Glenn R. , Rayle David L. , Jones Alan M. , Lomax Terri L.
Published: May 2006

Plasma membrane vesicles were isolated from zucchini (
Cucurbita pepo
) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure (
ca.
95%) plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-[7-
3
H]IAA ([
3
H]N
3
IAA), in a manner similar to the accumulation of [
3
H]IAA. The association of the [
3
H]N
3
IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of [
3
H]N
3
IAA to plasma membrane vesicles prior to exposure to UV light (15 sec; 300 nm) and detected by subsequent NaDodSO
4
/PAGE and fluorography. When the reaction temperature was lowered to - 196°C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Triton X-100 (0.1%) increased the specific activity of labeling and reduced the background, which suggests that the labeled polypeptides are intrinsic membrane proteins. The labeled polypeptides are of low abundance, as expected for auxin receptors. Further, the addition of IAA and auxin analogues to the photoaffinity reaction mixture resulted in reduced labeling that was qualitatively similar to their effects on the accumulation of radiolabeled IAA in membrane vesicles. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors.
Epididymis is a principal site of retrovirus expression in the mouse.
Authors: Kiessling A A , Crowell R , Fox C
Published: May 2006

High levels of retrovirus particles are present in the reproductive tract of male mice. In this report epithelial cells that line the lumen of the epididymis are shown to be a principal site of virus synthesis. Aggregates of free virus were evident in the epididymal lumen in addition to the sperm-associated virus previously reported. Large intraluminal cells with characteristics of macrophages and engorged with virus particles were also seen. Virus particles were not detected in testis, liver, brain, or spleen. Thus, the epididymal epithelium is a principal reservoir for retrovirus expression. The virus would be ejaculated as free, cell-associated, and sperm-bound particles. The high level of expression and the relative isolation of epididymal virus from the immune system may relate to venereal transmission of retrovirus infections in mice and humans.
Molecular cloning and primary structure of human glial fibrillary acidic protein.
Authors: Reeves S A , Helman L J , Allison A , Israel M A
Published: May 2006

Glial fibrillary acidic protein (GFAP) is an intermediate-filament (IF) protein that is highly specific for cells of astroglial lineage, although its tissue-specific role is speculative. Determination of the primary structure of this protein should be of importance for understanding the functional role it plays in astroglia. Therefore, we isolated a cDNA clone encoding this protein and determined its nucleotide sequence. The predicted amino acid sequence indicates that GFAP shares structural similarities--particularly in the central rod domain and to a lesser degree in the carboxyl-terminal domain--with other IF proteins found in nonepithelial cell types. Considerable sequence divergence in the amino-terminal region of GFAP suggests that the tissue-specific functions of this IF protein might be mediated through this region of the molecule. In contrast, conservation of structural characteristics and a moderate degree of sequence conservation in the carboxyl-terminal region suggest functional similarities. Blot hybridization analysis using the GFAP cDNA as a probe failed to detect GFAP mRNA in both normal and neoplastic human tissues in which IF proteins other than GFAP are known to be expressed.
Electrostatic fields in the active sites of lysozymes.
Authors: Sun D P , Liao D I , Remington S J
Published: May 2006

Considerable experimental evidence is in support of several aspects of the mechanism that has been proposed for the catalytic activity of lysozyme. However, the enzymatically catalyzed hydrolysis of polysaccharides proceeds over 5 orders of magnitude faster than that of model compounds that mimic the configuration of the substrate in the active site of the enzyme. Although several possible explanations for this rate enhancement have been discussed elsewhere, a definitive mechanism has not emerged. Here we report striking results obtained by classical electrodynamics, which suggest that bond breakage and the consequent separation of charge in lysozyme is promoted by a large electrostatic field across the active site cleft, produced in part by a very asymmetric distribution of charged residues on the enzyme surface. Lysozymes unrelated in amino acid sequence have similar distributions of charged residues and electric fields. The results reported here suggest that the electrostatic component of the rate enhancement is greater than 9 kcal.mol-1. Thus, electrostatic interactions may play a more important role in the enzymatic mechanism than has generally been appreciated.
Direct measurement of interstitial convection and diffusion of albumin in normal and neoplastic tissues by fluorescence photobleaching.
Authors: Chary S R , Jain R K
Published: May 2006

Macromolecular transport through the interstitial space of a tissue occurs by convection and diffusion. The convective component of transport results from interstitial fluid flow. There have been no direct measurements of the magnitude or direction of interstitial fluid flow in tissues to date. Using fluorescence recovery after photobleaching, we have measured interstitial fluid velocities and the diffusion coefficient of bovine serum albumin in normal and neoplastic tissues grown in a thin, transparent window in the ear of a rabbit. A well-defined laser beam was focused on a region within the interstitium of the fluorescence-bathed tissue. A short pulse of laser irradiation extinguished the fluorescence emanating from this selected region. The recovery of fluorescence due to diffusion and convection within the medium was monitored and analyzed to yield values of the diffusion coefficient and the fluid velocity. The average fluid velocity was about 0.6 microns/s, and albumin diffusion coefficients were 5.8 +/- 1.3 x 10(-7) cm2/s and 6.3 +/- 1.9 x 10(-7) cm2/s in normal and neoplastic tissues, respectively. The interstitial fluid flow, in general, was directed into postcapillary venules. The results obtained in this study should provide the impetus for further investigation into the diffusion and convection in various tissues under normal and pathological conditions.
Mouse Mos protooncogene product is present and functions during oogenesis.
Authors: Paules R S , Buccione R , Moschel R C , Vande Woude G F , Eppig J J
Published: May 2006

We have identified the mouse Mos-encoded protein product, p39mos, in maturing mouse oocytes and have shown that it is indistinguishable from the product expressed in Mos-transformed NIH 3T3 cells. p39mos is detected in oocytes arrested in the first meiotic prophase, during germinal-vesicle breakdown, metaphase I, anaphase I, and in ovulated eggs. We show that microinjection of three different Mos antisense (but not sense) oligodeoxyribonucleotides into germinal vesicle-stage oocytes prevents first polar-body emission and therefore interrupted the normal progression of meiosis. These results show that in mouse oocytes, as in the amphibian Xenopus [Sagata, N., Oskarsson, M., Copeland, T., Brumbaugh, J. & Vande Woude, G.F. (1988) Nature (London) 335, 519-525], the product of Mos is necessary for normal meiotic maturation.
Genetics of sexual isolation between two sibling species, Drosophila simulans and Drosophila mauritiana.
Authors: Coyne J A
Published: May 2006

Drosophila simulans and Drosophila mauritiana are sibling species that show substantial sexual isolation in one of their two reciprocal hybridizations. Genetic analysis reveals that in females this isolation is caused by at least one recessive gene on each autosome, while the X chromosome has little or no effect. Our results, combined with those of previous studies, show that in Drosophila the genetics of sexual isolation differs from that of postzygotic reproductive isolation, which invariably involves large effects of the X chromosome.
Cell proliferation and thymocyte subset reconstitution in sublethally irradiated mice: compared kinetics of endogenous and intrathymically transferred progenitors.
Authors: Penit C , Ezine S
Published: May 2006

After sublethal (6 Gy) whole-body irradiation, the C57BL/Ba (Thy-1.1) murine thymus regenerated in two waves, on days 3-10 and 25-32, separated by a severe relapse. The second phase of depletion-reconstitution reproduced the first one, in a less synchronous manner. The depletion affected all cell subsets, but CD4+ CD8- cells decreased later than immature cells. Cell proliferation, measured by BrdUrd incorporation, started on day 3 after irradiation and concerned CD4- CD8-, CD4- CD8+, and CD4+ CD8+ cells, sequentially. CD4+ CD8- cells never represented a significant percentage of cycling cells. When irradiation was immediately followed by an intrathymic injection of 10(5) C57BL/Ka (Thy-1.2) bone marrow cells, the relapse in thymus reconstitution was no longer observed. Detected with anti-Thy-1.2 antibodies, donor cells started cycling on day 14 and showed only one wave of proliferation. In these chimeras, recipient thymocytes behave exactly like thymocytes of solely irradiated mice. Intrathymically transferred CD4- CD8- thymocytes (10(5] showed the same proliferation kinetics as endogenous cells, with a peak in number on day 10 but completely disappeared from the thymus on days 14-21. These data reflect maturational differences between intrathymic and bone marrow precursor cells and suggest different radiosensitivities not linked to proliferative status. The resting state of the thymus immigrants was shown by the absence of Thy-1 acquisition by bone marrow cells continuously labeled for 10 days with BrdUrd in vivo before intrathymic transfer. When such labeled bone marrow cells were injected in the thymus, only the minor BrdUrd- subset gave rise to Thy-1+ cells.
Transgenic mouse model for human gastric carcinoma.
Authors: Koike K , Hinrichs S H , Isselbacher K J , Jay G
Published: May 2006

To understand the pathogenesis that may be induced by human adenovirus type 12 (Ad12), we have generated transgenic mice carrying the Ad12 early region 1 under control of the mouse mammary tumor virus long terminal repeat. Eleven of 11 male founder mice, but only 2 of 12 females, died between 3 to 4 mo of age. Death was associated with presence of tumors at or near the squamocolumnar junction of the stomach. Microscopically, these multifocal tumors appeared to arise from hyperplastic epithelium and showed features consistent with adenocarcinoma or adenosquamous carcinoma. High levels of expression of both the Ad12 E1A and E1B genes were seen in the tumor-bearing stomach. Various levels of expression were also detected in other tissues, although the stomach was the only organ with detectable pathology. These observations suggest an organ-specific action of the Ad12 early region 1 gene products. This transgenic mouse model provides an experimental system for studying the development of human carcinomas at sites of transition from squamous to columnar epithelium.
Constitutive expression of c-myc oncogene confers hormone independence and enhanced growth-factor responsiveness on cells transformed by human papilloma virus type 16.
Authors: Crook T , Almond N , Murray A , Stanley M , Crawford L
Published: May 2006

The effect of constitutive expression of the c-myc oncogene on the biological properties of cells transformed or immortalized by human papilloma virus type 16 (HPV16) was studied. Whereas transfection of HPV16 alone into primary baby mouse kidney (BMK) cells failed to generate any immortalized cell lines unless the tumor promoter phorbol 12-myristate 13-acetate was present, cotransfection of HPV16 with a plasmid that constitutively expresses murine c-myc (pSVc-myc-1) generated numerous rapidly growing colonies of cells. Cell lines transformed by HPV16 and pSVc-myc-1 did not require phorbol ester or steroid hormones for growth and were tumorigenic in syngeneic immunocompetent mice. Transfection of pSVc-myc-1 into established cell lines transformed by HPV16 and the v-fos oncogene increased the growth rate and saturation density of these lines severalfold, allowed growth in low-serum medium, and abolished the requirement of these cell lines for glucocorticoids or progestogens. Transfection of the EJ-ras oncogene into these lines did not significantly affect any of these properties.